AXL receptor tyrosine kinase as a promising anti-cancer approach: functions, molecular mechanisms and clinical applications

Cancer is not a single-cell disease but rather the result of complex interactions of tumour cells with surrounding matrix and immune cells. In recent years, molecular targeted therapy for cancer has been a research hotspot. Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor (PDGFR) have been evaluated in clinical trials with promising results, which prompted the search for additional diagnostic and prognostic biomarkers [1‐3]. AXL has emerged as a novel biomarker due to its role in biological processes and tumourigenesis [4]. AXL is a member of the TAM family that includes TYRO3, AXL and MER. Growth arrest-specific protein 6 (GAS6) serves as a ligand for AXL with high binding affinity. GAS6/AXL signalling functions as an important pathway driving cancer cell survival, proliferation, migration and invasion, which makes AXL a potential target in cancer treatment [5, 6].

The gene AXL, located at chromosome 19q13.2, was first identified in patients with chronic myeloid leukaemia (CML) [7]. The word AXL, coming from the Greek word “anexelekto”, means uncontrolled. The protein encoded by the gene AXL, called AXL (UFO, ARK, Tyro7, or JTK11), is a member of the TAM family of receptor tyrosine kinases (RTKs). AXL is composed of an extracellular, transmembrane and intracellular domain [8]. The extracellular structure consists of two immunoglobulin (Ig)-like repeats and two fibronectin type III (Fro III)-like repeats that resemble neural cell adhesion molecules (NCAMs) [9]. The Ig motifs are involved in the binding of AXL with its ligand Gas6 under regulation by Fro III [10]. The intracellular domain is critical for auto-phosphorylation and subsequent kinase activity [11]. The TAM family-specific KW(I/L)A(I/L)ES sequence resides within this intracellular domain, which shares homology with AXL-related RTKs, such as RET [12], and plays an important role in tyrosine kinase activity (Fig. 1a). The TAM family is widely expressed in normal cells and tissues, such as monocytes, platelets, endothelial cells, hippocampus, cerebellum, heart, and liver [13‐20], wherein it regulates cell survival, the non-inflammatory clearance of apoptotic cells by phagocytic cells, natural killer cell differentiation, platelet aggregation, etc. [10, 21‐23].

The synthesis of AXL is regulated at many levels. Five transcription factors act on the AXL promotor, including activator protein 1 (AP1) [24], Sp1/Sp3, YAP/TAZ/TEAD, hypoxia inducible factor 1α (HIF1α) [25] and myeloid zinc finger 1 protein (MZF-1) [26]. Activation of TLR signalling upregulates AXL mRNA in dendritic cells and macrophages [27, 28]. The transcription process is also feedback controlled by other RTKs. In non-small-cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), activated EGFR pathways and downstream MEK/ERK signalling promote AXL mRNA expression through the JUN transcription factor [29]. In urothelial carcinoma, AXL mRNA expression is induced after activation of MET and downstream MEK/ERK signalling [30]. AXL mRNA expression is inhibited by two microRNAs (miRNAs), miR-34a and miR-199a/b [31, 32]. In addition, AXL mRNA expression is subject to epigenetic modification, including histone acetylation and histone/DNA methylation [33, 34]. Although full-length AXL contains 894 amino acids and encodes a predicted protein of 98 kDa, the actual observed molecular weight ranges from 100 to 140 kDa due to post-transcriptional regulation of the activated AXL receptor by glycosylation, phosphorylation and multiple sites of monoubiquitination [35‐39].

In adults, AXL expression is relatively low [40, 41], but aberrant expression of Gas6/AXL has been shown in a number of human malignancies, including breast cancer, chronic lymphocytic leukaemia (CLL), NSCLC, pancreatic cancer, glioblastoma, melanoma, renal cell carcinoma (RCC), prostate cancer, and oesophageal cancer [4, 23, 42‐55], and this altered expression is associated with disease progression and shortened overall survival (OS). In various in vivo breast cancer models, AXL expression was found to be higher in metastatic nodules than in primary tumours, and downregulating AXL with miRNAs inhibited downstream AKT phosphorylation and diminished the motility, metastasis and invasion of tumour cells [47]. Given the role of AXL in cancer development, progression and drug resistance, AXL holds great promise as a prognostic biomarker and therapeutic target.

