Gene of the month: Axl.

Matthew Brown1, James R. M. Black1, Rohini Sharma1, Justin Stebbing1, David J. Pinato1

1. Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital

Campus, IRDB Building, Du Cane Road, W12 0HS, London (UK).

Competing interests: None to disclose.

Word Count: 3,834 Tables: 0 Figures: 1 References: 89

Running Title: Gene of the month: Axl.

Keywords: Axl, oncogene, cancer, therapy.

*To whom correspondence should be addressed:

Dr David J. Pinato, MD MRes MRCP PhD

NIHR Academic Clinical Lecturer in Medical Oncology

Imperial College London Hammersmith Campus, Du Cane Road, W12 0HS, London (UK)

Tel: +44 020 83833720 E-mail: david.pinato@imperial.ac.uk

ABSTRACT.

1

The interaction between Axl receptor tyrosine kinase and its main ligand Gas6 has been

implicated in the progression of a wide number of malignancies. More recently, overexpression

of Axl has emerged as a key molecular determinant underlying the development of acquired

resistance to targeted anticancer agents. The activation of Axl is overexpression-dependent and

controls a number of hallmarks of cancer progression including proliferation, migration,

resistance to apoptosis and survival through a complex network of intracellular second

messengers. Axl has been noted to influence clinically meaningful endpoints including

metastatic recurrence and survival in the vast majority of tumour types. With Axl inhibitors

having gained momentum as novel anticancer therapies, we provide an overview of the biologic

and clinical relevance of this molecular pathway, outlining the main directions of research.

INTRODUCTION.

2

Axl, previously known as UFO, is a receptor tyrosine kinase (RTK) that forms part of the

TAM family of RTKs together with Tyro3 and Mer. Evidence suggesting the oncogenic potential

of Axl has been ever-present from the point of its initial isolation from chronic myelogenous

leukaemia (CML).[1] Further work in to the functionality of this gene has shown its mechanistic

involvement in determining a wide variety of cancerous hallmarks including: proliferation,

survival, evasion from apoptosis, enhanced angiogenesis, invasiveness and, more recently,

resistance to targeted anticancer therapies.[2,3] Furthermore, Axl has been shown to influence

the clinical behaviour of a number of cancer histotypes, holding prognostic significance in

breast, lung, ovarian, renal, gastrointestinal cancers as well as many other solid and

hematopoietic malignancies.

STRUCTURE.

The Axl locus is located on chromosome 19 at position q13.2 and extends over 44 kb.

[1,4] The promoter region of the gene is rich in GC repeats, which are important for the

epigenetic control of Axl expression through methylation of guanine nucleotides.[5] The full

length receptor is encoded within 20 exons, however, two splice variants are observed differing

by the exclusion of the 27 bp exon 10.[4] Exon 10, although encoding part of the second

fibronectin type III repeat, would appear to have no functional relevance to the protein as both

variants hold transforming capabilities and are present within neoplastic cells.[1,6,7] Once

translated, the protein alone constitutes 104 kDa and when fully post-translationally modified,

at six N-linked glycosylation sites in the extracellular domain, a protein of 140 kDa is produced.

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[1]

Axl is a trans-membrane RTK, consisting of an extracellular ligand-binding domain and a

cytoplasmic kinase domain. The extracellular portion consists of two N-terminal

immunoglobulin-like domains followed by two fibronectin type III repeats giving it the

appearance of a cell adhesion molecule.[1,8–10] C-terminal to the short single pass

transmembrane domain, that follows the extracellular portion, is the kinase domain of Axl. An

interesting feature of the kinase domain of Axl is a conserved KWIAIE sequence as opposed to

the (K/T)W(T/M)APE motif usually characterising other RTKs.[1]

AXL LIGANDS.

There are two major ligands involved with activation of the TAM RTKs: Gas6 and Protein

S. Both Protein S and Gas6 are members of the vitamin K-dependent protein family and carry a

44% sequence homology.[11–13] When originally characterised, Gas6 was described as

containing four different regions, which were preserved within the Protein S structure.[11,12]

Region A at their N-terminal end is highly rich in γ-carboxyglutamic acid residues (Gla domain).

