Therapeutic Targeting of the Gas6/Axl Signaling Pathway in Cancer

Many signaling pathways are dysregulated in cancer cells and the host tumor microenvironment. Aberrant receptor tyrosine kinase (RTK) pathways promote cancer development, progression, and metastasis. Hence, numerous therapeutic interventions targeting RTKs have been actively pursued. Axl is an RTK that belongs to the Tyro3, Axl, MerTK (TAM) subfamily. Axl binds to a high affinity ligand growth arrest specific 6 (Gas6) that belongs to the vitamin K-dependent family of proteins. The Gas6/Axl signaling pathway has been implicated to promote progression, metastasis, immune evasion, and therapeutic resistance in many cancer types. Therapeutic agents targeting Gas6 and Axl have been developed, and promising results have been observed in both preclinical and clinical settings when such agents are used alone or in combination therapy. This review examines the current state of therapeutics targeting the Gas6/Axl pathway in cancer and discusses Gas6- and Axl-targeting agents that have been evaluated preclinically and clinically.


Introduction
Receptor tyrosine kinases (RTKs) are cell surface receptors that mediate a number of physiological responses and homeostasis. However, gene amplification, overexpression, and activating mutations of RTKs are often associated with cancer development, progression, and metastasis and have served as pharmacological targets in cancer treatment. Axl belongs to the TAM (Tyro3, Axl, MerTK) subfamily of the RTKs. Physiologically, the Gas6/Axl pathway plays an important role in platelet aggregation and vessel integrity [1][2][3]. Axl knockout in germ cells does not result in embryonic lethality [4]. The growth arrest specific 6 (Gas6) protein belongs to the vitamin K-dependent family of proteins and is a high affinity ligand for Axl. Overexpression and activation of Axl are widely observed in various cancer types and have been implicated in multiple steps of cancer pathogenesis. In addition, high Axl expression and activation are associated with poor prognosis, outcome, and resistance to therapy in cancer patients [5][6][7][8][9][10][11]. As such, the Gas6/Axl pathway has gained attention as a promising therapeutic target for drug development in multiple tumor types. In this review, we present an overview of the Gas6/Axl signaling pathway and highlight the Gas6 and Axl inhibitors that are being investigated preclinically and clinically.

