REVIEWS The TAM family: phosphatidylserinesensing receptor tyrosine kinases gone awry in cancer Douglas K. Graham1, Deborah DeRyckere1, Kurtis D. Davies1 and H. Shelton Earp2

Abstract | The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

University of Colorado Comprehensive Cancer Center, Department of Pediatrics, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado 80045, USA. 2 University of North Carolina Lineberger Comprehensive Cancer Center, Departments of Medicine and Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. Correspondence to H.S.E.  e‑mail: [email protected] doi:10.1038/nrc3847 1

The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs)1–6 was one of the latest to evolve 7 and one of the last to be identified, partly because they are not strong oncogenic drivers. Nonetheless, although genes encoding the TAM RTKs are infrequently amplified or mutated in human cancers, each was first identi‑ fied in neoplastic cell lines through a variety of PCR8, functional2 and bacterial cloning 1 strategies (see REF. 4 for a review of original papers identifying the three RTKs). Members of this family have a similar overall domain structure and are highly related by a unique KWIAIES conserved sequence in their kinase domain. TAM RTKs are ectopically expressed or overexpressed in a wide variety of human cancers in which they pro‑ vide tumour cells with a survival advantage4 (FIG. 1). In experimental models, AXL and MERTK can be oncogenic. AXL transforms NIH3T3 cells2; the virally transduced chicken orthologue of MERTK, EYK, is transforming 9; and transgenic expression of MERTK leads to lymphoid leukaemia in mice 10. Although MERTK and AXL can activate standard proliferative pathways (ERK, AKT and members of the signal trans‑ ducer and activator of transcription (STAT) family), their output generally promotes survival rather than

proliferation11–14. Much less is known about TYRO3, not necessarily because it is less important but because it has been understudied. TAM RTK activation mechanisms are unique, as maximal stimulation involves both an extra­cellular lipid moiety and a bridging protein ligand. The ligands are γ‑carboxylated proteins that bind to the recep‑ tor with their carboxy‑terminal domain and to the lipid phosphatidylserine (PtdSer) with their amino terminus 15–18 (FIG.  2) . The first such ligand, growth arrest-specific protein 6 (GAS6), was purified from conditioned media from normal lung and endo­ thelial cell lines15,16, and binds to all three TAM RTKs. A second γ‑carboxylated protein, vitamin K-dependent protein S (PROS1), binds only to MERTK and TYRO3 (REFS 19–21). Other tissue- and receptor-specific ligands have been identified more recently22–24. In the body, PtdSer is abundant but only available to activate TAM receptors when externalized on apoptotic cell mem‑ branes, aggregating platelets, exosomes and invading virus envelopes25–30. How the lipid moiety on a mem‑ brane external to the TAM RTK-expressing cell struc‑ turally interacts with the ligand receptor complex and whether engulfment requires oligomeric clustering of TAM RTKs is unknown.

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 769 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS TYRO3 NH2

AXL NH2

MERTK NH2

41–128

27–128

81–186

139–220

139–222

197–273

227–320

227–331

325–416 430

336–428 452

450

472

286–381 386–484 AXL cleavage site (V438–W452) 506 526

Y481

MER cleavage site (P490)

Y549

Y634 Y643 536–807

Y681* Y685* Y686*

p85 SRC

GRB2

Y804* Y828

LCK PLCγ

COOH (890) Ig-like domian

587–858

Y698* Y702* Y703* Y726 Y759 Y779* Y821*

Y872* Y929*

Y866*

COOH (894) FNIII domain

Y749* Y753* Y754*

p85 GRB2

518–790

COOH (999)

Kinase domain

Figure 1 | Schematic representation of the TAM family receptors. The TAM family Reviews receptors — TYRO3 (also known as DTK, SKY, RSE, BRT, TIF, ETK2),Nature AXL (also known| Cancer as UFO, ARK, JTK11 and TYRO7) and MERTK (also known as MER, EYK, RYK, RP38, NYK and TYRO12) — are shown. Conserved domains include two extracellular fibronectin type III (FNIII) and two immunoglobulin (Ig)-like domains, as well as a conserved kinase domain featuring the unusual KWIAIES sequence that is unique to this family of receptor tyrosine kinases (RTKs). The three members of the family were cloned and identified by several groups in the early and mid‑1990s (see REF. 4 for a review). Also depicted are validated tyrosine autophosphorylation sites and known SH2 domain-docking sites. Residue numbers correspond to the human sequence. Phosphorylated tyrosines are shown. Asterisks indicate autophosphorylation, which has been confirmed by experimental analysis or by sequence similarity. GRB2, growth factor receptor-bound protein 2; PLCγ, phospholipase Cγ.

M2‑polarized macrophage An alternatively activated macrophage that secretes immunosuppressive cytokines and growth factors to promote tissue repair.

Efferocytosis The process by which macrophages and epithelial cells ingest apoptotic material.

Retinitis pigmentosa An inflammatory process that occurs in the lining of the retina of the eye.

Involution The process that occurs after lactation ceases, which results in substantial cell death in the milk-producing epithelium of the mammary gland.

TAM RTK activation regulates cytoskeletal func‑ tions, intracellular signalling and gene expression. For example, in macrophages MERTK activation leads to engulfment of apoptotic material and sup‑ pression of the inflammatory cytokine response, as befits the ingestion of ‘self ’ material25. PtdSer on the surface of aggregating platelets activates MERTK, AXL and TYRO3, which collaboratively stabilize clot formation. These physiological roles do not implicate TAM RTKs as probable oncogenic drivers. However, TAM RTKs are ectopically induced or over‑ expressed in a wide range of neoplastic cells and their signals — which are activated by available autocrine or paracrine ligands and by the reservoir of PtdSer on apoptotic cells — promote survival, chemoresistance, motility and invasion. In addition, the normal roles of MERTK and AXL in preventing or terminating innate immune-mediated inflammation and natural killer (NK) cell responses are subverted in the tumour microenvironment. These actions can diminish anti‑ tumour immune reponses, allowing metastasis and

stimulating cell growth through the wound-healing nature of the MERTK-expressing M2‑polarized macrophage31–35. Thus, although the TAM family members are not classic, frequently mutated oncogenic driv‑ ers, they are aberrantly expressed in virtually every type of cancer, providing signals that are advanta‑ geous to the neoplastic cell, and they are therefore bona fide therapeutic cancer targets. In addition, their role in diminishing the innate immune response makes their inhibition a novel mechanism for revers‑ ing the immuno­suppressive tumour microenviron‑ ment. However, sustained TAM RTK inhibition may lead to inflammation and autoimmunity, and these adverse effects are intensified when two or three TAM receptors are chronically and simultaneously inhibited.

Physiological TAM functions Tissue repair and clearance of apoptotic material. The best-studied TAM RTK function is the role of MERTK in efferocytosis — the process by which apoptotic mat­ erial is cleared by both monocyte-derived and epithelial cells. The key role of MERTK in macrophage effero­ cytosis was discovered using Mertk−/− mice25. An epi‑ thelial role for MERTK was uncovered in genetic studies of retinitis pigmentosa in rats, which identified the Mertk mutation as causative36,37. Without MERTK, the pig‑ mented epithelial cells lining the retina cannot effi‑ ciently ingest the apoptotic material that is shed nightly by rods and cones, which leads to inflammation and scarring. Mertk−/− mice also show retinal degeneration38, and these rodent models are being used to test intraocu‑ lar MERTK gene therapy 39, as rare human families car‑ rying MERTK-inactivating mutations have variable age penetrance for retinitis37. Other specialized epithelial cells also rely on MERTK-dependent efferocytosis to clear damaged material, including mammary epithelial cells during weaning-induced involution40, podocytes in the renal glomerulus (which induce MERTK following nephrotoxic injury)41 and Sertoli cells in the testes42. In the brain, neurite endings that do not form productive synapses are cleared by MERTK-dependent microglial pruning 23,43,44. Most circulating monocytes lack expression of MERTK45–47. As they egress from the circulation and adhere in tissues, MERTK expression rapidly increases45. After encountering apoptotic cell PtdSer–ligand com‑ plexes, MERTK activation stimulates RHO family GTPases, which leads to engulfment48. MERTK signalling also alters macrophage gene expression, which suppresses inflammatory cytokine production and polarizes the macro­phage towards a wound-healing, anti-inflammatory M2 phenotype35,45. Thus, MERTK functions in macro­ phages to promote the rapid clearance of self antigens, to repair injured tissue and to suppress inflammation. In the absence of MERTK, inflammatory damage accu‑ mulates; for example, bleomycin-induced lung damage is greater 49, and experimental cardiac infarct results in a larger long-term decrement in cardiac function50 when infiltrating macrophages lack MERTK, as in Mertk−/− mice.

