Axl Kinase as a Key Target for Oncology: Focus on Small ... · Small Molecule Inhibitors To our...

Review

Axl Kinase as a Key Target for Oncology: Focus on SmallMolecule Inhibitors

Cl�emence Feneyrolles, Aur�elia Spenlinhauer, L�ea Guiet, B�en�edicte Fauvel, B�en�edicte Dayd�e-Cazals, PierreWarnault, Gw�ena€el Chev�e, and Aziz Yasri

AbstractReceptor tyrosine kinases (RTK) are transmembrane receptors that regulate signal transduction in cells. As a

member of the TAM (Tyro-3, Axl, Mer) RTK subfamily, Axl regulates key processes such as cell growth,

migration, aggregation, and apoptosis through several pathways. Its overexpression/overactivation has been

underlined in several conditions, especially cancers, and in both chemotherapy and targeted therapy

sensitivity loss. In this review, we propose to highlight the therapeutic implication of Axl, starting with the

pathways it regulates, validating its interest as a therapeutic target, and defining the tools available to develop

strategies for its inhibition.We especially focus on small molecule inhibitors, their structure, inhibition profile,

and development stages. Mol Cancer Ther; 13(9); 2141–8. �2014 AACR.

IntroductionTAM family and AxlAxl (Ark, Ufo) is an oncogene originally isolated from 2

patients with chronic myeloid leukemia (CML) and achronic myeloproliferative disorder in 1988 (1–3). Axl wasidentified as a member of the TAM family, a receptortyrosine kinase (RTK) subfamily comprising Tyro-3 (alsocalled Sky), Axl and Mer. Axl is an ubiquitous receptorpredominantly expressed in the brain (hippocampus andcerebellum), in immunity cells, blood platelets, endothelialcells, skeletal muscle, testis, heart, liver, and kidney (4).TAM receptors were initially found in cancer cells (2, 4),which guided the research in the field of oncology. TheseRTKs contribute to a carcinogenesis phenomenonvia over-expression or ectopia, regulating processes such as migra-tion, cell survival, andproliferation (3, 5).Theyare involvedin several cancers, such as pancreas, breast, prostate, non–small cell lungcancer (NSCLC),CML,andothercancers (6).Structurally, TAM receptors possess an extracellular, a

transmembrane, and an intracellular domain. The extra-cellular region is composed of 2 immunoglobulin-like and2 fibronectin domains that are involved in the binding ofthe natural ligand (7). The intracellular domain consists ofthe tyrosine kinase domain, which presents a conservedsequence KW-(I/L)-A-(I/L)-ES, a particularity of TAMreceptors (Fig. 1; ref. 8).

Axl signaling pathwaysIn 1995, the vitamin K–dependent protein named Gas6

(growth arrest–specific 6)was identifiedas thefirst knownendogenous ligand ofAxl (7). In 2005, the crystal structureof this ligand complexed to its receptor Axl (only theextracellular part) was solved (PDB ID code 2C5D), prov-ing that the receptor dimerization occurs without anyreceptor/receptor (on the extracellular domain) orligand/ligand contacts (9).

Although classical activation of Axl starts by the bind-ingof its natural ligandGas6onto its extracellular domain,several studies have also demonstrated that Axl could beactivated by a ligand-independent mechanism or by across-talk with other signaling pathways (10). The Gas6–Axl binding induces the dimerization of the RTK, allow-ing a transautophosphorylation of the cytoplasmicdomains. Three tyrosine residues (Y779, Y821, and Y866)are supposed to be phosphorylation sites (11), mostlybecause they can modulate the interaction of Axl withsignaling molecules such as phospholipase C, PI3K, Src,Lck, and Grb2 (9, 11). An analogy with the finding onMerwould underlie the possibility of 3 other phosphorylationsites that are conserved among theTAMfamily (Y698, Y702,and Y703; refs. 8, 12). However, it is interesting to note thatY866 of Axl is also suspected to be an inhibitor site ofphosphorylation (13).

Once activated, the catalytic capacity of Axl increases,enabling other cytoplasmic signaling substrates to bephosphorylated. Depending on the cell type and theenvironment, the activation of Axl can stimulate variouscellular functions (Fig. 1). Through the Src/MAPK/ERKpathway, Axl regulates cell survival and proliferation (14,15). Through the PI3K/Akt pathway, it regulates survivaland stimulates expression of antiapoptotic proteins, nota-bly through NF-kB modulation (16, 17). Through thePI3K/p38/MAPK pathway, Axl regulates cell migration

Authors' Affiliation: OriBase Pharma, Cap Gamma, Montpellier, France

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Aziz Yasri, OriBase Pharma, Cap Gamma, 1682rue de la Valsi�ere, CS17383, Montpellier 34189, France. Phone: 33-0-467727670; Fax: 33-0-467727679; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-1083

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 2141

on February 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 19, 2014; DOI: 10.1158/1535-7163.MCT-13-1083





http://mct.aacrjournals.org/



and proliferation. Activation of Axl, mainly by way ofatypical activation, also leads to cell aggregation (18) and aproangiogenic behavior (10, 19). Taken together, thesedata suggest that Axl is an important regulator of cellsurvival, proliferation, aggregation, migration, adhesionand is necessary for angiogenesis.

Mechanisms of negative control have been proposed,such as dephosphorylation by the C1-TEN phosphatase,which can bind Axl and whose overexpression correlateswith a downregulation of survival, proliferation, andmigration (20, 21). Ubiquitination of Axl by ubiquitinligase C, which leads to its downregulation, has also beenstudied. Among atypical activation mechanisms, Kamataand colleagues reported that behavior of numerouskinases and phosphatases was modified depending onthe intracellular oxidation rate (22). In particular, reactiveoxygen species (ROS) generation in cells by hydrogenperoxide adjunction resulted in Axl activation within ashort time (23). More importantly, it was shown that thisactivation did not induce ubiquitination of Axl by ubiqui-

tin ligase C, as it is the case of classical Gas6 activation. Asa matter of fact, oxidative stress might inhibit Axl ubiqui-tination and therefore downregulation through lysosomedegradation (24).

