AXL-Driven EMT State As a Targetable Conduit in Cancer Jane Antony1,2,3 and Ruby Yun-Ju Huang1,4,5

Home , EPHA6, EPHB3, Ephrin receptor, MST1R, TIE1

Published OnlineFirst June 30, 2017; DOI: 10.1158/0008-5472.CAN-17-0392

Cancer Review Research

AXL-Driven EMT State as a Targetable Conduit in Cancer Jane Antony1,2,3 and Ruby Yun-Ju Huang1,4,5

Abstract

The receptor tyrosine kinase (RTK) AXL has been intrinsically further elucidation to deﬁne targetable conduits. Therapeuti- linked to epithelial–mesenchymal transition (EMT) and pro- cally, as AXL inhibition has shown EMT reversal and resensi- moting cell survival, anoikis resistance, invasion, and metas- tization to other tyrosine kinase inhibitors, mitotic inhibitors, tasis in several cancers. AXL signaling has been shown to and platinum-based therapy, there is a need to stratify patients directly affect the mesenchymal state and confer it with aggres- based on AXL dependence. This review elucidates the role of sive phenotype and drug resistance. Recently, the EMT gradient AXL in EMT-mediated oncogenesis and highlights the recipro- has also been shown to rewire the kinase signaling nodes that cal control between AXL signaling and the EMT state. In facilitate AXL–RTK cross-talk, protracted signaling, converging addition, we review the potential in inhibiting AXL for the on ERK, and PI3K axes. The molecular mechanisms under- development of different therapeutic strategies and inhibitors. playing the regulation between the kinome and EMT require Cancer Res; 77(14); 3725–32. 2017 AACR.

Introduction was originally identified in 1991 as a transforming gene in chronic myeloid leukemia (CML; ref. 3). Under normal phys- AXL, which stems from the Greek word for uncontrolled, iologic conditions, it is ubiquitously expressed in several tissues "anexelekto," is a receptor tyrosine kinase (RTK) belonging to and organs, including but not limited to the hippocampus, the tumor-associated macrophage (TAM) family, comprising of cerebellum, macrophages, platelets, endothelial cells, heart, TYRO-3, AXL, and MER (Fig. 1). Structurally, the TAM receptors liver, kidney, and skeletal muscle. It was found to be over- comprise two Immunoglobulin-like (Ig) domains, two fibro- expressed in several cancers (6) such as breast, lung, liver, colon, nectin type III (FNIII) moieties in their extracellular domain, gastric, ovarian, pancreatic, and glioblastoma. and the conserved amino acid sequence KW(I/L)A(I/L)ES in their kinase domain (1). Among the RTK family, TIE and TEK are also known to contain both Ig and FNIII motifs on their AXL Signaling Pathways ectodomains. The Ig domains are common to the FGF, VEGF, Gas6 binds to the ectodomain of AXL, causing receptor and platelet-derived growth factor (PDGF) receptor families, dimerization with a 2:2 stoichiometry of Gas6 and AXL (7). whereas the FNIII is prevalent in the ephrin and insulin families. The proximity of the kinase domains of two AXL moieties in the The MET RTK family, comprising of cMET and RON, share high RTK–ligand complex enables trans-autophosphorylation of the degree of sequence similarity in the kinase domain (2). The residues on the cytoplasmic tails, where signaling molecules human AXL gene resides on chromosome 19q13.2 and is like phospholipase C-g (PLCg), PI3K, and growth factor recep- encoded by 20 exons to form the full-length protein comprising tor-bound protein 2 (Grb2) can dock (8, 9). The tyrosine of 894 amino acids. Though the expected molecular weight is 98 residues 698, 702, and 703 in the human sequence of AXL kDa, it is posttranslationally glycosylated to form either a 120 are conserved among the TAM receptors and are involved in the kDa (partially glycosylated form) or 140 kDa protein (complete functional activity of the kinase. The tyrosine residues 779, 821, glycosylation; ref. 3). It is activated by its ligand growth arrest and 866 are also potential autophosphorylation sites in the specific 6 (Gas6; ref. 4), which becomes biologically active upon C-terminal AXL domain (9). Gas6-independent AXL phos- vitamin K–dependent posttranslational modifications (5). AXL phorylation can also occur when the RTK is overexpressed, resulting in RTK dimerization (10, 11). – 1Cancer Science Institute of Singapore, National University of Singapore, In a cell type dependent context, Gas6/AXL signaling can Singapore. 2NUS Graduate School for Integrative Sciences and Engineering, mediate growth, survival, proliferation, motility, and invasion National University of Singapore, Singapore. 3Department of Surgery and by harnessing a diverse repertoire of signaling networks such Cancer, Imperial College London, London, United Kingdom. 4Department of as the Ras/Raf/MEK/ERK cascade, PI3K/Akt signaling path- Obstetrics and Gynecology, National University Health System, Singapore. ways. The MAPK/ERK cascade is usually involved in prolifer- 5 Department of Anatomy, Yong Loo Lin School of Medicine, National University ation, whereas PI3K activation signaling converges on cell of Singapore, Singapore. survival through the Akt/ribosomal s6 kinase (S6K) axis Corresponding Author: Ruby Yun-Ju Huang, National University Hospital, 1E (12). Gas6/AXL signaling also causes increased expression of Kent Ridge Road, NUHS Tower Block Level 12, Singapore 119228, Singapore. antiapoptotic proteins such as B-cell lymphoma 2 (Bcl-2) and Phone: 656-516-1148; Fax: 656-779-4753; E-mail: [email protected] B-cell lymphoma-extra large (Bcl-xL), phosphorylation and doi: 10.1158/0008-5472.CAN-17-0392 activation of NF-kB, phosphorylation and stabilization of 2017 American Association for Cancer Research. Bad, and inhibition of proapoptotic proteins such as caspase 3,