770 | DECEMBER 2014 | VOLUME 14

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS a

b GAS6

PROS1

c

PtdSer-presenting membrane GAS6

GLA1 gene product

PtdSer-presenting membrane

PROS1

GLA domain

Tyrosine kinase domain

P P AXL

MERTK

d

TYRO3

PtdSer-presenting membrane

MERTK

AXL

e

TYRO3

PtdSer-presenting membrane

f

GAS6 GAS6

MERTK or TYRO3

AXL

PROS1 NH2

GAS6 NH2 53–94

42–87

116–154 156–196 197–237 238–278

117–155 157–200 201–242 243–283

341–513

299–475

520–713

484–666

COOH (721) P

Podocytes Epithelial cells that wrap around the capillaries in the glomeruli of the kidney.

Sertoli cells The cells in the male gonad that nourish the sperm-producing processes.

M1 macrophage A macrophage that is activated by cytokines, or that is in contact with or has ingested foreign material, resulting in release of cytokines that initiate or prolong an inflammatory innate immune response.

P

P

P

GLA

EGF-like

COOH (676) Lamimin G-like

Figure 2 | Ligand-mediated activation of the TAM receptors.  Activation of TYRO3, AXL and MERTK (TAM) by growth Nature Reviews | Cancer arrest-specific protein 6 (GAS6) and vitamin K-dependent protein S (PROS1), the two best-characterized TAM ligands, is shown. Selectivity of the ligands for TAM receptors in the absence (part a) or in the presence (part b) of phosphatidylserine (PtdSer)-presenting membranes (for example, apoptotic cells, enveloped virus or PtdSer liposomes) is shown. The thickness of the arrows reflects the relative affinities and the degree of activation of the receptors by the ligands. In Ciona intestinalis, the GLA1 gene product encodes an extracellular PtdSer-binding γ‑carboxylglutamic acid-rich (GLA) domain and an intracellular tyrosine kinase domain (part c). This configuration suggests that coupling PtdSer sensing and intracellular signalling can occur via a single protein, and that separation into distinct ligands and receptors emerged later in or on a different pathway of evolution. A potential mechanistic explanation for the differential effects of PtdSer-presenting membranes on receptor activation by GAS6 is shown in parts d and e. GAS6 is able to bind to two different domains on separate AXL molecules, thus facilitating a 2:2 stoichiometry, receptor homodimerization and near-complete activation (part d). Only one GAS6‑binding domain is conserved on MERTK and TYRO3, which suggests that the presence of PtdSer-presenting membranes is required to increase local concentrations of the receptors and to promote dimerization and full activation (part e). A schematic representation of GAS6 and PROS1 with important protein domains is shown (part f; residue numbers correspond to the human sequences). Oligomeric forms of PROS1 may have roles in activation of TAM receptors. EGF, epidermal growth factor.

Innate immune control. A delicate balance of the immune system is required to respond to pathogens but guard against attacking ‘self ’ cells. MERTK and AXL have major roles in controlling this balance, using at least five cellular mechanisms to protect against an over­ zealous inflammatory response (FIG. 3). The first mecha‑ nism is efficient efferocytosis (discussed above), which rapidly eliminates intracellular antigens25. In the second

mechanism, during apoptotic cell ingestion, MERTK suppresses the M1 macrophage pro-inflammatory cytokine response (involving interleukin‑12 (IL‑12), IL‑6 and tumour necrosis factor (TNF)), partly by diminish‑ ing nuclear factor-ĸB (NF-ĸB) signalling 51–53, and also enhances M2 macrophage anti-inflammatory cytokine production — for example, IL‑10, transforming growth factor‑β (TGFβ), hepatocyte growth factor (HGF)

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 771 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS a Efficient clearance of

b Apoptotic cells polarize macrophages

intracellular antigens

towards M2

Macrophage

GAS6 MERTK

Apoptotic cell

M2

M1 Apoptotic cell debris with PtdSer

↓M1 macrophage IL-12

c AXL signalling dampens

TLR inflammatory response

d Activated T cell feedback inhibits innate immunity

AXL

PROS1

APC SOCS1 SOCS3

↑M2 macrophage IL-10

CD8+ ↓TLR

T cell

• ↓TH1 cell • ↓M1 macrophage

e TAM signalling inhibits NK cell anti-metastatic effects NK cell

AXL or MERTK

Lung metastasis

Figure 3 | Mechanisms of immunosuppression mediated by TAM RTKs in innate immune cells.  Innate immunosuppressive actions stimulated by ligand Nature Reviews | Cancer phosphatidylserine (PtdSer) complexes include MERTK-mediated rapid and efficient efferocytosis of apoptotic material by macrophages to clear intracellular antigens (part a). Efferocytosis-induced activation of MERTK in macrophages also alters the cytokine transcriptional profile, suppressing the inflammatory (M1) response and stimulating the expression of an immunosuppressive and wound-healing (M2) cytokine profile (part b). AXL suppresses pro-inflammatory Toll-like receptor (TLR) responses in antigen-presenting cells (APCs) (part c). AXL is induced following TLR signalling in APCs and, in turn, AXL feedback suppresses TLR signalling and pro-inflammatory cytokine release. Activated T cells transcriptionally upregulate vitamin K-dependent protein S (PROS1) and concomitantly externalize PtdSer (shown in red), which creates a complex ligand that binds to APCs and suppresses their activity through interaction with TAM receptor tyrosine kinases (RTKs) (part d). A recently described feedback mechanism mediated by TAM RTKs suppresses natural killer (NK) cell function, which allows enhanced metastasis (part e). By contrast, inhibition of TAM RTKs in this context decreases metastasis. GAS6, growth arrest-specific protein 6; IL, interleukin; SOCS, suppressor of cytokine signalling; TH1, T helper 1.

and IL‑4

Lipopolysaccharide (LPS). A bacterial product that is sensed by specific Toll-like receptors on innate immune cells, which triggers a robust inflammatory response.

(REFS 45,54–56).

In Mertk−/− mice, lipopoly­ saccharide (LPS) causes toxic shock and death at much lower doses than in wild-type mice as a result of exuber‑ ant production of M1 cytokines51. The third mechanism involves dendritic cells, which are more dependent on AXL than on MERTK21,34, to provide feedback that helps to terminate inflammatory Toll-like receptor (TLR) sig‑ nalling. In these antigen-presenting cells (APCs), TLR signalling results in activation of STAT1, which in turn induces AXL mRNA33. AXL functions together with the type I interferon (IFN) receptor to increase suppressor of cytokine signalling 1 (SOCS1) and SOCS3 expression, which helps to terminate inflammatory TLR signalling. The fourth mechanism is that activated T cells induce