Involvement in cancersKnowing the pathways regulated by Axl, altered activ-

ity of this RTK has been detected in a variety of cancertypes, such as pancreas, breast, prostate, NSCLC, CML,and other cancers (25). To date, no activating mutationsin the Axl gene have been implicated in malignanttransformations.

The Axl kinase is postulated to contribute to a carcino-genesis phenomenon via overexpression or ectopia. Inparticular, Axl overexpression has been reported in var-ious oncology indications, and the expression level of Axlis directly proportional to tumor grade and predicts poorpatient survival rate. In addition, Axl is involved inmetastasis as an essential regulator of the epithelial–mes-enchymal transition (EMT) process in cancer cells (26).

Figure 1. Schematic structure of aTAM receptor. A, the kinases ofthe TAM family are membranereceptors structurally composedof two immunoglobulin (Ig)-likedomains (yellow) and twofibronectin domains (pink) for theirextracellular part and of a well-conserved kinase domain (darkblue) with a particular highlyconserved sequence (KW-(I/L)-A-(I/L)-ES) within it. B, Gas6 (green)binding to Axl occurs with a 2:2stoichiometry, inducing thedimerization of the receptor.This dimerization induces theactivation of different pathways(light blue) leading to cellproliferation, migration, andsurvival.

Feneyrolles et al.

Mol Cancer Ther; 13(9) September 2014 Molecular Cancer Therapeutics2142




Since approximately the 2000s, several studies havedemonstrated a strong and positive impact of inhibitingAxl in cancer. The inhibition of Axl led to the decrease ofproliferation and survival in pancreatic cancer cell lines(27). In breast cancer, its inhibition decreased the migra-tion and invasion of cancer cells (28). In prostate cancer,migration, invasion, and proliferation were decreased bythe inhibition of Axl (29). In NSCLC, silencing of Axlinhibited tumor growth (30).Axl is a factor of poor prognosis when implicated in

cancer development (27, 31). Therefore, its overexpres-sion is related to high level of recurrence (32) as well asresistance mechanisms toward the effects of other anti-cancer drugs, such as imatinib (Glivec; ref. 33), erlotinib(Tarceva; refs. 34, 35), and lapatinib (Tyverb; ref. 36),on several cancers [leukemia, gastrointestinal stromaltumors (GIST), breast, and lung cancers].Taken together, all these data validate Axl as a thera-

peutic target to treat several cancers, and many researchscientists are developing strategies to inhibit this RTK.Different strategies have been elaborated, like inhibitingthe binding of Axl to its activator ligand, competing theATP to lower its activity, or reducing the expression of theprotein itself.

Biophysical and Statistical DataSo far, one X-ray tridimensional structure is available

for Axl (PDB ID code 2C5D). It concerns the extracellularpart binding its ligand Gas6, showing a ligand–receptor2:2 assembly (9). To date, there are no published dataavailable about the three-dimensional structure of thekinase domain of Axl.Kinases with a high identity score toward Axl, such as

same family kinases, could be expected to express aninhibition profile similar to Axl. The availability of thecrystal structure of Mer (37), Met (38), and IGF1R (39),which are closely related to Axl regarding both the ATP-binding site and the whole kinase domain, allowed Mol-lard and colleagues (40) to build a predictive homologymodel of Axl.In 2011,Metzand colleaguespublisheda comprehensive

kinome interaction network. More than 150,000 inhibitiondata of 3,800 ligands toward 172 kinases were collected(41). To determine whether 2 kinases can be classified asrelated or not, mathematical tools were used. Scaled Shan-non entropy (SSE; ref. 42) was used to measure the reli-ability of information, using the number of data and theirdispersion (Supplementary Table S1). The authors deter-mined an SSE of �0.4 as minimum threshold. Other toolswere used, such as Pij (Hopkins pharmacology interactionstrength; ref. 43), Rij (Pearson correlation coefficient), andTij (activityprofileTanimoto; ref. 44), to assess the relevanceof the interaction, using the number of ligandswith similarbioactivity on 2 kinases of the ligands tested on both thesekinases, and the linear correlation between the variables.The authors determined values on these indexes to estab-lish a strong pharmacologic relationship between 2 kinasesas Pij � 0.6, Rij � 0.45, and Tij � 0.55.

According to the authors,more than 90%of kinases thatshare sequence identity higher than 60% also have Pij �0.6. Regarding Axl, only 3 other kinases match theseparameters and can therefore be classified as related toAxl: Flt4, MAP4K5, and Ret (Supplementary Table S1).Furthermore, Tyro-3, which is the only kinase among the172 reported on the study that share more than 60%sequence identity with Axl, has a Pij of 0.18. The behaviorofAxl is different from themajority of kinases at least fromthat point of view. None of the kinases used to buildthepredictivemodelmatchmore thanone criterion exceptthe SSE. (Note that no data were collected on Mer on thatarticle.) Available data on Axl are not only rare but alsoquite contradictory.

Small Molecule InhibitorsTo our knowledge, no inhibitor clearly designed to

inhibit Axl is currently marketed. In particular, none ofthe listed molecules in the marketed drugs and clinicalphase II sections have been developed as preclinical orclinical Axl inhibitors. Most of the anti-Axl developeddrug candidates are in preclinical stages. However, someFDA-approved or clinical molecules, developed to inhibitother kinases, were evaluated against Axl given its inter-est. Some potent Axl kinase inhibitors in development aredescribed in the literature (45, 46). However, as few dataare available regarding their selectivity and their appli-cation as treatment, we do not describe them here. Here,we summarize properties of molecules that are FDAapproved, in clinical trial, or planned to be within thenext year. All the molecules listed below are shown inSupplementary Fig. S1.