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VEGFR TIE

InsulinR PDGFR EGFR α α FGFR HGFR EGFD IgD EPHR AXL α β CRD AB FNIII

β β

EGFR INSR PDGFR-α VEGFR1 FGFR1 MET EPHA1 AXL TIE ERBB2 IGF-1R PDGFR-β VEGFR2 FGFR2 RON EPHA2 MER TEK ERBB3+ IRR CSF-1R VEGFR3 FGFR3 EPHA3 TYRO3 ERBB4 KIT/SCFR EPHA4 FLK2/FLT3 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2 EPHB3 EPHB4 EPHB5 EPHB6

Figure 1. AXL belongs to the TAM family of RTKS. Legend for RTK subfamilies: InsulinR, insulin receptor; PDGFR, platelet-derived growth factor receptor; HGFR, hepatocyte growth factor receptor; EPHR, ephrin receptor; TIE, tyrosine kinase receptor in endothelial cells. CRD, cysteine-rich domain; FNIII, ﬁbronectin type III domain; IgD, Immunoglobulin-like domain; AB, acidic box; EGFD, epidermal growth factor-like domain. a and b denote distinct RTK subunits. NOTE: RTKs in bold font are implicated in human cancers. The "þ" on ERBB3 signiﬁes it is devoid of intrinsic kinase activity.

to induce prosurvival signaling (13, 14). Signaling through (22), can interact with AXL (23). There is evidence to suggest the PI3K/Ras/Rac axis causes actin reorganization and enables that the suppressor of cytokine signaling (SOCS-1) could serve migration (15, 16). The adapter protein Nck2, which comprises to negatively modulate AXL signaling (24, 25). The E3-ubiqui- of three tandem SH3 and one SH1 domains, is involved tin ligase Cbl-b has also been reported to counteract AXL in linking AXL with other signaling complexes. In particular, signaling by ubiquitination and subsequent degradation of the AXL–Nck2 interaction facilitates AXL-mediated modula- AXL (26, 27), enhancing the antitumor efﬁcacy of NK cells tion of the integrin-linked kinase (ILK), a major component in the immune system. The schematic representation of the of signaling platforms at focal adhesions, thereby enabling Gas6/AXL signaling is illustrated in Fig. 2. AXL to regulate cytoskeleton dynamics (6). Furthermore, AXL has also been directly linked to control of contractility by its The EMT State Rewires the AXL Signaling ability to phosphorylate tropomyosin 2.1 (17). AXL signaling in cancers has also been implicated in processes such as Modality epithelial–mesenchymal transition (EMT; refs. 6, 18, 19), In a panel of 643 human cancer cell lines, elevated AXL wherein polarized epithelial cells lose their junctional integrity expression correlated positively with the mesenchymal pheno- to become motile, invasive, mesenchymal cells (20, 21). type, particularly in NSCLC and breast cancer. It was further The modulation of AXL required to regulate signaling has demonstrated that AXL inhibition was synergistic with antimi- not been extensively documented. The phosphatase C1-TEN, totic drugs in EMTed systems that presented with resistance to which is known to prevent signaling downstream of Akt tyrosine kinase inhibitors(TKI;ref.28).

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Targeting AXL in Cancer

Gas6

AXL

Integrins Calcium PLCγ signaling

Shc ILK Nck2 C1-TEN PINCH-1 Grb2

Ras SRC

SOCS-1 Raf1 Rac PI3K

P38 Rac/ MEK1 MAPK Rho AKT

Pro-inﬂammatory ERK1/2 MAPKAP Pak cytokine Kinase 2 production Caspase-3 S6 Bad NFκB

Proliferation HSP25 JNK

Actin reorganization/ Survival cell migration

Figure 2. Gas6/AXL signaling schema. Axl activation by ligand Gas6 in a 2:2 stoichiometry results in phosphorylation of the Axl kinase domain. Signal transduction through the Ras/Raf/MEK/ERK pathway as well as Src converges on proliferation and migration phenotype. Signal transmission through the PI3K kinase axis involving S6, AKT, or JNK results in cell survival and protection from apoptosis. Gas6/AXL signaling also interlinks with integrin-linked kinase to affect actin reorganization through integrins. It has also been shown to modulate calcium signaling through PCLg and proinﬂammatory cytokine production through SOCS-1.