Gas6/Axl Pathway in Cancer
Axl was first isolated from chronic myelogenous leukemia cells in 1988 [12]. Structurally, Axl contains two immunoglobulin-like (IgL) domains, two fibronectin III (FNIII) domains, a transmembrane domain, and an intracellular kinase domain [13]. The Axl pathway can be activated by its ligand Gas6, which belongs to the vitamin K-dependent family of proteins. Gas6 contains the γ-carboxyglutamic acid (Gla) domain, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains [14,15]. The N-terminus Gla domain mediates binding to phosphatidylserine expressed on cell family of proteins. Gas6 contains the γ-carboxyglutamic acid (Gla) domain, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains [14,15]. The N-terminus Gla domain mediates binding to phosphatidylserine expressed on cell membranes, apoptotic cells, and debris [16,17]. Gas6 and Axl interact via the first LG-like domain of Gas6 and the two IgL domains of the Axl ectodomain [18]. Gas6 and Axl bind at a one-to-one receptor-to-ligand ratio and then dimerize with another Gas6-Axl complex to initiate a downstream signaling cascade.
The role of Gas6 and Axl in cancer has been reviewed previously [19][20][21][22]. Briefly, Axl is overexpressed in many cancer types and is associated with therapeutic resistance, poor clinical prognosis, and worse outcome [23][24][25][26]. Axl also mediates key components of the metastatic cascade, including, but not limited to, epithelial-to-mesenchymal transition, migration and invasion, proliferation, survival, stemness, angiogenesis, and immune evasion [19,20,22,27] (Figure 1). A study by Zdzalik-Bielecka and colleagues demonstrated that the Gas6/Axl pathway mediates actin cytoskeleton remodeling for cell migration and invasion via formations of peripheral ruffles and circular dorsal ruffles [28]. Such Gas6induced Axl activation contributes to focal adhesion turnover, cell spreading, and elongation through the activation of PI3K and RAC1 [28,29]. Another study similarly demonstrated that Axl mediates cell invasion through the regulation of lysosome peripheral distribution [30]. Figure 1. The Gas6/Axl pathway mediates multiple steps of the metastatic cascade. Upon Axl binding to its ligand growth arrest specific 6 (Gas6) protein, Axl dimerizes and autophosphorylates its tyrosine residues in the kinase domain. Axl activation regulates downstream signaling, including the JAK/STAT pathway, Ras/MEK/Erk1/2 pathway, and PI3K/Akt pathway. In turn, the Gas6/Axl pathway upregulates pro-tumorigenic functions, such as immune evasion, survival, proliferation, drug resistance, angiogenesis, epithelial-to-mesenchymal transition (EMT), migration, and invasion. Gas6 and Axl are also expressed by stromal cells, including endothelial cells, pericytes, and subsets of immune cells, to promote tumor progression and metastasis.
In the tumor immune microenvironment, Axl signaling in tumor cells polarizes tumor-associated macrophages toward the M2 phenotype via the Axl/Pi3K/Akt/NF-kB pathway [31]. Axl inhibition elicits a favorable immune response, characterized by a decreased infiltration of CD206 + macrophages and neutrophils and an increased infiltration of CD45 + immune cells, CD8 + T cells, CD4 + T cells, and I-A/I-E + macrophages [32]. The Gas6/Axl pathway mediates multiple steps of the metastatic cascade. Upon Axl binding to its ligand growth arrest specific 6 (Gas6) protein, Axl dimerizes and autophosphorylates its tyrosine residues in the kinase domain. Axl activation regulates downstream signaling, including the JAK/STAT pathway, Ras/MEK/Erk1/2 pathway, and PI3K/Akt pathway. In turn, the Gas6/Axl pathway upregulates pro-tumorigenic functions, such as immune evasion, survival, proliferation, drug resistance, angiogenesis, epithelial-to-mesenchymal transition (EMT), migration, and invasion. Gas6 and Axl are also expressed by stromal cells, including endothelial cells, pericytes, and subsets of immune cells, to promote tumor progression and metastasis.
In the tumor immune microenvironment, Axl signaling in tumor cells polarizes tumorassociated macrophages toward the M2 phenotype via the Axl/Pi3K/Akt/NF-kB pathway [31]. Axl inhibition elicits a favorable immune response, characterized by a decreased infiltration of CD206 + macrophages and neutrophils and an increased infiltration of CD45 + immune cells, CD8 + T cells, CD4 + T cells, and I-A/I-E + macrophages [32].
Additionally, it is becoming increasingly clear that the Gas6/Axl signaling axis also impacts non-neoplastic cell populations, which may be of particular relevance when viewed in the context of the tumor microenvironment. For example, pericyte FAK expression is a neg-MYD1 is an Axl decoy receptor with a high affinity for Gas6 (Table 1) [48]. MYD1 contains the major site of Axl Ig1 with four mutations to improve binding to Gas6 [48]. MYD1 inhibited Gas6-mediated Axl-, Akt-, and Erk-phosphorylation and decreased the number of peritoneal metastases in an ovarian cancer model [48].
MYD1-72 is a second-generation Axl decoy receptor that has a higher binding affinity than that of MYD1 (K D : 420 fM vs. 93 fM, respectively). In preclinical studies, MYD1-72 decreased Axl phosphorylation and impaired tumor growth and metastatic burden in vivo. A combination of MYD1-72 with gemcitabine significantly improved the survival of mice with pancreatic cancer to 57 days compared to 17 days in the control and MYD1-72 alone groups and 35 days in the gemcitabine group. A clinical evaluation of this compound has not been reported.
AVB-500 is undergoing Phase I/II clinical trials for patients with platinum-resistant or recurrent ovarian, fallopian tube, or peritoneal cancers as a combination therapy (Clinical Trial Identification #s: NCT03639246, NCT04019288). Three other studies are currently recruiting patients for ovarian cancer, renal cell carcinoma, and pancreatic adenocarcinoma to evaluate its efficacy as a combination therapy with paclitaxel, cabozantinib, and nab-paclitaxel/gemcitabine, respectively (Clinical Trial Identification #s: NCT04729608, NCT04300140, NCT04983407).