Marketed drugsBosutinib (SKI606, PF5208763, Bosulif; Pfizer) was

authorized by the FDA and the European MedicinesAgency (EMA) in 2012 for the treatment of patients withPhþ CML (Philadelphia chromosome-positive CML;refs. 47, 48). It is an inhibitor that mainly targets Bcr-Abl1and Src (IC50 ¼ 1 and 3.5 nmol/L, respectively) and, to alesser extent, Axl (IC50 ¼ 174 nmol/L; refs. 49, 50). Bosu-tinib is currently in a phase II clinical trial for glioblasto-mas and breast cancer.

Cabozantinib (XL184, Cometriq; Exelixis) was autho-rized by the FDAand the EMA in 2012 for the treatment ofmedullary thyroid cancer (51, 52). It is a multitargetedkinase inhibitor (MTKI) acting on VEGFR-2 (IC50 ¼ 0.035nmol/L),Met (IC50¼ 1.3 nmol/L), Ret (IC50¼ 5.2 nmol/L)as primary targets, as well as on Kit (IC50 ¼ 14.3 nmol/L),Flt-3 (IC50 ¼ 11.3 nmol/L), and Axl (IC50 ¼ 7 nmol/L;ref. 53). It is currently in a phase III clinical trial forhepatocellular, renal, and prostate cancers.

Sunitinib (SU11248, Sutent; Pfizer) was authorized bythe FDA in 2006 commonly for both indications,renal cell carcinomas and imatinib-resistant gastrointes-tinal stromal tumors, and in 2011 for pancreatic neuroen-docrine tumors. It is an inhibitor targeting Flt-3 (IC50¼ 0.5nmol/L), VEGFR-2 (IC50 ¼ 20 nmol/L), Kit (IC50 ¼ 22

Axl Kinase as an Emerging Key Target for Drug Discovery

www.aacrjournals.org Mol Cancer Ther; 13(9) September 2014 2143




nmol/L), as well as Axl (IC50 ¼ 5 nmol/L; ref. 54). It iscurrently in clinical trials for NSCLC and renal cellcarcinoma.

Clinical phase IIForetinib (XL880, GSK1363089; Exelixis, GlaxoSmithK-

line) is anMTKI targetingmainlyMet, VEGFR-2, Ron, Tie-2, and Kit (IC50 ¼ 0.4, 0.86, 3, 1.1, and 3.7 nmol/L,respectively). It also exhibits a significant activity towardAxl (IC50¼ 11 nmol/L; refs. 36, 55). It is a type II inhibitor,as illustrated by the X-ray crystal structure of foretinibwithin Met active site (PDB ID code 3LQ8). It is presentlyin a phase II clinical trial for NSCLC and in phase I/II forbreast cancers.

MGCD265 (Mirati Therapeutics) is a potent inhibitor ofMet (IC50¼ 1 nmol/L), Ron (IC50¼ 2 nmol/L), VEGFR 1/2/3 (IC50¼ 3, 3, and 4 nmol/L, respectively), Tie-2 (IC50¼7 nmol/L), and Axl (1 nmol/L � IC50 � 10 nmol/L;ref. 56). It is currently in phase I/II clinical trials forNSCLC and in phase I for advanced malignancies.

Clinical phase IBMS777607 (ASLAN002; Aslan Pharmaceuticals and

Bristol-Myers Squibb) is a type II (57) Met inhibitor (PDBID code 3F82; IC50 ¼ 3.9 nmol/L) that also acts on Ron(IC50 ¼ 1.8 nmol/L), Flt-3 (IC50 ¼ 16 nmol/L), andthe TAM family Tyro-3 (IC50 ¼ 4.3 nmol/L), Mer (IC50

¼ 14 nmol/L), and Axl (IC50 ¼ 1.1 nmol/L; ref. 58). It iscurrently in a phase I clinical trial for advanced or met-astatic solid tumors.

LY2801653 (Eli Lilly Company) is an inhibitor thatexhibits strong activity toward Ron (IC50 ¼ 0.8 nmol/L),Met (IC50¼ 4.7 nmol/L), Axl (IC50¼ 11 nmol/L), and Flt-3(IC50¼ 31 nmol/L), (59). It is currently in a phase I clinicaltrial for advanced cancers.

S49076 (Servier) is described as an MTKI exhibitingactivity toward Met (IC50 ¼ 2 nmol/L), FGFR-1 (IC50 ¼68 nmol/L), FGFR-2 (IC50 ¼ 95 nmol/L), FGFR-3 (IC50 ¼200 nmol/L), and Axl (IC50 ¼ 56 nmol/L). Its selectivitywas evaluated against 442 kinases, among which morethan 25 were identified as hits at 100 nmol/L (60). It iscurrently in a phase I clinical trial for advanced solidtumors.

BGB324 (R428; Rigel Pharmaceuticals, BergenBio) iscurrently one of the most promising molecules to targetAxl regarding both its activity (IC50 ¼ 14 nmol/L) andits selectivity. Indeed, the inhibition of Axl by thismolecule was 50 to 100 times lower than for the 133kinases tested, with the exception of Tie-2 (3 times), Flt-4 (5.5 times), Flt-1 (8 times), Ret (9 times), Abl (9.3times), and the other members of TAM family Tyro-3(14 times) and Mer (16 times; ref. 61). BGB324 justentered clinical phase I in 2013 for aggressive andmetastatic cancers.

Preclinical stageTP0903 (Tolero Pharmaceuticals) exhibits strong activ-

ity towardAxl (IC50¼ 27 nmol/L; ref. 40). It was screened

against a panel of 40 kinases at 200 nmol/L and exhibitedmore than 80% inhibition on 11 kinases, includingAuroraA, Jak2, Alk, and Abl, with higher inhibition percentagesthan Axl, as well as the rest of the TAM family. It iscurrently in preclinical stages for pancreatic cancer andglioblastomas (62).