Given the relationship between drug resistance, changes in sig- in protracted downstream phosphorylated ERK (pERK) tem- naling, and emergence of an invasive phenotype is well appre- poral response, motility, and invasion. This addiction to the ciated, EMT state-mediated rewiring of the RTK signaling nodes Gas6/AXL signaling node sensitizes the mesenchymal system has identiﬁed AXL as a key player. In triple-negative breast cancer to AXL inhibition. The epithelial systems present with linear (TNBC), AXL is transactivated by EGFR and is shown to diversify AXL signaling axis, are under regulation of cellular phospha- EGFR-mediated signaling and confer resistance to EGFR-TKI (29). tases such as dual speciﬁcity protein phosphatase 4 (DUSP4) In ovarian cancer, the EMT state has been shown to modu- that curtail pERK response, and are therefore less sensitive to late AXL signaling, with mesenchymal systems preferentially inhibition of AXL (Fig. 3; ref. 30). amplifying the Gas6/AXL signaling node (30). Exclusively EMT gradient-mediated rewiring of signaling circuitries in mesenchymal systems, AXL coclusters with, and activates has also been documented for the PI3K pathway. Epithelial EGFR, HER2 and tyrosine-protein kinase Met (cMET), resulting systems rely on HER3 signaling to activate PI3K signaling, and

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Membrane disruption + siDUSP4: Mesenchymal like signaling

AXL EGFR GAS6 cMET Epithelial system: Membrane modulation of AXL-RTK crosstalk and DUSP4 regulation of pERK

AXL EGFR cMET GAS6 ERK DUSP

EGFR GAS6 ERK Lipid raft AXL cMET DUSP

Epithelial ERK cells

Basement membrane Mesenchymal cells

Figure 3. Modulation of AXL signaling by the EMT state. Epithelial systems are under membrane modulation of RTK networks and DUSP regulation of pERK response, and loss of both these regulatory mechanisms will allow a signaling phenotype that is mesenchymal like. In particular, DUSP4, which is known to dephosphorylate ERK and modulate the MEK–ERK cascade in ovarian cancer, is signiﬁcantly lower in Mes subtype. Concurrently perturbing the integrity of the raft domains and silencing DUSP4 in Epi-A systems resulted in extensive AXL–RTK cross-talk as well as increased pERK response. Note: This ﬁgure originally appeared in Science Signaling (30).

mesenchymal systems rely on growth factor stimulation, and glioblastoma among others. In breast cancer, AXL over- p110a, and upregulated PI3K catalytic 110-KD alpha (PIK3CA) expression confers worse prognosis in patients, with AXL to harness the PI3K signaling node (31). expression being elevated in metastases. AXL was also identi- ﬁed as a downstream effector of EMT, which facilitated tumor AXL-Mediated EMT and Drug Resistance formation and invasiveness. Furthermore, AXL knockdown prevented the dissemination of highly metastatic breast can- in Cancer cer cell lines from mammary glands to the lymph nodes, and The role of AXL in EMT has been documented in literature other organs, thereby increasing survival. This study provided for breast, ovarian, non–smallcelllung,pancreatic,cancer, the ﬁrst in vivo evidence that directly links AXL to metastasis