Type I Kinase Inhibitors
Small molecular kinase inhibitors can be classified based on the structure of the protein-inhibitor complex [52]. The N-terminal Asp-Phe-Gly (DFG) motif of the activation loop is conserved and regulates kinase activity. Type I protein kinase inhibitors bind to the active protein kinase conformation or DFG-in conformation.
Bemcentinib is a highly specific and selective Axl inhibitor with an IC 50 of 14 nM in cellular assays (Table 2) [53,54]. This agent has been widely studied in the laboratory in a variety of cancer models, including breast, prostate, lung, pancreatic, and ovarian cancer, and has been shown to decrease tumor cell migration, invasion, and colony formation in vitro and impair primary tumor growth, immune cell infiltration, and metastasis in vivo [55][56][57][58][59][60][61][62]. Axl inhibition alters the expression patterns of EMT markers, upregulating epithelial markers, such as E-cadherin, and downregulating mesenchymal markers, such as N-cadherin, ZEB1, Snail, Slug, Twist, and MMP9 [63][64][65]. Furthermore, several studies have demonstrated that Axl inhibition reverses the therapeutic resistance of certain chemotherapies. Axl has also been associated with immune evasion, and the systematic treatment of tumor-bearing mice with bemcentinib has led to a reduction in tumor-infiltrating host cells, most notably cells of the myeloid lineage [66].  Bemcentinib entered clinical trials as the first Axl-specific inhibitor and is undergoing Phase I/II clinical trials for melanoma, non-small-cell lung cancer (NSCLC), mesothelioma, acute myeloid leukemia, glioblastoma, and pancreatic adenocarcinoma (Clinical Trial Identification Numbers: NCT02872259; NCT02922777; NCT03184571; NCT03654833; NCT03824080; NCT03965494; NCT03649321). A Phase II clinical trial of bemcentinib in combination with pembrolizumab for patients with triple-negative breast cancer has been completed, but the results are not yet available (Clinical Trial Identification #: NCT03184558).

Amuvatinib (MP-470)
Amuvatinib is a c-Kit/Axl kinase inhibitor (Table 2), with higher selectivity against c-Kit than against Axl (IC 50 < 1 µM in cellular assays). Amuvatinib has been studied in models of gastrointestinal stromal tumors, lung cancer, and prostate cancer in the context of overcoming therapeutic resistance [60,67,68]. In the gastrointestinal stromal tumor (GIST) model, Mahadevan and colleagues showed that an imatinib-resistant GIST cell line overexpressed Axl compared to its parental cell line [68]. Treatment with amuvatinib showed synergistic, cytotoxic effects with docetaxel [68]. In an erlotinib-resistant lung cancer model, Axl inhibition by amuvatinib restored sensitivity to erlotinib [67].
Clinically, amuvatinib is undergoing Phase I/II trials for small-cell lung cancer (SCLC) and other unresectable or metastatic solid tumors (Clinical Trial Identification Numbers: NCT01357395; NCT00894894; NCT00881166). In a Phase I, dose-escalation study of patients with advanced solid tumors, amuvatinib was well tolerated up to 1500 mg/day with no report of dose-limiting toxicities; indeed, the study did not reach the maximum tolerated dose [69]. In a Phase Ib study of amuvatinib in combination with standard of care therapies for adults with advanced solid tumors, 12% demonstrated partial response to amuvatinib combined with paclitaxel/carboplatin or carboplatin/etoposide in neuroendocrine, NSCLC, and SCLC tumors [70].
Dubermatinib has been studied as monotherapy and combination therapy. Preclinically, combination strategies with all-trans retinoic acid (ATRA), cisplatin, and VP16 demonstrated synergistic, cytotoxic effects [73]. Aveic and colleagues showed that a dubermatinib/ATRA combination in neuroblastoma cell lines was particularly effective against CD133 + cells, a marker associated with chemo-and radio-resistance [79][80][81]. Phase I/II studies are ongoing for patients with advanced metastatic or progressive solid tumors and AML to determine the dose, safety, pharmacokinetics, and pharmacodynamics of dubermatinib (Clinical Trial Identification Numbers: NCT02729298, NCT04518345, NCT03013998). A Phase I/II clinical trial investigating dubermatinib monotherapy and combination therapy with ibrutinib for patients with chronic lymphocytic leukemia was terminated (Clinical Trial Identification #: NCT03572634).
Crizotinib has been approved by the FDA to treat patients with ALK-or ROS1-positive NSCLC and pediatric patients with relapsed or refractory ALK-positive systemic anaplastic large-cell lymphoma (ALCL) [86,87]. There are 151 registered studies associated with crizotinib in the ClinicalTrials.gov database, and 44.4% (67 studies) are currently recruiting, enrolling by invitation, or active/not recruiting for monotherapy and combination therapy strategies. Acquisition of crizotinib resistance is common [88,89], typically developing within a few years for anaplastic lymphoma kinase-rearranged NSCLC [88]. Crizotinibresistant NSCLC cells showed overexpression of Axl, epithelial-to-mesenchymal transition, and the acquisition of cancer stem cell-like properties [88,90,91].
Bosutinib was approved by the FDA to treat patients with chronic, accelerated, or blast-phase Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia [97]. Bosutinib monotherapy and combination therapy with chemotherapy and other small molecule inhibitors are also being investigated in patients with breast cancer, glioblastoma, and other advanced solid cancers (Clinical Trial identification #s: NCT00319254, NCT01331291, NCT01001936).