SGI7079 (Tolero Pharmaceuticals) is an Axl inhibitor(IC50 � 30 nmol/L) that also exhibits a strong activitytoward other kinases, such as Flt-3,Mer,Met, TrkA and B,Ret, Yes, Jak2, VEGFR-2, JNK3, and Abl at the same range(IC50 � 30 nmol/L), as well as 12 others with IC50 below100 nmol/L (63). It is supposed to enter clinical trial phaseI in 2014 for NSCLC and other tumor type indications.

Suspended studiesAmuvatinib (SGI-0470-02, MP470; SuperGen, Inc.,

now known as Astex Pharmaceuticals, Inc.) is an MTKItargeting c-Kit, PDGFRa, Met, Flt-3 (IC50 ¼ 10, 40, and 81nmol/L, respectively). It was shown to inhibit the phos-phorylation of Axl in MDA-MB231 breast cancer cells(EC50¼ 1mmol/L). Itwas investigated in aphase II clinicaltrial for small cell lung cancer, but its clinical developmentwas discontinued in 2012 (64).

SNS314 (Sunesis Pharmaceuticals) is a pan-Aurorakinase inhibitor. It exhibits strong activity toward itsprimary targets Aurora A/B/C (IC50 ¼ 9, 31, and 3.4nmol/L, respectively), as well as toward 7 of 219 kinases(within 100-fold), including Axl (IC50 ¼ 84 nmol/L;ref. 65). It was investigated in a phase I clinical trial forsolid tumors but suspended in 2010 as a maximum tol-erated dose was not established in the trial, and noresponses were observed (66).

Medicinal ChemistryTheAxl inhibitors discussedabove canbe classified into

2 groups according to their chemical structures.The first scheme (Fig. 2A) includes inhibitors bearing a

hinge-binding heterocycle bearing a solubilizing group(N-methylpiperazine), linked to a hydrogen bond accep-tor–substituted phenyl group by a linker. Three inhibitorsof the precedent list obey this description.

As their structure is similar, it is interesting to look attheir inhibition profile toward their respected targetkinases (Table 1).

TP0903 andSGI7079 share a similar inhibitionprofile onthe target kinases of the first scheme (Fig. 2A). They alsopossess at least 2 hydrogen bond donor/acceptor on theirhinge-bonding part and a similar hydrophilic part. Bosu-tinib, however, possesses only one hydrogen bond accep-tor on its hinge-binding heterocycle. The major part of itsactivity is therefore related to affinity for less-conservedparts of the kinases, reducing the array of hit kinases, asAurora A, for instance.

Although these 3 inhibitors exhibit the same structuralskeleton as described in Fig. 2A, heterocycle and substit-uent nature induce a very specific inhibition profile foreach of them on the targeted kinases. It is difficult toestablish a relationship between structure and activity of

Feneyrolles et al.





these kinases; nevertheless, these 3 inhibitors exhibit 3IC50 lower than200nmol/Lon thepresentedkinasepanel,with a high inhibition of Axl, especially for TP0903 andSGI7079.The second scheme that can be observed (Fig. 2B)

presents 5 inhibitors with a hinge-binding heterocycle,with or without bearing a solubilizing group, linked to anoptionally ortho-fluoro substituted phenyl group by anoxygen linker, followed by a sequence of 2 to 4 hydrogendonors/acceptors linked to an optionally para-fluorosubstituted phenyl group. The oxygen linker is in paraposition of a nitrogen atom of the hinge-binding hetero-cycle, with the exception of LY2801653.Because the structure of inhibitors is highly similar, we

look at their inhibition profile toward their respectedtarget kinases (Table 1). Cabozantinib and foretinib struc-turally differ only by their solubilizing group and a fluor

substituent. These slight differences lead to significantvariation in the inhibition activity of VEGFR and Ronkinases (25-fold). MGCD265 is also structurally similar toforetinib and cabozantinib, with the notable exception oftheir hinge-binding heterocycle, the presence of a fluorineatom at the other edge of themolecule, and the position ofone of its hydrogen bond donor. However, these smalldifferences still confer MGCD265 an inhibition profilevery close to that of foretinib. BMS777607 and LY2801653mainly differ from their hinge-binding scaffold and,therefore, display a similar inhibition profile.

These 5 inhibitors strongly inhibit Met and Axl, with alow nanomolar activity. They exhibit similar kinase inhi-bition profiles on the targeted kinases with at least 3 IC50

less than 10 nmol/L, including Axl.Of all the previously cited Axl inhibitors, three inhibi-

tors are missing in the last 2 schemes: sunitinib, S49076,

Figure 2. Axl inhibitor structure schemes. A, the first scheme includes inhibitors of Axl: TP-0903, SGI7079, and bosutinib. B, the second scheme includesinhibitors of Axl: cabozantinib, foretinib, MGCD265, BMS 777607, and LY280165.






and BGB324. Their structures are quite different fromthose of the other inhibitors and from one another andwere, therefore, unsorted.

DiscussionAxl has been validated as a therapeutic target of choice

for several cancers aswell as implicated inmany resistancephenomena to accurate therapies. Despite its major inter-est, so far no Axl-specific inhibitor exists on the market.Moreover, none of the marketed or clinical phase II mole-cules have been designed to target Axl. Thus, it has to bestated thatBGB324, thefirst-in-classAxl inhibitor currentlyin clinical phase I, and TP0903, which is in preclinicaldevelopment, are possibly themost specificAxl inhibitors.The design of such an Axl inhibitor is a challenge due tothe absence of three-dimensional structure of its kinasedomain. Moreover, a statistical study showed that theinhibition profile of Axl cannot be related to sequenceidentity as could be done for 90% of all kinases.