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(32). It has been demonstrated that AXL acts in a positive eases. In the cancer setting, AXL is involved in tumor initiation feedback loop to induce EMT in normal and immortalized and progression, as well as negatively regulating antitumor human mammary epithelial cells, and regulates self-renewal immune responses (49). Tumor cells have been shown to and chemoresistance in breast cancer stem cells (18). Further- harness AXL signaling to directly promote growth and meta- more, EMT-driven breast cancer cell motility is dependent on stasis, as well as to dampen NK-cell activation and reduce AXL upregulation (33). innate cell-mediated antiimmune response (49). At present, AXL inhibition in ovarian cancer drastically abrogated inva- there are many bottlenecks of immunotherapy to target sion and matrix metalloproteinase activity. Furthermore, inhi- immune checkpoints such as CTLA-4 and PD-1/PD-L1 with biting AXL prevented initiation of metastatic dissemination and limited clinical responses in solid tumors. T-cell exclusion is a also prevented progression of established metastatic lesions common mechanism of immune resistance employed by tu- (34). The Gas6/AXL signaling node has also been shown to mors, and AXL has been implicated in sustaining oncologi- sustain the EMT state in ovarian cancer by inducing motility in cal pathways that contribute to this phenomenon. Indeed, the mesenchymal subtype (30), as well as invasion by con- targeting the Gas6/AXL pathway has been shown to enhance verging on the integrin b3 pathway (35). Importantly, AXL has anticancer immune response following radiotherapy (50). also been identified as a prognostic marker for ovarian cancer Therefore, targeting AXL would have the dual benefitofbeing patients by several groups (30, 35, 36). an anticancer therapeutic as well as synergizing the antitumor In esophageal squamous cell carcinoma, AXL overexpression immune response. correlated positively with tumor progression, and consequently adverse prognosis and distant metastasis (37). AXL knockdown in pancreatic cancer resulted in significant reduction in EMT Targeting AXL transcription factors SNAI1, SNAI2, and TWIST, and thereby Several small-molecule inhibitors have been developed to attenuated EMT-mediated invasiveness, migration, and metas- target AXL, and R428 (51), which is an AXL-specific inhibitor, tases (38). In malignant gliomas, AXL is implicated in brain was shown to block tumor dissemination and prolong survival tumor growth, invasion, and prolonged survival (39). in metastatic breast cancer mouse models. It is based on a In non–small cell lung cancer (NSCLC), AXL is shown to trisubstituted triazole core with nanomolar range potency confer mesenchymal cells with increased resistance to EGFR against AXL in biochemical assays. R428 prevented Gas6-medi- targeted therapy, with inhibition of AXL resensitizing the cells ated AXL and subsequently Akt phosphorylation, and also (40). Also, in EGFR-mutant lung cancer models, AXL over- suppressed invasion of cancer cells, in a dose-dependent man- expression conferred erlotinib resistance even in the absence ner. In mouse models, R428 suppressed angiogenesis and abro- of EGFR T790M mutation or cMET activation. Genetic or gated breast cancer metastasis, correlating with attenuation of pharmacologic inhibition of AXL restored sensitivity to erloti- Akt and ERK phosphorylation. Pharmacokinetic profiles in mice nib in these tumor models (41). It is of interest to note that AXL showed good plasma stability with a half-life of 13 hours. R428 confers resistance to EGFR-targeted therapies (29, 41, 42) by has also shown to reverse AXL-mediated resistance to ALK diversifying signaling, as well as to PI3K inhibitors due to inhibitors in neuroblastoma (47). A summary of R428 and association with EGFR (43). Inhibition of upstream regulator other AXL small-molecule inhibitors is given in Table 1 (52). of AXL, such as the YAP/Hippo pathway, has also been shown Another small-molecule inhibitor n-butylidenephthalide, to restore sensitivity to EGFR inhibitors in otherwise EGFR- loaded and delivered through an intracerebral gliadel wafer, has TKI–resistant lung cancers (44). Apart from NSCLC, AXL con- shown remarkable efficacy in down modulating AXL expression fers resistance to EGFR-targeted therapy in head and neck and tumor invasion in glioblastoma multiforme (53). In glio- cancer (42). blastoma, another small-molecule inhibitor BMS-777607 has AXL has also been implicated in acquired resistance to HER2- been shown to effectively reduce growth, migration, and invasion, targeted therapy in breast cancer through EMT-mediated mechan- both in vitro and in vivo (54). Recently, a series of 4-Oxo-1,4- isms (45). Gas6/AXL signaling has also been shown to confer dihydroquinoline-3-carboxamide derivatives were synthesized as chemoresistance in breast cancer cells through the Akt/GSK-3b/ highly potent AXL inhibitors. The lead compound 9im showed b-catenin, which converges on ZEB1 to regulate DNA damage inhibition against the AXL kinase domain at low nanomolar repair pathways and EMT (46). AXL-mediated EMT also results in concentrations and suppressed TGFb1-induced EMT, migration, resistance to anaplastic lymphoma kinase (ALK) inhibition in and invasion in breast cancer cells (55). neuroblastoma (47) and sunitinib resistance in renal cell carci- Aside from small-molecule inhibitors, biologics to target AXL noma (48). are in preclinical development. The YW327.6S2 antibody inhibits AXL activation by abrogating its binding with Gas6 as well as AXL Confers Resistance to Cancer down regulating the receptor (56). Also, AXL inhibition with Immunotherapy by Promoting Immune YW327.6S2 enhanced the efficacy of ERGR inhibitors in tumors resistant to EGFR-targeted therapy. YW327.6S2 also showed Evasion synergy with carboplatin/paclitaxel in the lung cancer xenograft Aside from their pro-oncogenic signaling in cancer cells, the model (56). The anti-AXL monoclonal antibody 20G7-D9 TAM receptors have also been previously described for their induced AXL degradation in TNBC cell lines as well as in role in modulating immune homeostasis. They function as patient-derived xenografts, and curtailed Gas6/AXL-dependent negative immune regulators primarily by dampening activation signaling events, particularly the EMT-related genes SNAIL, SLUG, of innate immune responses as well as clearance of apoptotic and VIM, which are mediated by the Gas6/AXL/FRA-1 axis (57). cells. Hence, deregulation of TAM signaling, particularly AXL, Although both these antibodies are promising therapeutic has been coupled with autoimmune and inflammatory dis- strategies, a potential drawback of this approach would be