the expression of PROS1 and externalize limited PtdSer patches on T cell membranes. This T cell-based ligand complex directly contacts innate immune cells, activat‑ ing MERTK and turning down inflammatory cytokine production57. The fifth mechanism is another feedback mechanism in NK cells. MERTK and AXL decrease NK cell antitumour activity, which paradoxically allows increased metastases32. Although genetic loss of any single TAM RTK results in autoimmunity 58,59 and potentially exacerbates chronic inflammation leading to neoplasia (for example, loss of MERTK may contribute to colitis-associated colon can‑ cer 60), the most dramatic example of TAM RTK control of the inflammatory response is the triple knockout mouse. Engineered loss of all three TAM RTKs is not embry‑ onically lethal but leads to a hyper-inflammatory state with profound autoimmunity, multiple organ defects and massive lymphoproliferation59 (see TABLE 1 for a summary of the phenotypes associated with disruption of TAM RTKs and their ligands in rodents). By contrast, TAM RTK-mediated suppression of the innate immune inflammatory response can sometimes be deleterious, for example, in cancers in which TAM RTKs contribute to an immunosuppressive tumour microenvironment. In a similarly deleterious man‑ ner, several viruses externalize PtdSer on their enve‑ lopes during budding and thereby bind to and activate TAM RTKs, in some cases allowing entry into cells (for example, Ebola (BOX 1)). This apoptotic mimicry helps the virus to avoid an initial robust innate immune response29. Determining whether TAM RTK inhibition could be used for antiviral or anticancer therapy will require analysis of the risks and benefits of removing a homeostatic check on inflammation; the answer may depend on the duration of therapy. Regulation of TAM RTK expression. TAM RTK induc‑ tion occurs in normal and neoplastic cells, but the mechanisms are understudied. Normal epithelial cells can induce MERTK expression by up to 20‑fold when induced to ingest apoptotic material, for example, in mammary epithelium during weaning and in injured renal epithelium40,41. Corticosteroids induce the expres‑ sion of Mertk mRNA in macrophages and dendritic cell subsets21,61. In macrophages, there is a feed-forward sys‑ tem whereby injury, stress and efferocytosis upregulate MERTK expression. Induction of MERTK in response to efferocytosis is mediated by intracellular modifica‑ tion of ingested membrane cholesterol, which yields steroid metabolites that activate the liver X receptor (LXR). Ligand-bound LXR directly binds to the Mertk promoter to enhance transcription62. MERTK activation during efferocytosis enhances IL‑10 secretion and IL‑10 functions in an autocrine manner to further increase macrophage Mertk mRNA45. There are clear differences in the control of MERTK and AXL expression; for example, in bone marrowderived macrophages dexamethasone increases MERTK expression and nearly obliterates AXL expres‑ sion, whereas TLR agonists increase AXL expression without altering MERTK expression21. Thus, although

772 | DECEMBER 2014 | VOLUME 14

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Chordates Animals that have a notochord, a dorsal nerve chord and gill slits at one or more stages of development. This includes the vertebrates.

MERTK and AXL are widely induced in cancer, their expression can be regulated by different pathways. Posttranscriptional processes can also regulate TAM protein levels; for example, ubiquitylation can regulate degra‑ dation32 and microRNAs (mi­RNAs) may alter produc‑ tion64-67. Alteration of expression that is regulated by mi­RNAs may be one way that TAM RTK expression is increased in the disordered neoplastic cell to pro‑ vide a survival advantage. Our understanding of both normal and neoplastic TAM RTK expression control mechanisms remains rudimentary. TAM RTK ligands. The homeostatic detection of PtdSer by RTKs occurred late in evolution (in chordates and vertebrates) with the appearance of a sin‑ gle TAM family member 68. The sea squirt Ciona intestinalis encodes one protein with a MERTK-like kinase domain, a transmembrane domain and a γ‑carboxylglutamic acid-rich (GLA) extracellular domain that is capable of binding to PtdSer, which in turn directly activates the intracellular kinase domain69 (FIG.  2) . In mammals, the PtdSer-sensing system is widespread, allowing macrophages and epithelial cells

to respond to exposed PtdSer; however, the PtdSerand TAM RTK-binding ligands in individual locales are not always the same. Two major ligands bridge PtdSer and TAM RTKs (FIG. 2), but the necessity for and the stoichiometry of the lipid:protein components are still being clarified. Although initially controversial, it is now clear that PROS1 is a true ligand of MERTK and TYRO3, but it does not bind to AXL (because of alterations in the third fibronectin type III (FNIII) extracellular domain). PtdSer substantially increases GAS6 and PROS1 activa‑ tion of MERTK and, to a lesser degree, TYRO3. AXL has the highest affinity for GAS6, and some evidence indicates that the affinity in vivo is high enough that AXL constitutively binds to GAS6, but that AXL is inac‑ tive until a source of PtdSer allows dimerization20. Other reports indicate that GAS6 can activate AXL without binding to PtdSer 19. However, the preponderance of evidence suggests that PtdSer does have a physiological role in AXL activation, and there is a general agreement that vitamin K‑dependent co‑carboxylation (which con‑ fers lipid binding) of GAS6 and PROS1 is needed for full activation of all three TAMs. In fact, there is some

Table 1 | Phenotypes associated with disruption of TAM RTKs and their ligands in rodents Knockout genes

Phenotype

Mertk

• Impaired clearance of apoptotic cells, hyperactivation of APCs and autoantibody production23,25,40,41,50,58,182,183 • Retinitis pigmentosa as a consequence of defective apoptotic cell clearance in the retina38 • Enhanced inflammation and endotoxic shock following LPS challenge because of increased TNF levels in the serum51 • Protection from thrombosis as a result of defects in platelet aggregation26,73 • Reduced tumour growth and metastasis because of the pro-inflammatory state in the tumour microenvironment31

Axl

• Hyperactivation of APCs and autoantibody production59 • More severe autoimmune encephalomyelitis and enhanced demyelination in a model of multiple sclerosis184 • Defects in apoptotic cell clearance and enhanced viral load in HSV1 infection185 • Protection from thrombosis as a result of defects in platelet aggregation73 • Decreased blood vessel integrity, resulting in increased permeability129

Tyro3

• Hyperactivation of APCs and autoantibody production59 • Protection from thrombosis as a result of defects in platelet aggregation73

Double knockouts of Tyro3, Axl or Mertk

• All combinations show autoantibody production to a greater degree than single knockouts59 • Axl−/−Mertk−/− double-knockout mice show increased pro-inflammatory cytokine levels and failure to clear apoptotic neutrophils in response to an exogenous insult186 • Axl−/−Mertk−/− double-knockout mice show impaired erythropoiesis187 • Axl−/−Tyro3−/− double-knockout mice show impaired neurological formation because of GNRH+ neuron dysfunction130

Tyro3, Axl and Mertk triple knockout

• Infertility in males because of enhanced inflammation and Sertoli cell dysfunction42,188 • Hyperproliferation of B cells and T cells, leading to increased spleen and lymphoid size, autoantibody production and development of severe autoimmunity59 • Increased inflammatory brain damage because of autoantibody deposition and autoreactive lymphocyte infiltration189 • Chronic hepatitis as a result of immune cell infiltration into the liver and elevated inflammatory cytokine levels190 • Impaired neurogenesis because of microglia hyperactivation43 • Impaired NK cell differentiation and function191 • Loss of negative regulation of TLR activation and cytokine production in dendritic cells33 • Impaired haemostasis, thrombocytopenia and defective megakaryocytopoiesis192

TAM ligands

• Gas6−/− mice show protection from thrombosis as a result of defects in platelet aggregation72 • Gas6−/− mice show increased demyelination and delayed remyelination following treatment with cuprizone193,194 • Gas6−/− mice show reduced liver inflammation and fibrosis following injury195 • Gas6−/− mice, Pros1‑conditional knockout mice, Tulp1−/− mice and Tub−/− mice show retinitis pigmentosa as a consequence of defective apoptotic cell clearance in the retina71,196,197 • Gas6−/− and Pros1+/− mice show decreased blood vessel integrity, which results in increased permeability129 • Tub−/− mice show weight gain197 • Lgals3−/− mice show reduced phagocytic clearance of apoptotic cells by macrophages198 • Lgals3−/− mice show decreased lung tumour incidence following administration of a chemical carcinogen199

APCs, antigen-presenting cells; Gas6, growth arrest-specific 6; GNRH, gonadotropin-releasing hormone; HSV1, herpes simplex virus 1; Lgals3, galectin 3; LPS, lipopolysaccharide; NK, natural killer; Pros1, vitamin K-dependent protein S; RTK, receptor tyrosine kinase; TAM, TYRO3, AXL and MERTK; TLR, Toll-like receptor; TNF, tumour necrosis factor; Tub, tubby; Tulp1, tubby-like protein 1.