Several molecules do inhibit Axl, but most of them areMTKIs, and Axl is an additional target, not the main one.Most of these molecules match 2 schemes that can berelated to type I inhibitors, targeting the active conforma-tion, called DFG-in, and type II inhibitors known to targetthe inactive conformation, called DFG-out. Thus, exper-imental evidence of such thermodynamically stabilizedDFG-out conformation has only been reported for a fewkinases, excluding Axl (67, 68). These patterns may pro-vide clues leading one to believe the existence of a DFG-out conformation for Axl.

Although it has not been addressed in this review, theexpression of Axl is upregulated in a variety of otherdiseases, including endometriosis and cardiovasculardiseases (69, 70). In addition, Axl appears to play acrucial role in innate immunity (71). Small moleculeinhibitors of the TAM receptors may be reasonable

candidates for potential therapies in acute sepsis andfor new generation of vaccine adjuvants for immuniza-tion (71). Also, it has to be mentioned that other ways toregulate the activation of Axl are being developed, suchas monoclonal antibodies targeting the kinase or itsligand Gas6, as well as aptamers to downregulate itsexpression (72, 73).

ConclusionSince the discovery of Axl in 1988, Axl has been studied

and validated as a therapeutic target of choice for severaldiseases. Despite the fact that no specific Axl inhibitor canbe found on the market today, there has been increasinginterest in Axl, leading to a better comprehension of thiskinase moving forward.

Design and development of Axl inhibitors is a challengedue to the absence of three-dimensional structure of itskinase domain, and only classical medicinal chemistryapproacheshave improvedscientificknowledgeaboutAxl.

Statistical studies have shown that the inhibition profileof Axl cannot be related to sequence identity as could bedone for 90% of kinases. Several molecules do inhibit Axl,but most of them are MTKIs, and Axl is an additionalsecondary target, not the main one.

There is no doubt that more studies will be carriedout, providing the data needed to achieve importantfindings about structural, chemical, pharmacological,and therapeutic aspects of this target. An Axl-specificinhibitor will be an efficient treatment for some aggres-sive diseases such as cancers and for counteracting Axl-induced resistance.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Received December 31, 2013; revised April 15, 2014; accepted May 5,2014; published OnlineFirst August 19, 2014.

Table 1. Inhibition profile of the inhibitors of schemes 1 and 2

Scheme 1 Kinase inhibition profile

IC50 Src Bcr-Abl Aurora A Axl

TP0903 (40) — >90% inhibitionat 200 nmol/L

100% inhibitionat 200 nmol/L

27 nmol/L

SGI7079 (63) 290 nmol/L �30 nmol/L 56.6 nmol/L �30 nmol/LBosutinib (74, 75) 3.5 nmol/L 1 nmol/L Kd > 10 mmol/L 174 nmol/L

Scheme 2 Kinase inhibition profile

IC50, nmol/L Met VEGFR Ret Ron Axl

Cabozantinib (76) 1.3 0.035 5.2 124 7Foretinib (77) 0.4 0.86 1 5 11MGCD265 (56) 1 3 — 2 1 � IC50 � 10BMS777607 (58) 3.9 180 — 1.8 1.1LY2801653 (59) 4.7 Nonactive 590 0.8 11

NOTE: The table includes the inhibition of the Axl's inhibitors among their main targeting kinases according to the scheme they rely on.

Feneyrolles et al.





References1. Liu E, Hjelle B, Bishop JM. Transforming genes in chronic myeloge-

nous leukemia. Proc Natl Acad Sci U S A 1988;85:1952–6.2. O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C,

et al. Axl, a transforming gene isolated from primary human myeloidleukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol1991;11:5016–31.

3. Janssen JW, Schulz AS, Steenvoorden AC, SchmidbergerM, Strehl S,Ambros PF, et al. A novel putative tyrosine kinase receptor withoncogenic potential. Oncogene 1991;6:2113–20.

4. Neubauer A, Fiebeler A, Graham DK, O'Bryan JP, Schmidt CA,Barckow P, et al. Expression of axl, a transforming receptor tyrosinekinase, in normal and malignant hematopoiesis. Blood 1994;84:1931–41.

5. Nielsen-Preiss SM, Allen MP, Xu M, Linseman DA, Pawlowski JE,Bouchard RJ, et al. Adhesion-related kinase induction of migrationrequires phosphatidylinositol-3-kinaseand ras stimulationof rac activ-ity in immortalized gonadotropin-releasing hormone neuronal cells.Endocrinology 2007;148:2806–14.

6. Paccez JD, Vogelsang M, Parker MI, Zerbini LF. The receptor tyrosinekinaseAxl in cancer: Biological functions and therapeutic implications:The role of Axl in cancer. Int J Cancer 2014;134:1024–33.

7. Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell YW, et al.Axl receptor tyrosine kinase stimulated by the vitamin K-dependentprotein encoded by growth-arrest-specific gene 6. Nature 1995;373:623–6.

8. Linger RMA, Keating AK, Earp HS, Graham DK. TAM receptor tyrosinekinases: biologic functions, signaling, and potential therapeutic target-ing in human cancer. Adv Cancer Res 2008;100:35–83.

9. Sasaki T, Knyazev PG, Clout NJ, Cheburkin Y, G€ohring W, Ullrich A,et al. Structural basis for Gas6-Axl signalling. EMBO J 2006;25:80–7.

10. Ruan GX, Kazlauskas A. Axl is essential for VEGF-A-dependent acti-vation of PI3K/Akt. EMBO J 2012;31:1692–703.

11. Braunger J, Schleithoff L, Schulz AS, Kessler H, Lammers R, Ullrich A,et al. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase ismediated mainly by a multi-substrate docking-site. Oncogene1997;14:2619–31.

12. Ling L, Templeton D, Kung HJ. Identification of the major autopho-sphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosinekinase. J Biol Chem 1996;271:18355–62.

13. Burchert A,Attar EC,McCloskeyP, Fridell YW,LiuET.Determinants fortransformation induced by the Axl receptor tyrosine kinase. Oncogene1998;16:3177–87.