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Table 1. Summary of AXL inhibitors Drug Target AXL inhibition Developer Phase of development

Foretinib (XL880, MET, VEGFR2, AXL, RON IC50 (in vitro) ¼ 11 nmol/L GSK Phase II, active, not GSK1363089) recruiting

Merestinib (LY2801653) MET, MST1R, DDR1, TIE1, MER, TYRO3, AXL IC50 (in vitro) ¼ 11 nmol/L Eli Lilly and Co. Phase I, active, recruiting NPS-1034 AXL, DDR1, FLT3, KIT, MEK, MET, ROS1, and TIE1 IC50 (in vitro) ¼ 10 nmol/L NeoPharm Preclinical LDC1267 MET, AXL, TYRO3 IC50 (in cells) ¼ 19 nmol/L Lead Discovery Centre Preclinical Bosutinib (SKI-606, BCR-ABL, ABL, SRC, YES, MEK, AXL, BMX IC50 (in vitro) ¼ 0.56 mmol/L Pﬁzer Approved for CML with marketed as Bosulif) resistance to treatment

S49076 MET and mutants, AXL, MER, FGFRs IC50 (in vitro) ¼ 7 nmol/L Institut de Recherches Phase I, active, recruiting Internationales Servier

BGB324 (R428) AXL (selective) IC50 (in vitro) ¼ 14 nmol/L Rigel Pharmaceuticals/ Phase I/II, active, BerGen BIO recruiting

binding of the antibody to soluble AXL (sAXL) in the blood to identify the presence of these clusters and thereby enrich for stream; this might severely reduce efficacy of the treatment. AXL-driven tumors (62). Downstream signaling molecules such MicroRNA sponges could also perform an alternate form of AXL as pERK could serve as surrogate pharmacodynamic readouts targeting, given miR-432 has been shown to down modulate AXL for the response of anti-AXL therapy. in lung adenocarcinoma. Other biological approaches would include engineering Conclusions sAXL to sequester Gas6 to prevent Gas6-mediated AXL activa- EMTed systems represent a challenge in the field of cancer tion (58). The high-affinity AXL decoy-receptor MYD1-72 biology wherein they equip cancer cells with aggressive pheno- hasbeenshowntobindGas6withfemtomolaraffinity and types, yet they cannot be effectively targeted. AXL represents a drastically inhibit disease progression in several preclinical novel class of RTKs, which is linked to, and reciprocally regulated models (59). Although this novel approach would ensure high by, the EMT state. Targeting AXL reverts the EMT phenotype and specificity and limited toxicity, Gas6-independent AXL dimer- resensitizes cancer cells to several small-molecule inhibitors and ization due to receptor overexpression could still promote chemotherapeutics. Given the increasing evidence that under- tumorigenesis (60). scores the intrinsic links between EMT and the AXL-addicted fi fi state, it would be bene cial to enrich the mesenchymal subtype AXL Inhibition in Strati ed Patient patients in a prospective AXL inhibitor trial. Using AXL signatures Populations or expression state could greatly benefit in stratifying patients for Given the heavy dependence of the mesenchymal state on potential AXL inhibitor trials, as well as biomarkers for disease AXL signaling, it would be beneficial to target specifically these status and progression. AXL-driven states. A potential design could be to select for patients having tumors with mesenchymal features using gene Disclosure of Potential Conflicts of Interest expression profiling. Gene expression profiling on formalin- No potential conflicts of interest were disclosed. fixed, paraffin-embedded diagnostic blocks has been validated in the clinical setting such as ovarian cancer (61). Intratumoral Grant Support heterogeneity would need to be taken into consideration when This work was supported by the National Medical Research Council (NMRC) applying this strategy. Therefore, multiple core biopsies taken of Singapore under its Center Grant scheme to National University Cancer Institute (NCIS) under the EMT Theme (R.Y.-J. Huang). J. Antony was funded by from the tumors would need to be adopted. Another strategy the Ovarian Cancer Action Centre and NUS Graduate School for Integrative could be to stratify patients based on the notion of observed Sciences and Engineering. AXL/RTK clustering in the mesenchymal tumors, thereby enriching for tumors amplifying the AXL signaling nodule. Received February 13, 2017; revised April 19, 2017; accepted May 22, 2017; Proximity ligation assays could be deployed in patient biopsies published OnlineFirst June 30, 2017.