This is followed by a loop region, which in protein S contains a thrombin-sensitive cleavage site

that is lacking from Gas6, reflecting the differential involvement of the two proteins in

haemostasis. Region C contains 4 epidermal growth factor-like repeats, and region D, at the C-

terminus, is a sex hormone binding globulin-like region, containing two tandem Laminin G-like

domains.[11–13]

Two regions of this ligand appear to hold high significance for its functionality. Firstly, the

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laminin G-like domains are vital for the ability of Gas6 to bind Axl and these domains alone are

sufficient to allow activation of RTK activity.[12] The second region which appears to bear

importance to the localisation of these ligands is the Gla repeats. These γ-carboxyglutamic acid

residues associate with 7-8 Ca2+ ions, which in turn mediate their ability to bind to negatively

charged phospholipids and clotting factors.[13,14]

Some more recently identified and lesser studied ligands are Tubby, Tubby-like protein 1

(Tulp-1) and Galectin-3.[2] There is a recognised differential affinity between this selection of

ligands and each member of the TAM family. In fact, Gas6 has a 3-10 fold higher affinity for Axl

compared to Mer and Axl appears to not be activated by the presence of Protein S despite the

sequence homology with Gas6.[2,13,15]

AXL ACTIVATION AND SIGNAL TRANSDUCTION.

The activation of Axl and its downstream signalling pathway relies on several different

mechanisms. Ligand-dependent homodimerisation is the standard method of activation in

physiological conditions; however, several ligand-independent mechanisms are possible and are

more relevant in cancer. These include homodimersation upon overexpression of Axl,

heterodimerisation with other TAM family RTKs: Axl and Tyro3 heterodimers have in fact been

observed in the absence of ligand in gonadotropin-releasing hormone (GnRH) secreting

neurons.[16,17] Heterodimerisation with non-TAM receptors can occur; interactions with

fibromyalgia syndrome-like tyrosine kinase 3 (FLT-3) and epidermal growth factor receptor

(EGFR) have been previously described in the literature.[7,18,19] Furthermore, Axl

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phosphorylation has also been reported to occur in the presence of reactive oxygen species in

rat vascular smooth muscle cells.[20,21]

Activation of Axl by Gas6 has been described as a two-step process. Firstly, there is the

formation of a high affinity 1:1 Gas6/Axl complex.[9] Lateral diffusion of these Gas6/Axl

complexes allows formation of a 2:2 Gas6/Axl complex leading to activation of Axl via trans-

auto-phosphorylation of several tyrosine residues in the intracellular domain of the protein.[9]

So far three tyrosine residues, Y821, Y866 and Y779, in the C-terminal kinase domain have been

identified as functionally relevant in the interaction of Axl with downstream signalling

molecules.[22]

Generally, activation of Axl in cancer is caused by overexpression as opposed to an

activating mutation. The methods by which overexpression occurs are not well understood and

may vary in different cellular settings, however, several potential mechanisms for

overexpression have been identified. Transcriptional control of the Axl gene occurs through

Sp1/Sp3 and Myeloid zinc finger 1 (MZF1) transcription factors.[5,23] The ability of Sp1/Sp3 to

bind the promoter region may be regulated via CpG methylation. This was shown in two

colorectal cell lines, Colo206f and WiDr, both have moderate expression of Sp1 and Sp3 but

exhibited very limited expression of Axl; demethylation of CG sites in these cell lines was found

to increase Axl expression in dose-dependent manner.[5]

Moreover, Axl expression is also regulated by three microRNAs (miRNAs), specifically

miR-34 and miR-199a/b,[23] through transcriptional repression via targeting of consensus

sequences within the 3’-untranslated region of nascent Axl mRNA. MiRNA expression is

epigenetically regulated by promoter methylation and unsurprisingly genomic hypermethylation

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verified across a panel of cell lines correlates inversely to Axl expression levels.[24]

Similarly to many other RTKs, Axl transmits a signal that is external to the cell through a

series of multiple networks of protein interactions within the cytoplasm. Key to the signal-

transducing properties of the receptor are a number phosphorylated tyrosine residues in its

kinase domain which act as a multi-substrate docking site.[22] This allows Axl to influence a

variety of different downstream pathways and processes, as illustrated in Figure 1. Two of the

substrates that can bind to phosphorylated Axl are p85α and p85β, two of the regulatory

subunits of PI3K.[22] These proteins bind at either tyrosine 821 or tyrosine 779, an interaction

that forms a major axis of Axl signalling by providing it with an influence over the PI3K/AKT

pathway, as can be seen in figure 1 the PI3K/AKT pathway plays a role in multiple aspect of Axl’s

oncogenic potential.