14. Fridell YW, Jin Y, Quilliam LA, Burchert A, McCloskey P, Spizz G, et al.Differential activation of the Ras/extracellular-signal-regulated proteinkinase pathway is responsible for the biological consequencesinduced by the Axl receptor tyrosine kinase. Mol Cell Biol 1996;16:135–45.

15. Stenhoff J, Dahlb€ack B, Hafizi S. Vitamin K-dependent Gas6 activatesERK kinase and stimulates growth of cardiac fibroblasts. BiochemBiophys Res Commun 2004;319:871–8.

16. Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phos-phatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axlmitogenic and survival activities in NIH 3T3 fibroblasts. Mol Cell Biol1997;17:4442–53.

17. Hasanbasic I, Cuerquis J, Varnum B, Blostein MD. Intracellular sig-naling pathways involved in Gas6-Axl-mediated survival of endothelialcells. Am J Physiol Heart Circ Physiol 2004;287:H1207–1213.

18. Bellosta P, Costa M, Lin DA, Basilico C. The receptor tyrosine kinaseARK mediates cell aggregation by homophilic binding. Mol Cell Biol1995;15:614–25.

19. Suleiman L, N�egrier C, Boukerche H. Protein S: a multifunctionalanticoagulant vitamin K-dependent protein at the crossroads of coag-ulation, inflammation, angiogenesis, and cancer. Crit Rev OncolHematol 2013;88:637–54.

20. Hafizi S, Alindri F, Karlsson R, Dahlb€ack B. Interaction of Axl receptortyrosine kinase with C1-TEN, a novel C1 domain-containing proteinwith homology to tensin. Biochem Biophys Res Commun 2002;299:793–800.

21. Hafizi S, Ibraimi F, Dahlb€ack B. C1-TEN is a negative regulator of theAkt/PKB signal transduction pathway and inhibits cell survival, prolif-eration, and migration. FASEB J 2005;19:971–3.

22. Kamata H, Hirata H. Redox regulation of cellular signalling. Cell Signal1999;11:1–14.

23. Konishi A, Aizawa T, Mohan A, Korshunov VA, Berk BC. Hydrogenperoxide activates the Gas6-Axl pathway in vascular smooth musclecells. J Biol Chem 2004;279:28766–70.

24. Valverde P. Effects of Gas6 and hydrogen peroxide in Axl ubiquitina-tion and downregulation. Biochem Biophys Res Commun 2005;333:180–5.

25. Verma A, Warner SL, Vankayalapati H, Bearss DJ, Sharma S.Targeting Axl and Mer kinases in cancer. Mol Cancer Ther 2011;10:1763–73.

26. Gjerdrum C, Tiron C, Høiby T, Stefansson I, Haugen H, Sandal T, et al.Axl is an essential epithelial-to-mesenchymal transition-induced reg-ulator of breast cancermetastasis and patient survival. Proc Natl AcadSci U S A 2010;107:1124–9.

27. Koorstra JBM, Karikari CA, Feldmann G, Bisht S, Rojas PL, OfferhausGJA, et al. The Axl receptor tyrosine kinase confers an adverseprognostic influence in pancreatic cancer and represents a newtherapeutic target. Cancer Biol Ther 2009;8:618–26.

28. Tai KY, Shieh YS, Lee CS, Shiah SG, Wu CW. Axl promotes cellinvasion by inducing MMP-9 activity through activation of NF-kappaBand Brg-1. Oncogene 2008;27:4044–55.

29. Paccez JD, VasquesGJ,CorreaRG, Vasconcellos JF, DuncanK,GuX,et al. The receptor tyrosine kinase Axl is an essential regulator ofprostate cancer proliferation and tumor growth and represents a newtherapeutic target. Oncogene 2013;32:689–98.

30. Li Y, Ye X, Tan C, Hongo JA, Zha J, Liu J, et al. Axl as a potentialtherapeutic target in cancer: role ofAxl in tumorgrowth,metastasis andangiogenesis. Oncogene 2009;28:3442–55.

31. Dunne PD,McArt DG, Blayney JK, KalimuthoM,Greer S,Wang T, et al.AXL is a key regulator of inherent and chemotherapy-induced invasionand predicts a poor clinical outcome in early-stage colon cancer. ClinCancer Res 2014;20:164–75.

32. Song X, Wang H, Logsdon CD, Rashid A, Fleming JB, Abbruzzese JL,et al. Overexpression of receptor tyrosine kinase Axl promotes tumorcell invasion and survival in pancreatic ductal adenocarcinoma. Can-cer 2011;117:734–43.

33. Mahadevan D, Cooke L, Riley C, Swart R, Simons B, Della Croce K,et al. A novel tyrosine kinase switch is a mechanism of imatinibresistance in gastrointestinal stromal tumors. Oncogene 2007;26:3909–19.

34. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activationof the AXL kinase causes resistance to EGFR-targeted therapy in lungcancer. Nat Genet 2012;44:852–60.

35. Giles KM, Kalinowski FC, Candy PA, Epis MR, Zhang PM, Redfern AD,et al. Axlmediates acquired resistance of headandneck cancer cells tothe epidermal growth factor receptor inhibitor erlotinib. Mol CancerTher 2013;12:2541–58.

36. Liu L, Greger J, Shi H, Liu Y, Greshock J, Annan R, et al. Novelmechanismof lapatinib resistance inHER2-positive breast tumor cells:activation of AXL. Cancer Res 2009;69:6871–8.

37. HuangX, Finerty P Jr,Walker JR, Butler-ColeC, VedadiM, SchapiraM,et al. Structural insights into the inhibited states of the Mer receptortyrosine kinase. J Struct Biol 2009;165:88–96.

38. Porter J, LumbS, Franklin RJ,Gascon-Simorte JM,CalmianoM, RicheKL, et al. Discovery of 4-azaindoles as novel inhibitors of c-Met kinase.Bioorg Med Chem Lett 2009;19:2780–4.