S49076
S49076 is a potent inhibitor of c-Met, as well as Axl, MerTK, and FGFR (Table 2). S49076 has been studied preclinically in gastric, lung, and liver cancer models [98,99] and has been shown to decrease the phosphorylation of Axl in a dose-dependent manner. S49076 impairs tumor cell viability, migration, and colony formation in vitro and tumor growth in vivo [98]. Clémenson and colleagues demonstrated that the mechanism of action of S49076 is dependent upon c-Met signaling dependency [100][101][102], where S49076 targets Aurora B kinase in c-Met-independent cells [99].
In a Phase I open-label study in patients with advanced solid tumors, a recommended once-daily dose of 600 mg was defined [103]. While 81.4% of patients had drug-related adverse events, the majority of the adverse events were grade I-II [103]. In a Phase I/II study in combination with bevacizumab in patients with recurrent glioblastoma, the combination did not improve patient survival outcomes [104]. The recommended Phase II dose was confirmed at 600 mg once daily, and no additional dose-limiting toxicity was observed [105,106].

Sunitinib (SU111248)
Sunitinib is an oral, multitargeted, small molecule receptor tyrosine kinase inhibitor that targets VEGFR2, PDGFRβ, c-Kit, and Axl (Table 2). Sunitinib is approved by the FDA to treat patients with gastrointestinal stromal cancer, advanced RCC, and progressive, well-differentiated pancreatic neuroendocrine tumors [107,108]. However, Axl overexpression is associated with sunitinib resistance in renal cell carcinoma cells [109,110]. In patients with renal cell carcinoma, elevated Axl expression is associated with shorter overall survival [109]. Genetic and pharmacologic Axl inhibition in sunitinib-resistant cell lines demonstrate decreased tumor cell migration, invasion, EMT, and angiogenesis [109].

SNS-314
SNS-314 is an aurora kinase inhibitor that also targets Axl, with an IC 50 of 84 nM in a cellular assay (Table 2). SNS-314 impairs colony formation, histone H3 phosphorylation, and tumor growth in vivo [111,112]. In 2007, SNS-314 entered a Phase I study for patients with advanced solid tumors to evaluate safety and tolerability (Clinical Trial Identification Number: NCT00519662). The maximum tolerated dose was not established, and no responses were observed [113].

Other Type I Axl Kinase Inhibitors Undergoing Preclinical Evaluation
NA80x1 is a 3-quinolinecarbonitrile that inhibits Axl phosphorylation, tumor cell migration, and invasion [94]. The IC 50 of NA80x1 against Axl is 4.11 µM in a cellular assay and demonstrates inhibitory activity against Src kinase [94,114]. SGI-7079 is a selective Axl inhibitor that reduces Gas6-induced Axl phosphorylation and impairs the growth of mesenchymal NSCLC xenograft tumors [115]. In addition, Axl inhibitor DP-3975 [116,117], a TAM kinase inhibitor UNC569 [118], and MerTK/Flt3 dual inhibitor UNC2025 [119] are also type I Axl inhibitors with promising preclinical results.