A second, equally important pathway affected by Axl signalling is the Ras/ERK pathway,

which is activated through the binding of GRB2 to tyrosine 821 of the Axl kinase domain.[22]

Tyrosine residue 821 is also functionally crucial to enable the interaction between Axl and Src,

Lck and PLCγ, although PLCγ can also bind through tyrosine 866.[22]

A number of emerging downstream targets are being characterised for their functional

interaction with Axl activation. For example, in migrating GnRH cells it has been shown that Axl

is involved with activation of the Rho family GTPase Rac.[25] In a yeast two-hybrid study several

downstream targets were observed to relate to Axl activation, including SOCS-1, Nck2, C1-TEN

and RanBMP.[26] Axl is also capable of lateral activation of Met, a paralog of Axl that is

implicated in the promotion of metastatic potential, through its direct interaction with Src.[27]

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FUNCTIONS.

Axl has a role in many aspects of cellular biology across multiple cell types, including

phagocytosis, cell migration, platelet aggregation and inflammation.[28] The expression of

phosphatidylserine on the surface of apoptotic cells allows binding of the Gla residues of Gas6

and Protein S, thereby activating Axl and recruiting macrophages and dendritic cells expressing

TAM receptors on their cell surfaces.[7,29] Inhibition of the pro-inflammatory response is

another key role played by Axl within a normal cellular environment. Axl expression is activated

by type 1 interferons which are produced downstream of Toll-Like Receptor signalling.[30] Once

active Axl then causes upregulation of Suppressor of Cytokine Signalling 1 and 3 (SOCS1 and

SOCS3) leading to attenuation of the inflammatory signal.[10] Due to its role in several major

cellular signalling pathways, aberrant activation of Axl in the context of cancer is capable of

influencing a number of features underlying the malignant phenotype.

Proliferation and Survival.

There is consolidated evidence to suggest that signalling through Axl is sufficient for the

promotion of survival in response to multiple pro-apoptotic stimuli including inorganic

phosphate, tumour necrosis factor and adenovirus type 5 E1A protein.[31–33] Evasion from

apoptosis relies upon the activation of several downstream effectors following the activation of

the PI3K/Akt pathway. Firstly, activation of S6K was shown to support cell survival, as does the

8

phosphorylation and consequent inactivation of Bad, a pro-apoptotic Bcl-2 family protein.

[34,35] Furthermore, Akt driven phosphorylation of inhibitor of nuclear factor κ-B kinase

subunit α (IKK-α), instigates phosphorylation and degradation of nuclear factor of kappa light

polypeptide gene enhancer in B-cells inhibitor alpha (IκBα).[36,37] Degradation of IκBα

removes inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)

allowing its translocation to the nucleus and subsequent induction of further anti-apoptotic

proteins, namely Bcl-2 and Bcl-xL.[36,38] Pre-conditioning with Gas6 has been shown to

suppress the activation of Caspase 3 in HUVEC cells, confirming the anti-apoptotic properties of

Gas6/Axl signalling.[38] Another potential pro-survival pathway activated by PI3K is the c-Jun N-

terminal Kinase (JNK) pathway, involving a cascade initiated by PI3K activation of Rac and Rho,

Rho-family GTPases. This proceeds through PAK, p21-activated kinase, which has been known

to lead to activation of JNK.[7,35]

A proliferative effect is observed in some cell lines as a result of Axl signalling, although

it is not as commonly seen as the pro-survival effect. The role of PI3K in proliferative signalling

appears to be variable, with it being required in some studies but dispensable in others,[34,39]

however, the Ras/ERK pathway has been more heavily implicated as a vital factor in governing

the mitogenic capabilities of Axl. Activation of several components of this pathway including

Ras, Raf-1, MEK-1 and ERK occurs through the Axl/Grb2 interaction and concurrent activation of

all these components is necessary to elicit a mitogenic response.[35,39]

Invasion and Metastasis.

9

The role of Axl in promoting the invasive potential of malignant cells is well documented

by a number of studies showing a positive correlation between Axl expression and invasiveness.

[40] Furthermore, disruption of Axl signalling through shRNA reduces cellular motility and

invasion in experimental models.[41] Axl increases cellular motility and invasiveness through

the promotion of the epithelial to mesenchymal transition (EMT), a process by which epithelial

cells lose cell-cell contacts, polarity and switch to a more mobile mesenchymal phenotype. This

process is often associated with expression of EMT-inducing transcription factors such as Twist,

Snail and Slug. Whilst the precise molecular features underlying these phenotypic changes are

not completely characterised, Axl appears to be involved in a form of positive feedback loop as

several of the quintessential EMT transcription factors are both induced by and can themselves

induce the expression of Axl.[41] Interestingly, vimentin, an intermediate filament that is

present in mesenchymal cells, has also been shown to up-regulate expression of Axl.[42]

Further work has shown that Axl may play a role in the initial induction of EMT as its expression

in epithelial cells is capable of down-regulating E-cadherin and leading to the up-regulation of

N-cadherin, Slug and Snail.[43] This suggests Axl may be involved with both the induction and

maintenance of EMT signalling.