39. Favelyukis S, Till JH, Hubbard SR, Miller WT. Structure and autore-gulation of the insulin-like growth factor 1 receptor kinase. Nat StructBiol 2001;8:1058–63.

40. Mollard A, Warner SL, Call LT, Wade ML, Bearss JJ, Verma A, et al.Design, synthesis and biological evaluation of a series of novel Axlkinase inhibitors. ACS Med Chem Lett 2011;2:907–12.

41. Metz JT, JohnsonEF,SoniNB,MertaPJ, KifleL,HajdukPJ.Navigatingthe kinome. Nat Chem Biol 2011;7:200–2.






42. Godden JW,Bajorath J. An information-theoretic approach to descrip-tor selection for database profiling and QSAR modeling. QSAR CombSci 2003;22:487–97.

43. Paolini GV, Shapland RHB, van Hoorn WP, Mason JS, Hopkins AL.Global mapping of pharmacological space. Nat Biotechnol 2006;24:805–15.

44. Bamborough P, Drewry D, Harper G, Smith GK, Schneider K.Assessment of chemical coverage of kinome space and itsimplications for kinase drug discovery. J Med Chem 2008;51:7898–914.

45. Su�arez RM, Chevot F, Cavagnino A, Saettel N, Radvanyi F, Piguel S,et al. Inhibitors of the TAM subfamily of tyrosine kinases: synthesis andbiological evaluation. Eur J Med Chem 2013;61:2–25.

46. Rho JK, Choi YJ, Kim SY, Kim TW, Choi EK, Yoon SJ, et al. MET andAXL inhibitor NPS-1034 exerts efficacy against lung cancer cellsresistant to to EGFR kinase inhibitors due to MET or AXL activation.Cancer Res 2014;74:253–62.

47. TraskPC,CellaD,BessonN,Kelly V,Masszi T, KimDW.Health-relatedquality of life of bosutinib (SKI-606) in imatinib-resistant or imatinib-intolerant chronic phase chronic myeloid leukemia. Leuk Res2012;36:438–42.

48. Khoury HJ, Cortes JE, Kantarjian HM, Gambacorti-Passerini C, Bac-carani M, Kim D-W, et al. Bosutinib is active in chronic phase chronicmyeloid leukemia after imatinib and dasatinib and/or nilotinib therapyfailure. Blood 2012;119:3403–12.

49. Levinson NM, Boxer SG. Structural and spectroscopic analysis of thekinase inhibitor bosutinib and an isomer of bosutinib binding to the Abltyrosine kinase domain. PloS ONE 2012;7:e29828.

50. Boschelli F, Arndt K, Gambacorti-Passerini C. Bosutinib: a review ofpreclinical studies in chronic myelogenous leukaemia. Eur J Cancer2010;46:1781–9.

51. Nagilla M, Brown RL, Cohen EEW. Cabozantinib for the treatment ofadvanced medullary thyroid cancer. Adv Ther 2012;29:925–34.

52. Hart CD, De Boer RH. Profile of cabozantinib and its potential in thetreatment of advanced medullary thyroid cancer. OncoTargets Ther2013;6:1–7.

53. Yakes FM,ChenJ, Tan J, Yamaguchi K, Shi Y, YuP, et al. Cabozantinib(XL184), a novel MET and VEGFR2 inhibitor, simultaneously sup-presses metastasis, angiogenesis, and tumor growth. Mol CancerTher 2011;10:2298–308.

54. Kitagawa D, Yokota K, Gouda M, Narumi Y, Ohmoto H, Nishiwaki E,et al. Activity-based kinase profiling of approved tyrosine kinaseinhibitors. Genes Cells 2013;18:110–22.

55. Qian F, Engst S, Yamaguchi K, Yu P, Won KA, Mock L, et al. Inhibitionof tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880,GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosinekinases. Cancer Res 2009;69:8009–16.

56. LifeSciAdvisors. Mirai Therapeutics [Initation report]. Available from:http://lifesciadvisors.com/clients/mirati/ [accessed November 15,2013]. 2013 Jul. p. 51.

57. Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G. TargetingMET in cancer: rationale and progress. Nat Rev Cancer 2012;12:89–103.

58. Schroeder GM, An Y, Cai ZW, Chen XT, Clark C, Cornelius LAM, et al.Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxa-mide (BMS-777607), a selective and orally efficacious inhibitor of theMet kinase superfamily. J Med Chem 2009;52:1251–4.

59. Yan SB, Peek VL, Ajamie R, Buchanan SG, Graff JR, Heidler SA, et al.LY2801653 is an orally bioavailable multi-kinase inhibitor with potentactivity against MET, MST1R, and other oncoproteins, and displays

anti-tumor activities in mouse xenograft models. Invest New Drugs2013;31:833–44.

60. BurbridgeMF, BossardCJ, SaunierC, Fejes I, BrunoA, L�eonceS, et al.S49076 is a novel kinase inhibitor of MET, AXL, and FGFR with strongpreclinical activity alone and in association with bevacizumab. MolCancer Ther 2013;12:1749–62.

61. Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, aselective small molecule inhibitor of Axl kinase, blocks tumor spreadand prolongs survival in models of metastatic breast cancer. CancerRes 2010;70:1544–54.

62. ToleroPharmaceuticals, Inc–TP-0903 [Internet]. 2013 [cited2013May7].Available from: http://www.toleropharmaceuticals.com/TP-0903.html.

63. Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. Anepithelial-mesenchymal transition (EMT) gene signature predicts resis-tance to EGFR and PI3K inhibitors and identifies Axl as a therapeutictarget for overcoming EGFR inhibitor resistance. Clin Cancer Res2013;19:279–90.

64. Astex Pharmaceuticals. Astex pharmaceuticals discontinues amuva-tinib clinical development program [Internet]. 2012 [cited 2013Nov 18]. Available from: http://investor.astx.com/releasedetail.cfm?ReleaseID¼708646.