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 773 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Box 1 | TAM family RTKs and viral pathogenesis The innate immune system recognizes invading viral pathogens by detecting foreign proteins, glycoproteins and nucleic acids using both extracellular and intracellular sensors. The response entails inflammatory cytokine release and intracellular processes to prevent viral replication or to destroy infected cells. In the evolutionary tug of war, viruses use cellular receptors for entry and have found various methods to evade innate immune detection and response. Multiple virus families — including important human pathogens such as Ebola, Dengue and HIV — externalize phosphatidylserine (PtdSer) during budding and are known to bind and potentially to activate all three TAM family kinases. Studies have shown that viral entry is permitted or accelerated by binding to TYRO3, AXL and MERTK (TAM) receptor tyrosine kinases (RTKs) in the presence of a PtdSer-binding ligand30,178–181. This process of using PtdSer to bind to TAM RTKs and thereby to facilitate access to the cell was termed ‘apoptotic mimicry’ (REF. 29). Several cell surface receptors in addition to TAM RTKs may also be used. Furthermore, activation of TAM RTKs in macrophages in response to viral particles expressing PtdSer stimulates production of an anti-inflammatory cytokine profile that mimics the response to ingestion of apoptotic material and that inhibits the antiviral immune response30. These observations suggest that TAM RTK inhibition may be useful for the treatment of clinically important viruses, particularly if provided during early infection.

evidence that warfarin, which depletes vitamin K, has an anti-neoplastic effect by inhibiting TAM RTK acti‑ vation32. GAS6 binds to AXL and TYRO3 with much higher affinity than to MERTK19,70, which reinforces the idea that MERTK is the family member that is most dependent on PtdSer. There is also selectivity of function; for example, GAS6 and PROS1 both function as ligands for effero­ cytosis in the pigmented retinal epithelial cell71, but only GAS6 functions as a ligand for platelet activation (discussed below), even though PROS1 is abundant in the blood. The ability of activated T cells to feedback to APCs as described above requires the induction of PROS1 expression, patches of PtdSer and physical con‑ tact between the two cells33. These examples suggest that the physiological effects of ligand–PtdSer complexes may require differential local availability and direct compartmentalized contact. However, in the neoplas‑ tic milieu — from bone marrow stroma in leukaemia to the solid tumour microenvironment — autocrine (originating from tumour cells) or paracrine (originat‑ ing from infiltrating cells) ligands, as well as apoptotic cells, exosomes and platelet microaggregates, are freely available to activate TAM RTKs. Platelet aggregation. Platelets externalize PtdSer and release GAS6 from granules during the initial phase of aggregation, and Gas6−/− mice show defects in the final phases of platelet aggregation 26,72. Moreover, genetic deletion of any single TAM RTK in mice has been shown to prevent GAS6 from inducing integ‑ rin tyrosine phosphorylation 73. Therefore, in plate‑ lets TAM RTKs are not redundant; all are needed to stimulate outside‑in signalling via αIIbβ3 integrin. Whether this relates to the need for oligomers with enhanced activity or individual signalling by the three RTKs is unknown. The absence of coordinated TAM signalling decreases adherence to fibrinogen, platelet spreading, degranulation, ADP secretion and

the secretion of von Willebrand factor (VWF) 26,73. Notably, disruption of GAS6–TAM RTK signalling did not affect initial platelet monolayer formation at the site of vascular injury. As a result, there was no significant change in tail bleeding time in Gas6- or TAM RTK-knockout mice, which suggests that TAM RTK inhibition might provide a novel approach for short-term reduction of thrombosis without increased bleeding risk.

TAM RTK expression in human cancer Expression of TAM family kinases and ligands in cancer. TAM receptor and ligand overexpression have been shown in a wide range of solid and haemato‑ logical tumours, and correlate with poor prognosis in a variety of tumour types (TABLE 2; see Supplementary information S1 (table)). For example, ectopic expres‑ sion of MERTK was first observed in human lympho‑ blastic and myeloid leukaemia cells1, and MERTK is absent from mature lymphocytes and in lymphoid and myeloid precursor cells1,47,74,75. MERTK is aber‑ rantly expressed in 30–50% of samples from paediat‑ ric patients with B cell acute lymphoblastic leukaemia (B-ALL) and T-ALL, in 70–90% of samples from paediatric and adult patients with acute myeloid leukaemia (AML), and in samples from a subset of patients with multiple myeloma1,47,74–76. By contrast, AXL expression is less commonly observed in ALL1,77 but is frequent in AML77–81 and is observed in chronic lymphoid leukaemia (CLL)82 and chronic myeloid leu‑ kaemia (CML)77,81. In addition, TYRO3 mRNA is over‑ expressed in some leukaemia samples80,83,84, and GAS6 is overexpressed in subsets of human leukaemia76,81. Mechanisms of TAM family kinase expression and activation in cancer. The mechanisms that mediate the overexpression of TAM RTKs in so many tumour types are currently unknown, but some may mimic the mechanisms of expression in normal cells. In macro­ phages, MERTK transcription is stimulated by LXR and LXR ligands such as 27‑hydroxycholesterol. This sterol is produced by both tumour cells and cells in the tumour microenvironment 62,85. Epstein–Barr virus (EBV) lytic transcription factors substantially increase MERTK mRNA levels, and mechanistic studies might provide some insights into neoplastic induction86. With respect to AXL, hypoxic conditions in the tumour increase the expression of hypoxia-inducible factor 1α (HIF1α), which directly promotes AXL transcrip‑ tion87. Epigenetic regulation of expression is observed in relapsed AML and in cancer cell lines; for example, hypomethylation of the AXL promoter correlates with increased AXL expression88–90. Post-transcriptional reg‑ ulation of TAM expression by miRNAs also occurs63–66, and miR‑355, which can be suppressed in breast cancer, negatively regulates MERTK67. Genes encoding TAM RTKs are rarely mutated or amplified; however, chromosome 9q13.1 amplifi‑ cation results in increased AXL expression in some glioblastoma and gastric cancers 91,92. Infrequently, point mutations (for example, MERTK‑P802S in a

774 | DECEMBER 2014 | VOLUME 14

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Table 2 | Expression and function of TAM family kinases and ligands in specific tumour types* TAM kinase or ligand‡

Ectopic expression or overexpression

Prognostic importance§

Functional roles||

Metastatic roles¶

Roles in chemoresistance#

AXL

AML, CML, B‑CLL, lung cancer, glioblastoma, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, oesophageal cancer, melanoma, squamous cell skin cancer, prostate cancer, endometrial cancer, ovarian cancer, oral squamous cell carcinoma, thyroid cancer, bladder cancer, renal cancer, schwannoma, mesothelioma, Kaposi’s sarcoma and osteosarcoma

AML, lung cancer, glioblastoma, osteosarcoma, oral squamous cell carcinoma, breast cancer, head and neck cancer, colorectal cancer, pancreatic cancer, oesophageal cancer, ovarian cancer, gastric cancer and bladder cancer

Prostate cancer, ovarian cancer, breast cancer, thyroid cancer, lung cancer, pancreatic cancer, melanoma, hepatocellular carcinoma, glioblastoma, mesothelioma, osteosarcoma, schwannoma, Kaposi’s sarcoma and oesophageal cancer

Breast cancer, lung cancer, melanoma, prostate cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, thyroid cancer, bladder cancer, Kaposi’s sarcoma, mesothelioma, oesophageal cancer, glioblastoma, colorectal cancer, cervical cancer, neuroblastoma and osteosarcoma

AML, CML, breast cancer, lung cancer, ovarian cancer and oesophageal cancer

MERTK

AML, ALL, lung cancer, glioma, melanoma, prostate cancer, schwannoma, mantle cell lymphoma and rhabdomyosarcoma

Gastric cancer

AML, ALL, glioma, lung cancer and melanoma

Glioblastoma and melanoma

B‑ALL, T‑ALL, glioma, lung cancer, pancreatic cancer and breast cancer

TYRO3

AML, multiple myeloma, lung cancer, melanoma, prostate cancer, endometrial cancer, thyroid cancer and schwannoma