As well as leading to the upregulation of EMT transcription factors, activation of Axl has

been implicated in influencing re-modelling of the actin cytoskeleton in GnRH neuronal cells.

This re-modelling was found to be facilitated by a signalling cascade involving the Rho family

protein Rac, p38 MAPK, MAPKAP kinase 2 and finally HSP25.[25] HSP25, and analogue of

human heat shock protein 27, is capable of capping actin-filaments and is involved in cortical

actin remodelling and membrane ruffling which are vital steps preceding cell migration.[25]

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Angiogenesis.

In a normal cellular environment Axl is involved with repair of vascular injury. Expression

of Gas6 along with the presence of reactive oxygen species (ROS) activates Axl in vascular

smooth muscle cells which leads to increased resistance to apoptosis and migration of these

cells.[44,45] In a neoplastic setting, Axl overexpression has been linked with increased

angiogenesis,[46] this may be unsurprising especially as the tumour microenvironment is rich in

ROS which may enhance activation of Axl. Consequently, multiple studies have shown Axl

knockdown leads to reduced endothelial vessel formation, both in vitro and in vivo.[47,48]

Through profiling the changes in mRNA expression during Axl knockdown two potential

downstream pathways have been identified. The first is DKK3, a member of the Dickkopf family

usually involved with Wnt signalling, which was downregulated.[47] DKK3 has been shown to

regulate endothelial tube formation and stable overexpression of this protein in the C57/BL6

melanoma model led to increased microvessel density.[49] The second pathway was identified

through the upregulation of Ang-2, part of the angiopoietin pathway. Ang-2 acts to inhibit the

interaction of Ang-1 and Tie2, which together promote endothelial cell survival.[47] Therefore

activation of Axl leads to downregulation of Ang-2, freeing Ang-1 and Tie2 allowing their pro-

angiogenic activity.

THE ROLE OF AXL IN CANCER PROGRESSION.

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Considering the broad involvement of Axl in various aspects of cellular signalling and its

implication in wide variety of hallmarks of cancer, it is unsurprising that the activation of Axl has

been confirmed as a clinically meaningful trait across different solid and haematopoietic

malignancies.[2] Furthermore, the degree to which Axl influences phenotypic events that can

be lethal to the patient, such as progression to metastatic disease and resistance to treatment,

makes Axl a prognostically appealing molecular marker in the clinical setting.

Lung carcinoma.

Axl expression is seen in approximately 60% of human non-small cell lung cancer

(NSCLC) cell lines, and its overexpression directly correlates to cell migration and invasion

capacity.[50,51] Modulation of cytoskeletal rearrangement through loss of the downstream

target of Axl, Rac1 has linked cell motility with sensitivity to anticancer treatment.[52]

Accumulating evidence suggests Axl expression to correlate with more advanced clinical

stage at presentation[51] and poorer survival in lung adenocarcinoma.[53] In a study of 109

cases, the prevalence of Axl overexpression was 60% in patients with Epidermal Growth Factor

Receptor (EGFR) wild-type and 50% of EGFR-mutant disease.[54] In particular, in NSCLC, Axl is a

molecular determinant of sensitivity to EGFR inhibitors including erlotinib and gefitinib[55] and

mechanistic evidence shows that induction of Axl expression correlates positively with Vimentin

and N-cadherin and negatively with E-cadherin expression, indicating inhibition of Axl as a

mechanism to modulate reversal of EMT and drug resistance.[56]

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Breast carcinoma.