References 1. Linger RM, Keating AK, Earp HS, Graham DK. TAM receptor tyrosine K-dependent proteins related to protein S, a negative coregulator in the kinases: biologic functions, signaling, and potential therapeutic targeting blood coagulation cascade. Mol Cell Biol 1993;13:4976–85. in human cancer. Adv Cancer Res 2008;100:35–83. 6. Verma A, Warner SL, Vankayalapati H, Bearss DJ, Sharma S. Targeting Axl 2. Robinson DR, Wu YM, Lin SF. The protein tyrosine kinase family of the and Mer kinases in cancer. Mol Cancer Ther 2011;10:1763–73. human genome. Oncogene 2000;19:5548–57. 7. Sasaki T, Knyazev PG, Clout NJ, Cheburkin Y, Gohring W, Ullrich A, et al. 3. O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, et al. Structural basis for Gas6-Axl signalling. EMBO J 2006;25:80–7. Axl, a transforming gene isolated from primary human myeloid leuke- 8. Fridell YW, Jin Y, Quilliam LA, Burchert A, McCloskey P, Spizz G, mia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991; et al. Differential activation of the Ras/extracellular-signal-regulated 11:5016–31. protein kinase pathway is responsible for the biological conse- 4. Varnum BC, Young C, Elliott G, Garcia A, Bartley TD, Fridell YW, et al. Axl quences induced by the Axl receptor tyrosine kinase. Mol Cell Biol receptor tyrosine kinase stimulated by the vitamin K-dependent protein 1996;16:135–45. encoded by growth-arrest-specific gene 6. Nature 1995;373:623–6. 9. Braunger J, Schleithoff L, Schulz AS, Kessler H, Lammers R, Ullrich A, et al. 5. Manfioletti G, Brancolini C, Avanzi G, Schneider C. The protein encoded by Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated a growth arrest-specific gene (gas6) is a new member of the vitamin mainly by a multi-substrate docking-site. Oncogene 1997;14:2619–31.