Not reported

Melanoma and thyroid cancer

Melanoma and thyroid cancer

Not reported

Galectin 3**

AML, CML, ALL, lymphoma, multiple myeloma, thyroid cancer, parathyroid cancer, pituitary tumours, gastric cancer, pancreatic cancer, hepatocellular carcinoma, colorectal cancer, renal cancer, bladder cancer, glioma, melanoma, breast cancer, prostate cancer, ovarian cancer, lung cancer, osteosarcoma, neuroblastoma, haemangioblastoma and other carcinomas

AML, ALL, lymphoma, pituitary tumours, hepatocellular carcinoma, colorectal cancer, renal cancer, bladder cancer, glioblastoma, melanoma, ovarian cancer and other carcinomas

CML, ALL, lymphoma, multiple myeloma, thyroid cancer, gastric cancer, pancreatic cancer, hepatocellular carcinoma, melanoma, breast cancer, prostate cancer and cholangiocarcinoma

Pituitary tumours, gastric cancer, pancreatic cancer, hepatocellular carcinoma, colorectal cancer, bladder cancer, glioma, melanoma, breast cancer, prostate cancer, lung cancer, osteosarcoma and other carcinomas

CML, ALL, lymphoma, multiple myeloma, thyroid cancer, gastric cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer and ovarian cancer

GAS6

AML, ALL, CML, multiple myeloma, glioblastoma, breast cancer, gastric cancer, endometrial cancer, ovarian cancer, thyroid cancer, renal cancer and schwannoma

AML, lung cancer, glioblastoma and renal cancer

Lymphoma, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, thyroid cancer, schwannoma, gastric cancer, osteosarcoma and renal cancer

Breast cancer, prostate cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, osteosarcoma and renal cancer

B‑ALL

PROS1

AML, thyroid cancer, colorectal Prostate cancer cancer, pancreatic cancer, brain tumours, lung cancer, prostate cancer, ovarian cancer and osteosarcoma

Not reported

Prostate cancer

Not reported

ALL, acute lymphoid leukaemia; AML, acute myeloid leukaemia; B‑CLL, B cell chronic lymphoid leukaemia; CML, chronic myeloid leukaemia; GAS6, growth arrest-specific protein 6; PROS1, vitamin K-dependent protein S; TAM, TYRO3, AXL and MERTK. *See Supplementary information S1 (table) for references. ‡ Relevant studies of recently identified ligands tubby and tubby-like protein 1 (TULP1) have not been reported. §Expression of TAM family kinase or ligand is associated with poor prognosis. ||TAM family kinase or ligand mediates increased tumour cell proliferation, survival or colony-forming potential in cell culture assays, or promotes tumorigenesis in murine xenograft models. ¶TAM family kinase or ligand promotes tumour cell migration, invasion or epithelial-to‑mesenchymal transition or increases metastasis in murine xenograft models. #TAM family kinase or ligand promotes resistance to cytotoxic chemotherapies in cell culture or murine xenograft models. **Decreased galectin 3 expression relative to normal cells or adjacent tissues has also been reported in multiple tumour types, including colorectal, breast, prostate, ovarian and skin cancers. Similarly, increased galectin 3 expression has been associated with a more favourable prognosis in some tumour types, including colorectal, bladder, gastric, breast cancers and neuroblastoma.

patient with melanoma93) and translocations creat‑ ing fusion genes (for example, AXL–MAP3K12 in a patient with lung adenocarcinoma 94) have been detected, although the functional importance of these mutations is not clear (see TABLE 3 and Supplementary information S2 (table) for other examples). In some

cases, ectopic or over­expression of TAM RTKs alone is sufficient to promote oncogenic phenotypes. For example, introduction of MERTK into non-neoplastic breast epithelial cells was shown to confer the ability to efferocytose and be activated by apoptotic material, leading to increased motility and chemoresistance95.

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 775 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Non-oncogene addiction Increased dependence of the neoplastic cell on the induction of endogenous cellular survival factors needed to prevent apoptosis in response to the hyperproliferative and altered metabolic states driven by oncogene expression or tumour suppressor loss.

TAM receptor signalling and function in neoplasia The biology of TAM RTKs in tumour cells. To be such a common occurrence, TAM RTK induction must provide a generic advantage to neoplastic cells. Oncogenes and loss of tumour suppressors promote unregulated growth, but neoplastic cells become dependent on non-oncogenes for survival during stress (that is, non-oncogene addiction). For example, although MERTK knockdown by short hairpin RNA (shRNA) only modestly promotes apoptosis and slows proliferation in cell cultures, the effect is more pro‑ nounced when combined with serum starvation47, and MERTK knockdown markedly reduces growth under more stressful conditions in soft agar and in xeno‑ grafts47,96. TAM survival signals may be particularly important in the tumour microenvironment, in which limited oxygen and nutrient supplies exacerbate the proteotoxic and genotoxic conditions. An example of this pathophysiology is that GAS6 and PtdSer on exosomes from bone marrow stromal cells and osteo‑ blasts provide survival signals for cancer cells in the bone marrow niche97,98. The roles of TAM RTKs in survival were first shown in leukaemia cells in which ligand stimula‑ tion of chimeric or endogenous MERTK, AXL or TYRO3 prevented apoptosis following growth fac‑ tor withdrawal11,97,99–101. AXL tyrosine kinase inhibi‑ tors (TKIs) induced apoptosis in B‑CLL cells82, and shRNA-mediated MERTK knockdown promoted apoptosis in AML cells in response to serum starva‑ tion47. Inhibition of MERTK or AXL in leukaemia cells using shRNA also decreased colony-forming poten‑ tial and chemoresistance47,74,75,79. In animal models, transgenic MERTK expression in haematopoietic cells resulted in the development of T cell malignancy 10, and shRNA-mediated MERTK knockdown in leu‑ kaemia cells increased survival in orthotopic ALL and AML xenograft models47,74,75. A recent study similarly implicates AXL in myeloid leukaemogenesis79, and studies using small interfering RNA (siRNA), shRNA

and dominant-negative constructs have shown similar roles for TAM RTKs in a wide range of solid tumours (TABLE 2; see Supplementary information S1 (table)). Do AXL and MERTK have different roles in tumour cells? In a non-small-cell lung cancer (NSCLC) cell line, AXL inhibition decreased chemoresistance to a greater degree and in response to a wider range of agents than did MERTK inhibition96. Conversely, MERTK inhibition almost completely blocked tumorigenesis in an NSCLC xenograft model, whereas AXL inhibition only had a partial effect 96. In glioblastoma models, MERTK inhi‑ bition reduced migration, whereas inhibition of AXL did not 102. In samples from patients with melanoma, differential expression of AXL and MERTK was prefer‑ entially associated with migratory and proliferative gene expression signatures, respectively 93. Similarly, shRNAmediated knockdown of MERTK or TYRO3 suppressed soft agar growth in different subsets of melanoma cell lines93,103. The TAM RTK (or RTKs) expressed in indi‑ vidual tumours or in different tumour types are not nec‑ essarily redundant; each may connect to downstream survival or motility signalling in slightly different ways. Whether the TAM RTK dependencies in tumour cell lines represent the heterogeneity of the patient tumour from which the cell lines were derived or adaptations in culture will only be truly determined by biomarker driven clinical trials of TAM RTK inhibitors. Survival signalling pathways activated by TAM RTKs. As with other RTKs, ligand binding induces homo­ dimerization and subsequent activating autophos‑ phorylation, which results in stimulation of a range of pathways, including those involving ERK, AKT, p38, RHO family proteins, NF-ĸB, Janus kinase (JAK)–STAT and SRC family kinases (FIGS 1,4) (reviewed in REF. 4). In certain instances, particularly when overexpressed, TAM receptors may heterodimerize or at least crossphosphorylate104. Chronic GAS6 treatment was also shown to reduce MERTK expression on the cell sur‑ face and to increase MERTK bound to chromatin in

Table 3 | Somatic TAM family kinase mutations in cancer* TAM RTK

Extracellular and transmembrane domain mutations

Cytosolic domain mutations

AXL

C24G (ovarian cancer), P36L (melanoma), N43T (colon cancer), R236C (melanoma), P238L (skin cancer), V289M (gastric cancer), R295W (lung cancer), T343M (breast cancer), A358V (ovarian cancer), R368Q (prostate cancer), G413W (melanoma), L423Q (lung cancer), E431K (melanoma) and A451T (melanoma)