Activation of the Gas6/Axl signalling pathway holds complex clinical implications in

breast cancer. Analysis of a panel of human breast cancer cell lines has shown significant Axl

upregulation in triple-negative/basal B phenotype compared with luminal or basal A cells.[57]

In endocrine-sensitive disease, initial reports have suggested Gas6 to be under the

transcriptional regulation of the progesterone receptor and correlate with a number of

favourable prognostic factors including a higher rate of lymph-node negativity, lower grade and

smaller size of the tumours.[58] However, in a separate study, Axl expression was shown to

positively correlate with estrogen receptor positivity, higher proliferation index and more

advanced stage, to suggest that ligand-independent up-regulation of Axl might confer survival

advantage to the progressive ER+ malignant clones.[59]

In triple-negative breast cancer Axl is functionally required to diversify EGFR-mediated

cell motility through coupled trans-activation of both pathways, suggesting a therapeutically

appealing enrichment of Axl expression in receptor-negative breast cancer for which targeted

approaches are lacking.[19]

In Her-2 positive disease, Axl influences the development of lapatinib-resistance[60] and

modulates Her-3 expression, a cognate RTK that mediates part of Her-2-driven signalling

through heterodimerisation with Her-2.[61]

Across a panel of breast cancer cell lines, Axl expression has been correlated with

invasiveness[40] and subsequent studies have shown the independent effect of Axl in adversely

affecting patient’s survival.[41]

13

In keeping with evidence gathered in other tumour types, the relationship between Axl

expression, invasion and poorer clinical outcome seems to be stemming from the EMT-

modulating effects of Axl in cancer cells. Vimentin has been shown to directly induce and

sustain Axl expression and promote invasiveness[42] and recent evidence suggests that Axl is

implicated in regulating self-renewal and chemoresistance of the breast cancer stem cells

compartment.[43]

Gastrointestinal Malignancies.

Aberrant activity of Axl signalling is documented across various gastrointestinal cancers.

In a large study of 223 colorectal cancer patients overexpression of Axl and Gas-6 was seen in

>70% of the specimens. This correlated with a poorly differentiated phenotype, with Gas6

expression associating with nodal involvement and advanced tumour stage.[62] Dunne et al

showed increase in Axl within colorectal cancer cell lines that were able to invade and colonise,

and confirmed higher Axl mRNA or protein level within the tumour to prelude to poorer overall

survival.[63] Importantly, they provided mechanistic evidence to suggest that fluoropirimidine

treatment may enhance the invasive potential of tumour cells by up-regulation of Axl, with

genetic and pharmacologic inhibition of this target being capable of reversing the phenotype in

experimental models.[62]

Axl expression has also been identified in oesophageal adenocarcinoma (EAC) and

squamous cell carcinoma (ESCC). Axl overexpression is common in 50% of human EAC and

correlates with lower sensitivity to cisplatin in vitro.[64] Recently, Axl was found to be

14

overexpressed in various ESCC cell lines. Genetic silencing of Axl with siRNA prevented

proliferation, migration, invasion and tumour growth. Mechanistically, these properties related

to the inhibition of a number of key genes involved in the NF-κβ pathway, the induction of

glycogen kinase synthase 3β activity, and the reversal of EMT,[37] highlighting a potential role

for Axl inhibition in cancer therapy.

Similar roles for Axl have been established in hepato-biliary malignancies. The

prevalence of Axl positivity is common in 50-70% of pancreatic ductal adenocarcinomas (PDAC),

having a key role in maintaining cell proliferation and invasion.[65,66] Proteomic profiling of

primary and metastatic PDAC cell line has shown Axl up-regulation as a key event in driving the

metastatic diffusion.[67]

In hepatocellular cancer, Axl knockdown not only decreased cellular migration, invasion

and metastatic colonisation, but also impaired the ability of cancer cells to resist TGF-β-

mediated growth suppression, through to 14-3-3ζ, a master regulator of intracellular kinases

activity. In patient samples, Axl expression corresponded clinically with higher proportion of

microvascular invasion and shorter recurrence-free and overall survival.[68]

Ovarian cancer.

Whilst absent in normal ovarian epithelium, the expression of Axl has been linked to

metastatic progression and poor prognosis in ovarian cancer; a disease where Axl expression

seems to be under tight epigenetic regulation through expression levels of the tumour-

suppressive miR-34a.[69,70]

15

Clinical studies have shown that higher Gas-6 and Axl immuno-labelling deteriorate

patients’ prognosis through co-expression with p130Cas, an adaptor protein related to integrin

β3 and therefore involved extracellular matrix adhesion and invasion.[71] As proven in other

cancers, genetic knockdown of Axl is sufficient to prevent metastatic spread in experimental

models.[69]

Renal cell carcinoma.

The progression of renal cell cancer (RCC) invariably follows inherited or acquired

functional loss in the Von-Hippel-Lindau (VHL) locus, which enables unopposed pro-angiogenic

signalling. Axl is known to be indirectly under transcriptional control of VHL through hypoxia-

inducible factors.[27] Recent work has implicated Axl in the acquisition of resistance to anti-

angiogenic therapy, and revealed its contribution in driving metastatic spread and poorer

survival.[3,72] Recent large-scale phase III trials of TKI endowed with Axl inhibitory properties

have confirmed Axl as a therapeutic target in RCC.[73]

Other cancer types.