Common ligands for the TAM family include Gas6, protein S, Tubby, Tubby-like protein 1 (TULP-1) and Galectin-3 [56, 57]. Studies on Gas6 and protein S are abundant, as these two ligands were the first to be discovered. Gas6 can bind to all three members of the TAM family, whereas protein S binds only to MER and TYRO3. The affinity of Gas6 for AXL is 3–10 times higher than that for the other two members in the family [8, 58]. Gas6 is encoded by growth arrest-specific gene 6 (Gas6) and belongs to the vitamin K-dependent protein family [59]. Upon high-affinity binding to its ligand Gas6, the AXL receptor undergoes homo-dimerization and subsequent trans-autophosphorylation within the intracellular kinase domain, thus recruiting adaptor molecules and effector proteins containing Src homology 2 (SH2) or other phosphotyrosine-binding domains (PTBs) and activating downstream signalling pathways [60, 61]. Six phosphorylation sites have been found in AXL, Tyr698, Tyr702, Tyr703, Tyr779, Tyr821 and Tyr866. Three N-terminal tyrosine residues, 779, 821 and 866, are related to auto-phosphorylation and AXL activation, while the other three C-terminal sites are rather conserved among the TAM receptors and indispensable for complete functions of the kinase [10]. A study on a EGFR-AXL chimeric receptor has revealed that the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signalling pathway is induced by binding of Tyr779 and Tyr821 to p85, and the MEK/ERK cascade is activated by binding of phosphorylated Tyr821 to GRB2 [62]. In addition, phosphorylated Tyr821 and Tyr866 can bind to SRC, LYK and PLCα and activate protein kinase C and STAT [4, 10, 15, 61]. Therefore, the PI3K/AKT/mTOR, JAK/STAT, NF-κB, and RAS/RAF/MEK/ERK signalling pathways function as important downstream pathways for the Gas6/AXL axis and play major roles in tumour cell survival, anti-apoptosis signalling, mitogenesis, migration, invasion, drug resistance, angiogenesis and the tumour-host relationship [63‐66] (Fig. 1b).

In addition to Gas6-dependent AXL activation, AXL might also be activated via Gas6-independent mechanisms [62]. Overexpression of AXL leads to aggregation of AXL extracellular domains or ligand-independent homo-dimerization, thereby leading to receptor activation [13, 53]. Moreover, the broad homology AXL shared with other TAM family members, as well as with other non-TAM RTKs such as fibroblast growth factor receptor (FGFR), EGFR, PDGFR and MET [4], encourages the formation of heterodimers and the activation of AXL-dependent signalling [67, 68] (Fig. 1c). In multiple tumour tissues, aberrant AXL expression is related to the extent of malignancy, metastasis and a poor prognosis.

Because of the pleiotropic role of AXL in tumour pathophysiology and drug resistance, AXL represents a promising therapeutic target in the management of cancer. AXL inhibitors have been shown to potentiate tumour cell apoptosis and suppress migration and invasion [43]. Targeting AXL enhances the therapeutic efficacy of chemotherapy and other small-molecule inhibitors, such as VEGF, EGFR, PI3K, PARP, and HER2 inhibitors [54, 123, 139]. Different therapeutic agents have been developed over the decades, including small molecule inhibitors, anti-AXL mAbs, nucleotide aptamers, soluble receptors, and several natural compounds (Fig. 2).

Although AXL-targeted therapies appear promising in treating malignancies, many questions remain unanswered. AXL small molecule inhibitors have pronounced therapeutic effects; nonetheless, their inherent limitations, the off-target effects, might give rise to the inhibition of additional kinases and subsequent unexpected toxicities, limiting their clinical use. A variety of side effects have been reported for molecular targeted therapies, especially for multi-targeted AXL TKIs [148, 151‐153, 210, 220, 235‐237].

In addition, many patients do not respond to anti-AXL treatment or acquire resistance to these agents. Therefore, it is beneficial to select the optimal patients who could draw clinical benefits from AXL inhibitors and to avoid potential toxicities. Reliable integrated biomarker design is warranted to guide treatment strategies in predicting response and overcoming therapeutic resistance.

AXL inhibitors, either as single agents or in combination with conventional chemotherapy or other inhibitors such as immune checkpoint inhibitors, angiogenesis inhibitors and other TKIs, are likely to improve the survival of many patients. However, rational combination approaches, the sequence of administration, and the right time of incorporation of anti-AXL agents into treatment regimens should be taken into consideration before clinical use. Dissection of AXL molecular signalling networks and further investigations into the relationships between AXL and other kinases need to be performed to improve antitumour therapies and personalized cancer treatment.