• E484D (pancreatic cancer), R492C (gastric cancer), K526N (lung cancer), E535K (melanoma), G829E (melanoma) and S842F (gastric cancer) • Mutations in the kinase domain: M580K (colon cancer), S599F (lung cancer), I610V (melanoma), P636H (gastric cancer), A666T (melanoma), L684P (colon cancer), S685F (melanoma), P742T (kidney cancer), E745K (breast cancer), S747R (breast cancer) and R784Q (melanoma)

MERTK

P40S (melanoma), S159F (lung cancer), E204K (urinary tract cancer), S428G (gastric cancer), I431F (lung cancer), A446G (kidney cancer), N454S (liver cancer), W485S/C (lymphoma) and V486I (melanoma)

• L586F (urinary tract cancer), V873I (liver cancer), S905F (lung cancer), K923R (melanoma), P958L (kidney cancer), D983N (liver cancer) and D990N (colon cancer) • Mutations in the kinase domain: G594R (breast cancer), S626C (urinary tract cancer), P672S (lung cancer), L688M (colon cancer), A708S (head and neck cancer), N718Y (lung cancer), R722‡ (colon cancer), M790V (lung cancer) and P802S (melanoma)

TYRO3

Q67‡ (melanoma) and E340‡ (lung cancer)

• R462Q (melanoma), R514Q (pancreatic) and G809D (colon) • Mutations in the kinase domain: M592I (colon), N615K (lung), W708fs*5 (melanoma) and A709T (brain)

RTK, receptor tyrosine kinase; TAM, TYRO3, AXL and MERTK. *See Supplementary information S2 (table) for references. Published TAM family kinase mutations that have been confirmed to be somatic are shown. The disease in which the mutation was identified is shown in parentheses. ‡Indicates mutations that encode a premature stop codon.

776 | DECEMBER 2014 | VOLUME 14

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Receptor crosstalk and cooperation AXL

FLT3

MERTK TYRO3

EGFR HER2

Cyclin D MEK

p38

BCL-2 BCL-XL

PI3K

NF-κB

PUMA

CREB

JAK

ATF1

STAT3

MCL1 Survivin ERK

AKT

FAK1 RHO BAD

mTOR

Anti-apoptosis and survival

RAC RALA

STAT5

MMP9 RALB

STAT6

TWIST SNAIL SLUG EMT

MITF

?

SRC family

Cell cycle progression ACK1

WWOX

Nucleus Cellular growth and proliferation

Invasion and migration

Oncogenic transformation

Tumour growth

Figure 4 | Signalling by TAM family RTKs.  Multiple investigators have studied signalling by ligand-activated, overexpressed or chimeric TYRO3, AXL and MERTK (TAM) receptor tyrosine kinases (RTKs) using primary, normal and Nature Reviews | Cancer neoplastic cell lines. Crosstalk with numerous other RTKs has been described and is depicted at the cell surface. Almost every oncogenic pathway is regulated downstream of TAM RTKs in some contexts. The specific response depends on the TAM RTK and ligand, and different subsets of pathways are activated in different cell types (not shown). Survival signalling and invasion are most prominent but, in some tumour cell lines, proliferation is observed in response to TAM activation. The function of nuclear MERTK is unknown (indicated by a question mark). ACK1, activated CDC42 kinase 1; ATF1, activating transcription factor 1; BAD, BCL‑2‑associated agonist of cell death; CREB, cAMP-responsive element-binding protein; EGFR, epidermal growth factor receptor; EMT, epithelial-to‑mesenchymal transition; FAK1, focal adhesion kinase 1; FLT3, FMS-like tyrosine kinase 3; JAK, Janus kinase; MCL1, myeloid cell leukaemia 1; MITF, micropthalmia-associated transcription factor; MMP9, matrix metalloproteinase 9; NF‑κB, nuclear factor‑κB; PUMA, p53 upregulated modulator of apoptosis; STAT, signal transducer and activator of transcription; WWOX, WW domain-containing oxidoreductase.

the nucleus105. The proportion of MERTK bound to chromatin was related to the characteristics of recep‑ tor glycosylation, which changed with chronic stimula‑ tion. The functional consequences of nuclear MERTK are unknown. Acute GAS6 stimulation activates PI3K–AKT signal‑ ling in multiple cancers, and this effect can be abrogated by inhibition of TAM RTKs47,74,75,88,96,103,106–115. The RAS– RAF–MEK signalling pathway is similarly activated by GAS6 and, again, TAM inhibition by genetic or pharma‑ cological means blocks both ligand-stimulated and basal pathway activation in tumour cells10,12,74,75,80,88,96,97,103,114–116. STATs are implicated in tumour cell proliferation and survival, and STAT tyrosine phosphorylation and tran‑ scriptional activity are regulated downstream of MERTK (in the cases of STAT3, STAT5 and STAT6) or AXL (in the case of STAT3) in multiple tumour types47,66,75,103,117. One potentially crucial distinction between MERTK signalling in normal and neoplastic cells revolves around the NF-ĸB pathway. In tumour cells, MERTK activates the NF-ĸB pathway 99,118–122 and potentially its anti-apoptotic mechanisms. By contrast, when suppressing the innate immune inflammatory response, MERTK and AXL inhibit NF-ĸB signalling and subsequent inflamma‑ tory cytokine production51–53. This context-dependent reversal of NF-ĸB signalling may be a mechanism by which TAM activity in tumour cells is subverted to

enhance survival. In addition, AXL or MERTK inhibi‑ tion decreased expression of the anti-apoptotic proteins BCL‑2, BCL‑XL, myeloid cell leukaemia 1 (MCL1) and survivin in tumour cells80,82,96, and induced the expression or the activation of the pro-apoptotic pro‑ teins BCL‑2‑associated agonist of cell death (BAD), BAX, BCL‑2 homologous killer (BAK) and p53 upregu‑ lated modulator of apoptosis (PUMA; also known as BBC3)74,80,123. GAS6 treatment increased levels of BCL‑2 in AML cells88, and the pro-apoptotic protein BAD was phosphorylated and inactivated by GAS6‑induced AKT activation106. Similarly, MERTK activates acti‑ vated CDC42 kinase 1 (ACK1) in prostate cancer cells, which results in ubiquitylation and degradation of the multifunctional pro-apoptotic tumour suppressor WW domain-containing oxidoreductase (WWOX)48. Activation of ACK1 may also influence other survival pathways124. Each of these actions, mediated by TAM RTKs in different tumour contexts, functions to promote tumour cell survival in response to apoptotic stimuli.

TAM function in tumour angiogenesis TAM receptors and ligands are expressed in endothelial cells, pericytes and vascular smooth muscle cells, and the GAS6–AXL pathway has been implicated in vas‑ culogenesis125. AXL knockdown was shown to impair endothelial tube formation108, and AXL-dependent

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 777 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Tingible body macrophages Macrophages in the lymph node germinal centres that help to maintain tolerance to ‘self’ antigens.

activation of AKT is thought to promote tumour angio‑ genesis in conjunction with vascular endothelial growth factor A (VEGFA) and VEGF receptor 2 (VEGFR2)126. By contrast, PROS1 stimulation of MERTK was shown to inhibit the VEGFA-induced activation of VEGFR2, diminishing endothelial tube formation and endothelial cell migration127,128. Axl−/− mice have defects in vessel per‑ meability and integrity, but Mertk−/− and Tyro3−/− mice do not, which again emphasizes the different roles of TAM RTKs, with AXL promoting angiogenesis in the vasculature129.