3730 Cancer Res; 77(14) July 15, 2017 Cancer Research

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10. Bellosta P, Costa M, Lin DA, Basilico C. The receptor tyrosine kinase ARK 32. Gjerdrum C, Tiron C, Hoiby T, Stefansson I, Haugen H, Sandal T, et al. Axl is mediates cell aggregation by homophilic binding. Mol Cell Biol 1995; an essential epithelial-to-mesenchymal transition-induced regulator of 15:614–25. breast cancer metastasis and patient survival. Proc Natl Acad Sci U S A 11. Konishi A, Aizawa T, Mohan A, Korshunov VA, Berk BC. Hydrogen 2010;107:1124–9. peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells. 33. Vuoriluoto K, Haugen H, Kiviluoto S, Mpindi JP, Nevo J, Gjerdrum C, et al. J Biol Chem 2004;279:28766–70. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and 12. Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phospha- migration by governing Axl expression in breast cancer. Oncogene 2011; tidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mito- 30:1436–48. genic and survival activities in NIH 3T3 fibroblasts.MolCellBiol1997; 34. Rankin EB, Fuh KC, Taylor TE, Krieg AJ, Musser M, Yuan J, et al. AXL is an 17:4442–53. essential factor and therapeutic target for metastatic ovarian cancer. Cancer 13. Demarchi F, Verardo R, Varnum B, Brancolini C, Schneider C. Gas6 anti- Res 2010;70:7570–9. apoptotic signaling requires NF-kappa B activation. J Biol Chem 2001; 35. Rea K, Pinciroli P, Sensi M, Alciato F, Bisaro B, Lozneanu L, et al. Novel Axl- 276:31738–44. driven signaling pathway and molecular signature characterize high-grade 14. Hasanbasic I, Cuerquis J, Varnum B, Blostein MD. Intracellular signaling ovarian cancer patients with poor clinical outcome. Oncotarget 2015;6: pathways involved in Gas6-Axl-mediated survival of endothelial cells. 30859–75. Am J Physiol Heart Circ Physiol 2004;287:H1207–13. 36. Lozneanu L, Pinciroli P, Ciobanu DA, Carcangiu ML, Canevari S, Tomas- 15. Allen MP, Xu M, Linseman DA, Pawlowski JE, Bokoch GM, Heidenreich setti A, et al. Computational and immunohistochemical analyses highlight KA, et al. Adhesion-related kinase repression of gonadotropin-releasing AXL as a potential prognostic marker for ovarian cancer patients. Antican- hormone gene expression requires Rac activation of the extracellular signal- cer Res 2016;36:4155–63. regulated kinase pathway. J Biol Chem 2002;277:38133–40. 37. Hsieh MS, Yang PW, Wong LF, Lee JM. The AXL receptor tyrosine kinase is 16. Nielsen-Preiss SM, Allen MP, Xu M, Linseman DA, Pawlowski JE, Bouchard associated with adverse prognosis and distant metastasis in esophageal RJ, et al. Adhesion-related kinase induction of migration requires phos- squamous cell carcinoma. Oncotarget 2016;7:36956–70. phatidylinositol-3-kinase and ras stimulation of rac activity in immortal- 38. Koorstra JB, Karikari CA, Feldmann G, Bisht S, Rojas PL, Offerhaus GJ, et al. ized gonadotropin-releasing hormone neuronal cells. Endocrinology The Axl receptor tyrosine kinase confers an adverse prognostic influence in 2007;148:2806–14. pancreatic cancer and represents a new therapeutic target. Cancer Biol Ther 17. Yang B, Lieu ZZ, Wolfenson H, Hameed FM, Bershadsky AD, Sheetz MP. 2009;8:618–26. Mechanosensing controlled directly by tyrosine kinases. Nano Lett 39. Vajkoczy P, Knyazev P, Kunkel A, Capelle HH, Behrndt S, von Tengg- 2016;16:5951–61. Kobligk H, et al. Dominant-negative inhibition of the Axl receptor tyrosine 18. Asiedu MK, Beauchamp-Perez FD, Ingle JN, Behrens MD, Radisky DC, kinase suppresses brain tumor cell growth and invasion and prolongs Knutson KL. AXL induces epithelial-to-mesenchymal transition and survival. Proc Natl Acad Sci U S A 2006;103:5799–804. regulates the function of breast cancer stem cells. Oncogene 2014;33: 40. Byers LA, Diao L, Wang J, Saintigny P, Girard L, Peyton M, et al. An 1316–24. epithelial-mesenchymal transition gene signature predicts resistance 19. Linger RM, Keating AK, Earp HS, Graham DK. TAM receptor tyrosine to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target kinases: biologic functions, signaling, and potential therapeutic targeting for overcoming EGFR inhibitor resistance. Clin Cancer Res 2013;19: in human cancer. Adv Cancer Cell 2008;100:35–83. 279–90. 20. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads 41. Zhang Z, Lee JC, Lin L, Olivas V, Au V, LaFramboise T, et al. Activation of the of development and tumor metastasis. Dev Cell 2008;14:818–29. AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat 21. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Genet 2012;44:852–60. Nat Rev Cancer 2002;2:442–54. 42. Giles KM, Kalinowski FC, Candy PA, Epis MR, Zhang PM, Redfern AD, et al. 22. Hafizi S, Ibraimi F, Dahlback B. C1-TEN is a negative regulator of the Akt/ Axl mediates acquired resistance of head and neck cancer cells to the PKB signal transduction pathway and inhibits cell survival, proliferation, epidermal growth factor receptor inhibitor erlotinib. Mol Cancer Ther and migration. FASEB J 2005;19:971–3. 2013;12:2541–58. 23. Hafizi S, Alindri F, Karlsson R, Dahlback B. Interaction of Axl receptor 43. Elkabets M, Pazarentzos E, Juric D, Sheng Q, Pelossof RA, Brook S, et al. AXL tyrosine kinase with C1-TEN, a novel C1 domain-containing protein with mediates resistance to PI3Kalpha inhibition by activating the EGFR/PKC/ homology to tensin. Biochem Biophys Res Commun 2002;299:793–800. mTOR axis in head and neck and esophageal squamous cell carcinomas. 24. Kinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, Ohishi M, et al. Cancer Cell 2015;27:533–46. SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. 44. Lee JE, Park HS, Lee D, Yoo G, Kim T, Jeon H, et al. Hippo pathway effector Immunity 2002;17:583–91. YAP inhibition restores the sensitivity of EGFR-TKI in lung adenocarcino- 25. Nakagawa R, Naka T, Tsutsui H, Fujimoto M, Kimura A, Abe T, et al. SOCS-1 ma having primary or acquired EGFR-TKI resistance. Biochem Biophys Res participates in negative regulation of LPS responses. Immunity 2002;17: Commun 2016;474:154–60. 677–87. 45. Creedon H, Gomez-Cuadrado L, Tarnauskaite Z, Balla J, Canel M, MacLeod 26. Gioia R, Tregoat C, Dumas PY, Lagarde V, Prouzet-Mauleon V, Desplat KG, et al. Identification of novel pathways linking epithelial-to-mesenchy- V, et al. CBL controls a tyrosine kinase network involving AXL, SYK and mal transition with resistance to HER2-targeted therapy. Oncotarget LYN in nilotinib-resistant chronic myeloid leukaemia. J Pathol 2015; 2016;7:11539–52. 237:14–24. 46. Wang C, Jin H, Wang N, Fan S, Wang Y, Zhang Y, et al. Gas6/Axl axis 27. Paolino M, Choidas A, Wallner S, Pranjic B, Uribesalgo I, Loeser S, et al. The contributestochemoresistanceand metastasis in breast cancer E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural through Akt/GSK-3beta/beta-catenin Signaling. Theranostics 2016;6: killer cells. Nature 2014;507:508–12. 1205–19. 28. Wilson C, Ye X, Pham T, Lin E, Chan S, McNamara E, et al. AXL inhibition 47. Debruyne DN, Bhatnagar N, Sharma B, Luther W, Moore NF, Cheung NK, sensitizes mesenchymal cancer cells to antimitotic drugs. Cancer Res et al. ALK inhibitor resistance in ALK(F1174L)-driven neuroblastoma is 2014;74:5878–90. associated with AXL activation and induction of EMT. Oncogene 29. Meyer AS, Miller MA, Gertler FB, Lauffenburger DA. The receptor 2016;35:3681–91. AXL diversifies EGFR signaling and limits the response to EGFR- 48. Zhou L, Liu XD, Sun M, Zhang X, German P, Bai S, et al. Targeting MET and targeted inhibitors in triple-negative breast cancer cells. Sci Signal AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. 2013;6:ra66. Oncogene 2016;35:2687–97. 30. Antony J, Tan TZ, Kelly Z, Low J, Choolani M, Recchi C, et al. The GAS6-AXL 49. Paolino M, Penninger JM. The role of TAM family receptors in immune cell signaling network is a mesenchymal (Mes) molecular subtype-specific function: implications for cancer therapy. Cancers (Basel) 2016;8. therapeutic target for ovarian cancer. Sci Signal 2016;9:ra97. 50. Aguilera TA, Giaccia AJ. Molecular pathways: oncologic pathways and 31. Salt MB, Bandyopadhyay S, McCormick F. Epithelial-to-mesenchymal their role in T-cell exclusion and immune evasion- A new role for the transition rewires the molecular path to PI3K-dependent proliferation. AXL receptor tyrosine kinase. Clin Cancer Res 2017 Mar 13. [Epub Cancer Discov 2014;4:186–99. ahead of print].