The range of malignancies where Axl has been highlighted as a putative oncogenic driver

is in continuous expansion. In acute myeloid leukaemia (AML), Axl-positive neoplastic cells

educate the stroma to secrete Gas6 and maintain an endocrine auto-stimulatory loop.[74] In

CML, the disease where Axl was originally characterised, this RTK has been recently implicated

16

in resistance to imatinib treatment.[75] In prostate cancer, Axl is overexpressed and associated

with migration and invasiveness, regulating genes involved in promoting cancer cell survival.[76]

A prognostic role for Axl has been documented in several other malignancies including gliomas,

[77] sarcomas,[78,79] and melanoma.[80]

AXL INHIBITORS.

The expanding knowledge over the role of Axl in influencing several domains of the

neoplastic phenotype has justified a growing interest over the development of Axl-targeting

therapeutics, a direction of research that has been further strengthened by the understanding

of the central role of this oncogene in the development of acquired resistance to a plethora of

targeted anticancer agents.

A number of studies have suggested Axl inhibition as an additive or synergistic

therapeutic strategy in combination with conventional targeted therapies. In NSCLC, for

example, genetic or pharmacological inhibition of Axl has been shown to restore sensitivity to

erlotinib in resistant cells.[81] Similarly, promotion of Axl degradation has proven cytotoxic in

gefitinib-resistant cells.[55] Restoration of sensitivity to treatment through Axl inhibition in

previously drug-resistant cells has also been documented in lapatinib-treated HER-2 positive

breast cancer cells;[60] sunitinib in glioblastoma and renal cell carcinoma;[72,82] taxol in

ovarian cancer cells;[83] and PI3K-α inhibitors in squamous cell carcinomas.[84]

A number of strategies to pharmacologically inhibit Axl are currently in clinical

development, the most significant examples of this are anti-Axl TKIs, monoclonal antibodies

17

(mAbs), Axl decoy receptors.

Anti-Axl TKIs often possess off-target effects and frequently exert concurrent effects on

the Met pathway, such as cabozantinib, crizotinib and foretinib. Cabozantinib, initially

developed as a Met and VEGFR2 inhibitor, has been proven clinically effective in RCC[73]

through joint inhibition of Axl and Met.[72]

BGB-324, also known as R428, is a specific small molecule TKI which blocks auto-

phosphorylation of Axl, thus preventing subsequent activation of downstream signalling.

Following studies showing its broad anticancer effects across a wide range of malignancies it is

currently in early phase clinical trials as a monotherapy for chemoresistant cancers.[85] Another

Axl-specific TKI, DP3975, is currently in preclinical development and has been shown to prevent

proliferation and cell migration in pre clinical models of malignant mesothelioma.[86]

The anti-Axl mAbs YW327.6S2 and hMAb173 have also been tested pre-clinically.

hMAb173 was recently shown to induce apoptosis and inhibit growth in renal cancer cells in

vivo.[87] As an alternative strategy, the ‘decoy receptor’ MYD1 Fc can inhibit the Gas6/Axl

interaction through ligand sequestration, with proven antitumour effects in vivo.[88]

Functional inhibition of Gas6 through suppression of the hepatic gamma-carboxylation

of glutamate residues has recently emerged as a novel potential approach. The use of warfarin

at doses that were sub-therapeutic for anticoagulation has been proven effective in reducing

Gas6-mediated Axl activation, translaing in sustaiend reduction of migration, invasiveness and

improved sensitivity to chemotherapy.[89]

Whilst not exhaustive, these examples show that Axl-targeted therapies are gaining

momentum in the clinical arena. It remains unknown, however, how patients should be best

18

selected for anti-Axl therapies, a clinical context where no stratifying biomarker currently exists.

Whilst immunohistochemical analysis has been the preferred method to quantify

overexpression of Axl in clinical samples and in pre-clinical work, future studies should address

its clinical utility allowing for optimal patient selection.

ACKNOWLEDGEMENTS.

DJP is supported by grant funding from Action Against Cancer, the Academy of Medical Sciences

and the Imperial BRC.

FIGURE LEGEND.

Figure 1. A schematic representation illustrating the downstream signalling molecules

that are influenced by Gas6/Axl activation, as well as the cellular processes they effect. Spear

headed arrows indicate activation, whilst flat headed arrows indicate repression.

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