Type II Kinase Inhibitors
Unlike type I protein kinase inhibitors, type II protein kinase inhibitors prefer to bind the inactive, DFG-out conformation.
Cabozantinib has been approved by the FDA for patients with advanced renal cell carcinoma and sorafenib-resistant hepatocellular carcinoma. In January 2021, combination therapy of cabozantinib with nivolumab was approved as first-line treatment for patients with advanced renal cell carcinoma. The efficacy of the combination therapy versus sunitinib alone was evaluated in CHECKMATE-9ER, a randomized, open-label trial in patients with previously untreated advanced renal cell carcinoma [122] (Clinical Trial Identification: NCT03141177). The trial demonstrated significant improvement in progression-free survival, overall survival, and confirmed overall response rate for patients in the combination therapy arm compared with those who received sunitinib monotherapy [122].
Clinically, foretinib has been studied as a monotherapy and a combination therapy. In a Phase I dose-escalation study in patients with advanced solid tumors, the recommended maximum tolerated dose of foretinib was identified [127] (Clinical Trial Identification #: NCT00742131). Stable disease and partial responses were identified in 22 and 3 cases, respectively [127]. In a Phase I study of foretinib in combination with lapatinib in patients with HER2-positive metastatic breast cancer, no responses or improvement in progressionfree survival were observed [123].

Sitravatinib (MGCD516)
Sitravatinib is a multitargeted, small molecule receptor tyrosine kinase inhibitor that inhibits c-Kit, PDGFRα/β, c-Met, and Axl (Table 3). Sitravatinib has been shown to decrease Axl phosphorylation, colony formation, angiogenesis, and proliferation in sunitinib-or axitinib-resistant cells, as well as impairing tumor growth inhibition in vivo [128][129][130]. Du and colleagues demonstrated that sitravatinib alters the immune landscape of tumors by reducing the number of tumor-associated immunosuppressive myeloid cells and increasing the number of CD4 + T cells and proliferating CD8 + T cells [130]. Combination treatment of sitravatinib and PD-1 blockade demonstrated complete remission in 2 out of 14 mice with E0771 tumors [130].
In the clinic, sitravatinib is being investigated for use as monotherapy and combination therapy. A Phase I study is currently ongoing for patients with advanced cancer to evaluate its safety, pharmacokinetic, metabolism, pharmacodynamic, and clinical activity profiles (Clinical Trial Identification #: NCT02219711). In addition, several Phase I/II studies are underway to evaluate sitravatinib in combination with immune checkpoint inhibitors (Clinical Trial Identification #s: NCT03015740, NCT04734262, NCT03666143, NCT02954991).

Glesatinib (MGCD265)
Glesatinib is an oral, multitargeted tyrosine kinase inhibitor that targets c-Met, VEGFR, Ron, Tie-2, and Axl (Table 3). Glesatinib impairs tumor angiogenesis and upregulates the expression of PD-L1 [130]. Du and colleagues demonstrated that combination therapy of glesatinib with anti-PD1 therapy delayed tumor growth [130]. As such, a Phase II study of glesatinib in combination with nivolumab in patients with non-small-cell lung cancer is ongoing (Clinical Trial Identification #: NCT02954991).

Rebastinib (DCC-2036)
Rebastinib is a Bcr-Abl inhibitor that also impairs Src family proteins, c-Met, Tie-2, and Axl (Table 3). In a preclinical study utilizing breast cancer models, rebastinib impaired Axl phosphorylation, colony formation, cell cycle distribution, migration, invasion, and tumor growth and pulmonary metastasis in vivo [131]. Phase I/II studies are ongoing for patients with advanced metastatic or progressive solid tumors, breast cancer, and chronic myeloid leukemia to evaluate its safety, pharmacokinetics, and pharmacodynamics as monotherapy (Clinical Trial Identification Number: NCT00827138) or combination therapy with paclitaxel (NCT03601897, NCT02824575), eribulin (NCT02824575), and carboplatin (NCT03717415).