TAM RTKs in metastasis Metastatic roles for TAM RTKs in tumour cells. Most patients with solid tumours die of metastatic disease rather than from the primary tumour. AXL in particu‑ lar has been implicated in metastasis in multiple tumour types ( TABLE  2; see Supplementary information  S1 (table)). First, AXL has a role in normal directed motility in the nervous system during the migration of gonado­ tropin-releasing hormone (GNRH)+ neurons to the hypothalamus130,131. Second, in patient samples and cell lines, AXL expression correlates with migration and metastasis (TABLE 2; see Supplementary information S1 (table)). Third, metastasis often requires epithelialto‑mesenchymal transition (EMT), which is facili‑ tated by AXL. Canonical EMT-inducing gene products TWIST, SNAIL (also known as SNAI1) and SLUG (also known as SNAI2) are induced by AXL overexpression or through GAS6 stimulation. TWIST and SNAIL can also stimulate AXL expression, reinforcing EMT88,110,121,132. AXL and MERTK have also been linked to motility, migration and invasion through their regulation of focal adhesion kinase 1 (FAK1), RHO, RAC, RALA, RALB, cell division cycle 42 (CDC42) and the GTP exchange factor VAV1 (REFS 48,56,93,102,133,134). These actions are presumably a by-product of physio­logical TAM RTK functions controlling the cytoskeleton. Inhibition of AXL or MERTK using shRNA, a MERTK-blocking antibody, dominant-negative AXL or an AXL TKI decreased cancer cell motility and invasion in culture (TABLE 2; see Supplementary information S1 (table)). Furthermore, GAS6 sequestration using AXL decoy receptors decreased metastases in animal models87,135. MERTK and AXL also enhance expression of matrix metalloproteinase 9 (MMP9), which promotes tissue remodelling and invasion119. TAM RTKs suppress antitumour immunity Tumour-associated macrophages and other tolerant innate immune cells are keys to the immunosuppres‑ sive tumour microenvironment 136,137. The fact that tumours are products of a patient’s self tissue is not in doubt, so it is not surprising that the immune ‘edu‑ cation’ that occurs early in life needs to be overcome to mount an antitumour immune response. Although cancer cells do have mutant gene products that could be antigenic, immune surveillance fails in most progress‑ ing tumours. Cancer immunotherapy has focused on tolerance in the adaptive immune response, and cur‑ rent successful approaches include immune checkpoint

therapeutics that aim to reactivate the T cell. However, the innate immune system has built‑in TAM RTKmediated safeguards to prevent prolonged, injurious inflammation138. For example, as a variation on its normal role, MERTK-expressing tumour-associated macrophages are stimulated by apoptotic material to promote an immunosuppressive, wound-healing M2‑like phenotype31,32. MERTK is highly expressed in a subset of tingible body macrophages in lymph node germinal centres, which promote the maintenance of B cell tolerance 139. When MERTK is eliminated, apop­ totic cells languish, which allows the proliferation of non-tolerant B cells, enhanced CD4 + T helper cells and the release of inflammatory cytokines. If chronic, this would result in autoimmunity (which occurs in Mertk−/− mice). In tumour-associated macrophages, MERTK inhibition might therefore lead to enhanced antitumour immunity. The tumour-associated macrophage and its less wellstudied counterpart, the monocytoid myeloid-derived suppressor cell, are derived from monocyte lineage cells that express little or no MERTK140. However, in tissues, differentiated subsets induce the expression of MERTK. One major MERTK-expressing macrophage subtype, M2c, is differentiated in response to macrophage colonystimulating factor (M‑CSF; also known as CSF1). In the tumour microenvironment, macrophage exposure to glucocorticoids, LXR ligand provided by efferocytosis, and even autocrine GAS6 and IL‑10 further stimulate MERTK expression45,62,141. Continued MERTK acti‑ vation by dying cells suppresses macrophage NF-ĸB signalling and the downstream induction of inflam‑ matory cytokines (for example, IL‑12 and IFNγ), and MERTK-mediated increases in IL‑10 and GAS6 ensue31. When syngeneic breast and melanoma tumour cells were orthotopically implanted into Mertk−/− mice, tumour growth was suppressed in the antitumour innate immune environment that results from MERTK abla‑ tion, in contrast to the immunosuppressive cytokine milieu that is present in wild-type mice31. Moreover, reconstitution of lethally irradiated wild-type mice with bone marrow from Mertk−/− mice resulted in an M1‑polarized macrophage phenotype and slowed the growth of mammary tumours in polyoma middle T antigen (PyMT)-transgenic mice, which indicates that the antitumour response was generated from the haematopoietic compartment. In other immune-competent models of breast cancer and glioblastoma, inhibiting the macrophage colonystimulating factor receptor (MCSFR; also known as CSF1R) pathway with an MCSFR monoclonal antibody or a small-molecule MCSFR kinase inhibitor diminished immunosuppressive phenotypes in tumour-associated macrophages and led to slower tumour growth142,143. Tumours that were implanted into Gas6−/− mice also grew more slowly, possibly as a result of eliminating a ligand for both MERTK on macrophages and TAM RTKs on tumour cells144. Taken together, these data indicate that MERTK signalling in macrophages can suppress the antitumour immune response. AXL and MERTK also suppress other innate immune cell types

778 | DECEMBER 2014 | VOLUME 14

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Postpartum breast cancer Breast cancer diagnosed in women after delivery of offspring. For reasons that have not been elucidated, the prognosis of postpartum breast cancer is worse than that of breast cancer diagnosed during pregnancy, even though both groups of women are premenopausal.

by dampening TLR signals in APCs, terminating APC activity by direct interaction of T cells with upregu‑ lated ligand (for example, PROS1) on their cell surface and reducing the anti-metastatic function of tumourinfiltrating NK cells (discussed below)25,32,33,45,54,55,57,145. TYRO3 is also expressed in innate immune cells, but its role remains to be defined. TAM RTKs and innate immune control of metastasis. In addition to their direct roles in tumour cells in metastasis and motility, as well as their general role in the immuno­ suppressive tumour microenvironment, TAM RTKs may have additional functions that promote metastasis. Metastases of syngeneic implanted breast, colon and melanoma tumour cells were reduced in Mertk−/− mice, partly because, in the absence of Mertk, the metastasispromoting M2 leukocyte cytokine profile was replaced with an M1 inflammatory profile31. Similarly, postpar‑ tum metastases were dramatically increased in a mouse mammary tumour virus (MMTV)–PyMT ortho‑ topic mouse model of breast cancer, which mirrors the poor prognosis of human postpartum breast cancer. In this model, metastasis was diminished by 70–80% in Mertk−/− mice146. AXL, MERTK and TYRO3 are targets of the E3 ubiquitin ligase CBLB32. Genetic deletion of Cblb in NK cells prevented TAM RTK ubiquitylation and degradation, prolonging TAM RTK signalling and thereby inhibiting NK cell activity, which allowed more frequent metastasis. This was shown when NK cell Cblb was genetically deleted in mice, which led to increased melanoma lung metastases. The enhanced metastatic state was reversed (even in mice in which Cblb was selectively knocked out in NK cells) by pharmacologi‑ cal TAM RTK inhibition, which indicates a crucial role for TAM RTKs in NK cell suppression32.

TAM RTKs in therapeutic resistance TAM RTKs can contribute to therapeutic resistance by at least three mechanisms: intrinsic survival signalling in tumour cells, induction of TAM RTKs as an escape mechanism for tumours that have been treated with oncogene-targeted agents and immunosuppression in the tumour microenvironment. TAM RTKs in intrinsic chemoresistance. Consistent with their importance in survival under conditions of cell stress, TAM RTKs promote resistance to cytotoxic chemotherapies in leukaemia cells and solid tumour cells ( TABLE  2 ; see Supplementary information S1 (table)). Transgenic lymphocytes ectopically express‑ ing MERTK were more resistant to dexamethasone than wild-type lymphocytes 10, and stimulation of B‑ALL cells with GAS6 increased resistance to cyta‑ rabine97. AXL is induced in AML cells that have been treated with cytotoxic chemotherapies, and it mediates increased chemoresistance88. Chemotherapy-resistant CML cell lines have upregulated levels of AXL, and shRNA-mediated knockdown of AXL increases chemosensitivity in CML cells and xenograft mod‑ els 147. Similarly, shRNA-mediated MERTK knock‑ down sensitizes B‑ALL and T‑ALL cells to a range of