65. Oslob JD, Romanowski MJ, Allen DA, Baskaran S, Bui M, Elling RA,et al. Discovery of a potent and selective aurora kinase inhibitor. BioorgMed Chem Lett 2008;18:4880–4.

66. Sunesis Pharmaceuticals Inc – Form 10-K- March 31, 2010 [Internet].getfilings.com; 2013. Available from: http://www.getfilings.com/sec-filings/100331/SUNESIS-PHARMACEUTICALS-INC_10-K/.

67. Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of thetyrosine kinase domain of the human insulin receptor. Nature1994;372:746–54.

68. HubbardSR.Crystal structure of the activated insulin receptor tyrosinekinase in complex with peptide substrate and ATP analog. EMBO J1997;16:5572–81.

69. Berk BC. Vascular smooth muscle growth: autocrine growth mechan-isms. Physiol Rev 2001;81:999–1030.

70. Angelillo-Scherrer A, Burnier L, FloresN, Savi P, DeMolM, Schaeffer P,et al. Role of Gas6 receptors in platelet signaling during thrombusstabilization and implications for antithrombotic therapy. J Clin Invest2005;115:237–46.

71. Lemke G, Rothlin CV. Immunobiology of the TAM receptors. Nat RevImmunol 2008;8:327–36.

72. YeX, Li Y, Stawicki S,CoutoS, Eastham-Anderson J,KallopD, et al. Ananti-Axl monoclonal antibody attenuates xenograft tumor growth andenhances the effect of multiple anticancer therapies. Oncogene2010;29:5254–64.

73. Cerchia L, Esposito CL, Camorani S, Rienzo A, Stasio L, Insabato L,et al. Targeting Axl with an high-affinity inhibitory aptamer. Mol TherJ Am Soc Gene Ther 2012;20:2291–303.

74. Remsing Rix LL, Rix U, Colinge J, Hantschel O, Bennett KL, Stranzl T,et al. Global target profile of the kinase inhibitor bosutinib in primarychronic myeloid leukemia cells. Leukemia 2009;23:477–85.

75. Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, et al.Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol2011;29:1046–51.

76. YouWK, Sennino B, Williamson CW, Falc�on B, Hashizume H, Yao LC,et al. VEGF and c-Met blockade amplify angiogenesis inhibition inpancreatic islet cancer. Cancer Res 2011;71:4758–68.

77. Shi H, Liu L, Greger J, Gilmer T. Abstract #1746: GSK1363089 inhibitsMET and synergizes with HER targeted agents in MET amplified/over-expressed and HER1/HER2 amplified tumor cells. AACR Meet Abstr2009;2009:1746.


Feneyrolles et al.




Correction

Correction: Axl Kinase as a Key Target forOncology: Focus on Small Molecule Inhibitors

In this article (Mol Cancer Ther 2014;13:2141–8), published in the September2014 issue ofMolecular Cancer Therapeutics (1), Figure 2 and Supplementary Figure1 remained from an unrevised version of the manuscript and present incorrectstructures for compounds BMS777607, MGCD265, SGI7079 and S49076. Thecorrect structures of these compounds are presented below in Figure 2 andSupplementary Figure 1. The authors apologize for the errors.

Reference1. Feneyrolles C, Spenlinhauer A, Guiet L, Fauvel B, Dayd�e-Cazals B, Warnault P, et al. Axl kinase as a

key target for oncology: focus on small molecule inhibitors. Mol Cancer Ther 2014;13:2141–8.

Published OnlineFirst May 21, 2015.doi: 10.1158/1535-7163.MCT-15-0297�2015 American Association for Cancer Research.

Oxygenbond

N

N NH

Cl

NH

NN

SO

N

N

NNH

NH

N

N

CNF

N

OON

N

NH

OCl

Cl

CN

O O

NH

O F

NH

O

N

OO

O

O

NH

O F

NH

O

N

O

FN

O

N

O

NH

FClNH2

N

OF

O

O

OF

NH

O

N

OF

NN

NNH

N

OF

NH

S

NH

O

S

N N

TP0903ToleroPharmaceu�cals

SGI7079ToleroPharmaceu�cals

Bosu�nib,Bosulif®Pfizer

Hinge bondingheterocycle

with asolubilizing

group

Hydrogenbond

acceptorsubs�tutedphenyl group

Bond Hinge bondingheterocyclewith orwithout asolubilizing

group

(p-fluoro)phenylgroup

Cabozan�nib, Cometriq®Exeliris

Fore�nibExeliris, GlaxoSmithKline

MGCD265Mira� Therapeu�cs

BMS777607Aslan Pharmaceu�cals,Bristol-Myers Squibb

LY2801653Eli Lilly Company

Hydrogendonor andacceptorsequence

A B

(o-fluoro)phenylgroup

Figure 2.

MolecularCancerTherapeutics

Mol Cancer Ther; 14(6) June 20151518

2014;13:2141-2148. Published OnlineFirst August 19, 2014.Mol Cancer Ther Clémence Feneyrolles, Aurélia Spenlinhauer, Léa Guiet, et al. InhibitorsAxl Kinase as a Key Target for Oncology: Focus on Small Molecule

Updated version

10.1158/1535-7163.MCT-13-1083doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2015/05/21/1535-7163.MCT-13-1083.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/13/9/2141.full#ref-list-1

This article cites 73 articles, 23 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/13/9/2141.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/13/9/2141To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-1083

http://mct.aacrjournals.org/content/suppl/2015/05/21/1535-7163.MCT-13-1083.DC1

http://mct.aacrjournals.org/content/13/9/2141.full#ref-list-1

http://mct.aacrjournals.org/content/13/9/2141.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/13/9/2141


Axl Kinase as a Key Target for Oncology: Focus on Small ... · Small Molecule Inhibitors To our...

Documents

Transcript of Axl Kinase as a Key Target for Oncology: Focus on Small ... · Small Molecule Inhibitors To our...