Merestinib (LY2801653)
Merestinib is an oral, multitarget kinase inhibitor that impairs c-Met, Axl, Ron, and Flt3 (Table 3) [132]. Merestinib impairs cell proliferation, angiogenesis, and tumor growth in vivo [132,133]. Merestinib is being investigated clinically in multiple indications, including advanced or metastatic solid tumor, biliary tract cancer, non-small-cell lung cancer, and chronic myeloid leukemia, as a monotherapy or combination therapy (Clinical Trial Identification #s: NCT02711553, NCT02920996, NCT03125239). A Phase I study of merestinib with or without cetuximab, cisplatin, or gemcitabine identified the recommended dose at 120 mg once daily [134] (Clinical Trial Identification #: NCT01285037). One complete response and three partial responses were observed in patients with cholangiosarcoma [134].

RXDX-106
RXDX-106 is an oral, small molecule inhibitor targeting the TAM receptors and c-Met (Table 3). RXDX-106 has been characterized in colorectal and breast cancer models and has been shown to impair Axl, Tyro3, MerTK, Akt, and Erk phosphorylation; tumor growth in vivo; and infiltration of leukocytes, macrophages, NK cells, and CD8 + T cells [141]. While an early Phase I study to evaluate RXDX-106 safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity was planned, the study was terminated in 2018 (Clinical Trial Identification #: NCT03454243).

Other Type II Axl Kinase Inhibitors Undergoing Preclinical Evaluation
LDC1267 is a selective TAM kinase inhibitor that provides anti-metastatic potential by enhancing NK cell activity in vivo [42]. In addition, NPS-1034 is a c-Met/Axl inhibitor that is efficacious against EGFR-TKI resistant non-small-cell lung cancer cells with c-Met and Axl activation [142].

Axl-107-MMAE
Axl-107-MMAE is an antibody drug conjugate, containing human Axl antibody linked to the microtubule-disrupting agent monomethyl auristatin E (Table 1) [145]. Axl-107-MMAE induces cytotoxicity in vitro and impairs tumor growth in cervical cancer, lung cancer, and melanoma models [145]. In addition, combination treatment with MAPK pathway inhibitors cooperatively inhibited melanoma patient-derived xenograft growth [145]. The clinical safety and efficacy of Axl-107-MMAE are currently being evaluated in a Phase I/II clinical study for patients with advanced solid tumors (Clinical Trial Identification #: NCT02988817).

BA3011
BA3011 is an anti-Axl humanized monoclonal antibody conjugated to monomethyl auristatin E using a cleavable linker (CAB-Axl-ADC) ( Table 1). BA3011 is capable of reversibly binding to recombinant Axl and Axl-expressing cells in the tumor microenvironment but not in normal tissues. Preclinical studies demonstrate that BA3011 induces cytotoxicity in vitro and impairs tumor growth in lung, prostate, and pancreatic xenograft models [146]. The clinical safety and efficacy of BA3011 are currently being evaluated in Phase I/II and II clinical studies for patients with solid tumors, NSCLC, and ovarian cancers (Clinical Trial Identification #s: NCT03425279, NCT04681131, NCT04918186).

Other Monoclonal Axl Antibodies Undergoing Preclinical Evaluation
YW327.6S2 is a phage-derived monoclonal antibody that recognizes both human and murine Axl [147]. In NSCLC and breast cancer models, YW327.6S2 impaired xenograft tumor growth, reduced breast cancer metastasis, and enhanced the efficacy of erlotinib as well as chemotherapy when used in combination [147]. Mab173 has been demonstrated to induce Axl degradation and impair tumor cell migration, invasion, apoptosis, and xenograft tumor growth in a Kaposi sarcoma model [148].

Axl-Specific Chimeric Antigen Receptor (CAR) T Cells
T cells with a chimeric antigen receptor (CAR) consisting of a single-chain variable fragment against Axl have been developed and evaluated preclinically. In a triple-negative breast cancer model, Axl-CAR-T cells inhibited tumor growth and showed association with increased secretion of antitumor cytokines, including TNFα, IFNγ, IL-2, IL-6, and IL-17A (Table 1) [149,150]. Axl-CAR.C7R (Axl-targeting CAR-T cells) that co-express constitutively activated the IL-7 receptor (C7R), showed enhanced activation of Axl-CAR-T cells in vitro, and impaired xenograft tumor growth (Table 1) [151].
In the clinical landscape, CCT301 CAR-T is undergoing a Phase I/II study for patients with renal cell carcinoma to assess safety, tolerability, and anti-tumor activity (Table 1) (Clinical Trial Identification #: NCT03393936). A Phase I study for Axl-CAR-T cell therapy for patients with lung cancer is also being initiated to evaluate safety, tolerability, and preliminary efficacy (Clinical Trial Identification #: NCT03198052).  (Table 1). VAE treatment decreased Axl expression and phosphorylation and impaired colony formation and tumor cell proliferation of naïve cells as well as cisplatinand erlotinib-resistant cells [152].