www.aacrjournals.org Cancer Res; 77(14) July 15, 2017 3731

Antony and Huang

51. Holland SJ, Pan A, Franci C, Hu Y, Chang B, Li W, et al. R428, a selective enhances the effect of multiple anticancer therapies. Oncogene 2010;29: small molecule inhibitor of Axl kinase, blocks tumor spread and pro- 5254–64. longs survival in models of metastatic breast cancer. Cancer Res 57. Leconet W, Chentouf M, Du Manoir S, Chevalier C, Sirevnt A, Ait-Arsa I, 2010;70:1544–54. et al. Therapeutic activity of anti-AXL antibody against triple-negative 52. Myers SH, Brunton VG, Unciti-Broceta A. AXL inhibitors in can- breast cancer Patient Derived Xenografts and metastasis. Clin Cancer Res. cer: a medicinal chemistry perspective. J Med Chem 2016;59: 2016 Dec 6. [Epub ahead of print]. 3593–608. 58. Kariolis MS, Miao YR, Jones DS 2nd, Kapur S, Mathews II, Giaccia AJ, et al. 53. Yen SY, Chen SR, Hsieh J, Li YS, Chuang SE, Chuang HM, An engineered Axl `decoy receptor' effectively silences the Gas6-Axl et al. Biodegradable interstitial release polymer loading a novel signaling axis. Nat Chem Biol 2014;10:977–83. small molecule targeting Axl receptor tyrosine kinase and 59. Kariolis MS, Miao YR, Diep A, Nash SE, Olcina MM, Jiang D, et al. reducing brain tumour migration and invasion. Oncogene 2016; Inhibition of the GAS6/AXL pathway augments the efficacy of chemothera- 35:2156–65. pies. J Clin Invest 2017;127:183–98. 54. Onken J, Torka R, Korsing S, Radke J, Krementeskaia I, Nieminen M, et al. 60. Chen L, Kong G, Zhang C, Dong H, Yang C, Song G, et al. MicroRNA-432 Inhibiting receptor tyrosine kinase AXL with small molecule inhibitor functions as a tumor suppressor gene through targeting E2F3 and AXL in BMS-777607 reduces glioblastoma growth, migration, and invasion in lung adenocarcinoma. Oncotarget 2016;7:20041–53. vitro and in vivo. Oncotarget 2016;7:9876–89. 61. Leong HS, Galletta L, Etemadmoghadam D, George J, Australian 55. Tan L, Zhang Z, Gao D, Luo J, Tu ZC, Li Z, et al. 4-Oxo-1,4-dihydroquino- Ovarian Cancer S, Kobel M, et al. Efficient molecular subtype classifi- line-3-carboxamide derivatives as new Axl kinase inhibitors. J Med Chem cation of high-grade serous ovarian cancer. J Pathol 2015;236:272–7. 2016;59:6807–25. 62. Smith MA, Hall R, Fisher K, Haake SM, Khalil F, Schabath MB, et al. 56. Ye X, Li Y, Stawicki S, Couto S, Eastham-Anderson J, Kallop D, et al. An anti- Annotation of human cancers with EGFR signaling-associated protein Axl monoclonal antibody attenuates xenograft tumor growth and complexes using proximity ligation assays. Sci Signal 2015;8:ra4.

3732 Cancer Res; 77(14) July 15, 2017 Cancer Research

AXL-Driven EMT State as a Targetable Conduit in Cancer

Jane Antony and Ruby Yun-Ju Huang

Cancer Res 2017;77:3725-3732. Published OnlineFirst June 30, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-0392

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