chemotherapies74,75. In solid tumours, overexpression of AXL or MERTK promotes chemoresistance, and shRNA-mediated inhibition sensitizes cells to treat‑ ment with cytotoxic chemotherapies 96,113,116,118,147–150. These data support roles for MERTK and AXL in intrinsic tumour cell chemoresistance. Recent studies also implicate TAM RTKs in tumour cell autophagy, which can promote survival in conditions of nutrient deprivation and following chemotherapy 116. AXL in acquired TKI resistance. In contrast to intrin‑ sic chemoresistance, examples of acquired resistance are currently limited to AXL. AXL is upregulated in imatinib-resistant CML and gastrointestinal stromal tumour (GIST) cell lines and tumour samples151–153, and siRNA-mediated knockdown of AXL restored imatinib sensitivity to resistant cell lines152. Similarly, AXL is induced in lapatinib-resistant HER2 (also known as ERBB2)-positive breast cancer cell lines, and AXL inhibition restored lapatinib sensitivity 154. AXL has been associated with acquired resistance to epidermal growth factor receptor (EGFR) TKIs and therapeutic antibodies in triple-negative breast cancer 155 and head and neck cancer 66 cell lines, as well as with resistance to inhibitors targeting other kinases, including fibroblast growth factor receptor (FGFR)156, anaplastic lymphoma kinase (ALK)157 and insulin-like growth factor 1 recep‑ tor (IGF1R)158. Perhaps most germane for human clini‑ cal trials, AXL is upregulated in NSCLC cell lines and xenografts that are resistant to EGFR TKIs and antibody drugs (cetuximab and erlotinib)159,160, and it is induced in 20% of matched tumour samples taken from patients with NSCLC after development of resistance to erlotinib (an EGFR TKI)160.

TAM RTKs as dual therapeutic targets Although TAM RTKs are not oncogenic drivers, their intrinsic and acquired therapeutic resistance properties and immunosuppressive actions suggest that they could be effective cancer targets for inhibition using smallmolecule kinase inhibitors, specific monoclonal anti‑ bodies or extracellular domains that function as ‘ligand traps’. The best approach could depend on the context; for example, small molecules that inhibit both MERTK and AXL may be most useful for the treatment of lung cancer, in which both are expressed and can have inde‑ pendent roles in tumour progression96. By contrast, the use of a selective agent may minimize therapy-associated side effects; for example, a MERTK-selective inhibitor or antibody may be best in paediatric ALL in which MERTK is often expressed but AXL is not 74,75. MERTKselective agents would also narrowly target the immuno­ suppressive macrophage phenotype. Alternatively, it may be necessary to inhibit several TAM RTKs to affect a range of innate immune cells and to effectively stimulate antitumour immunity. Selective biological entities are also attractive because they might have fewer off-target effects; for example, the AXL extracellular domain ligand trap that was engineered to have very high affinity for GAS6 (REF. 135) would selectively bind to GAS6 but not to PROS1. The future clinical development of inhibitors

NATURE REVIEWS | CANCER

VOLUME 14 | DECEMBER 2014 | 779 © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Table 4 | TAM family small-molecule kinase inhibitors in development* Compound

Indications

Target profile Primary targets AXL activity

MERTK activity TYRO3 activity Other targets

Cabozantinib

Thyroid, prostate, kidney, ovarian, lung and breast cancers

MET and VEGFR2

IC50 = 7 nM; IC50 = 42 μM‡

–

–

RET, KIT, FLT3 and TIE2

Approved

Bosutinib (also known as SKI‑606 and PF-5208763)

Breast carcinoma, glioblastoma and Ph+ CML

SRC and ABL

Kd = 52 nM; IC50 = 0.6 nM; IC50 = 340 nM‡

Kd = 110 nM

Kd = 61 nM

AXL

Approved

Crizotinib (also known as PF‑2341066)

NSCLC

MET

Kd = 7.8 nM; IC50 = 300 nM

Kd = 3.6 nM

Kd = 800 nM

ALK, RON and AXL

Approved

Vandetinib

Thyroid cancer and NSCLC

VEGFR2, VEGFR3 and EGFR

Kd = 250 nM

Kd = 1400 nM

Kd = 93 nM

RET

Approved

Sunitinib

RCC and GIST

PDGF and VEGF

Kd = 9 nM; IC50 = 259 nM

Kd = 26 nM IC50 = 12 nM

Kd = 49 nM IC50 = 251 nM

RET and FLT3

Approved

Lestaurtinib (also AML and ALL known as CEP‑701)

FLT3

Kd = 35 nM

Kd = 32 nM

Kd = 650 nM

JAK2, TRKA, TRKB and TRKC

Phase III

Neratinib

Breast cancer

HER2

Kd = 190 nM

Kd = 400 nM

Kd > 3000 nM

–

Phase III

AT9283

Multiple myeloma and leukaemia

AURKA, AURKB and JAK

–

IC50 ≤10 nM

–

–

Phase II

R406

Rheumatoid arthritis and lymphoma

SYK

Kd = 82 nM

Kd = 170 nM

Kd = 1900 nM

–

Phase II

Foretinib (also known as GSK1363089 and XL880)

Triple-negative metastatic breast cancer, HER2+ breast cancer and NSCLC

MET and VEGFR2

Kd = 0.1 nM; IC50 = 11 nM

Kd = 0.3 nM

Kd = 2 nM

RON, PDGFRβ, KIT, FLT3, TIE2 and AXL

Phase Ib/II

MK‑2461

Advanced solid tumours

MET

–

IC50 = 24 nM

–

RON and VEGFR

Phase I/II

BMS‑777607 (also known as ASLAN002)

Advanced solid tumours

MET

IC50 = 1.1 nM

IC50 = 14 nM

IC50 = 4.3 nM

RON, AURKB, FLT3 and AXL

Phase I

LY2801653

Advanced cancers

MET

IC50 = 11 nM; IC50 = 2 nM‡

IC50 = 0.8 nM

IC50 = 1200 nM

RON, FLT3 and AXL

Phase I

SU‑14813

Advanced solid tumours

FLT3 and VEGFR

Kd = 84 nM

Kd = 66 nM

Kd = 2400 nM

PDGFR and KIT

Phase I

S49076

Advanced solid tumours

MET and FGFR

IC50 = 7 nM

IC50 = 2 nM

–

–

Phase I

BMS‑796302

Not specified

MET

Not reported§

Not reported§

Not reported§

FLT3, RON, VEGFR2 and AXL

Phase I

BGB324 (also known as R428)

Breast cancer

AXL and RET

IC50 = 14 nM; IC50 = 14 nM‡

IC50 = 220 nM IC50 = 700 nM‡

IC50 = 200 nM IC50 > 1400 nM‡

VEGFR, FLT3, ABL and TIE2

Phase I

Amuvatinib (also known as MP‑470)

Neuroendocrine, KIT and FLT3 lung and endometrial cancers

IC50 = 1 uM‡

–

–

KIT, MET, RET, PDGFR, RAD51 and AXL

Development discontinued

JNJ‑28312141

Solid tumours and AML

MCSFR and FLT3 Kd = 5.3 nM; IC50 = 12 nM

Kd = 86 nM

Kd >3000 nM

KIT, TRKA and LCK

Preclinical

GSK2606414

Solid tumours

PERK

IC50 = 2700 nM

IC50 = 470 nM

–

–

Preclinical

Ki‑20227

Breast cancer MCSFR and autoimmune disorders

Kd = 140 nM

Kd = 460 nM

Kd = 2000 nM

PDGFR and KIT

Preclinical

780 | DECEMBER 2014 | VOLUME 14

Development phase

www.nature.com/reviews/cancer © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Table 4 (cont.) | TAM family small-molecule kinase inhibitors in development* Compound

Indications

6g

Not specified

Target profile

Development phase

Primary targets AXL activity

MERTK activity TYRO3 activity Other targets

AXL, MERTK and MET

Kd = 39 nM

Kd = 42 nM

Kd = 200 nM

TYRO3

Preclinical

Diaminopyrimidine Pancreatic cancer

AXL

IC50 = 27 nM IC50 = 220 nM‡

IC50