Celastrol
Celastrol is a pentacyclic triterpenoid extracted from Tripterygium wilfordii Hook F and Celastrus regelii, L., which has anti-Axl activities in non-small-cell lung cancer cells (Table 1). Celastrol reduces Axl expression and impairs tumor cell migration and proliferation in both naïve and gefitinib-resistant non-small-cell lung cancer cells [153].

Yuanhuadine
Yuanhuadine is a daphnane diterpenoid from the flowers of Daphne genkwa, which has previously shown anti-proliferative effects in human lung cancer cells (Table 1) [154]. Yuanhuadine decreases Axl expression in both naïve and gefitinib-and osimertinib-resistant nonsmall-cell lung cancer cells [155]. Yuanhuadine suppresses tumor growth in vivo [155] and synergistically inhibits the growth of EGFR-TKI-resistant cells in vitro and in vivo [156].

Conclusions
Receptor tyrosine kinases have served as pharmacological targets in cancer treatment due to their frequent dysregulations, including gene amplifications, overexpression, and activating mutations, which promote tumor development, progression, and metastasis. The Gas6/Axl signaling pathway has gained significant attention as a therapeutic target due to its implications in cancer progression, metastasis, and therapeutic resistance [19][20][21]. In addition, studies demonstrate that the Gas6/Axl signaling pathway also modulates the tumor microenvironment [22]. Gas6 is secreted by both cancer and stromal cells [76,[157][158][159][160][161][162][163], and studies demonstrate that Axl is also expressed by cancer and stromal cells [22]. Therefore, targeting the Gas6/Axl pathway offers an attractive and promising approach to impair tumor progression and dissemination.
In addition, Axl overexpression is associated with resistance to conventional anticancer treatments, such as chemotherapy and radiotherapy, as well as targeted therapies [60,67,77,109,110,115,126,155,[164][165][166][167][168][169][170]. Studies demonstrate that the Axl targeting of resistant tumors can restore sensitivity. Hence, combination therapy with Axl inhibitors offers a rational treatment strategy to overcome therapeutic resistant tumors. Furthermore, Axl inhibition has been shown to modulate the tumor immune microenvironment by multiple mechanisms [22]. Chimeric antigen receptor T cells against Axl have been developed and studied preclinically [149][150][151], and a number of clinical studies are ongoing to evaluate the combination therapy of small molecule Axl inhibitors with immune checkpoint inhibitors. Since Axl is involved in multiple facets of the hallmarks of cancer, results from the ongoing clinical trials will highlight its implications in therapeutic resistance, immune evasion, and metastasis while also demonstrating its safety, tolerability, and efficacy.
Overall, a number of strategies have been employed to target Gas6 and Axl, including small molecule inhibitors, soluble receptors, monoclonal antibodies, CAR-T cell therapy, and natural compounds. These inhibitors have been tested as a monotherapy as well as combination therapies. While targeting the Gas6/Axl pathway has shown promising preclinical and clinical efficacy, targeting Gas6 or Axl as a monotherapy raises a concern about a possible narrow therapeutic index due to its expression and regulation of tumor growth in various cell types of the tumor. The Gas6/Axl pathway has been implicated in drug resistance and immune evasion, and preclinical and clinical studies have demonstrated that combination therapy may exhibit synergistic antitumor activity and drug selectivity. Hence, rational combination approaches and the selection of an appropriate patient population remain a necessity.
Although Axl overexpression is associated with poor clinical prognosis and outcome in multiple cancer types [20], the histological confirmation of Axl expression or activation is often absent in clinical study inclusion criteria. Furthermore, as Gas6 mRNA expression may not predict breast cancer outcomes [171], the assessment of Gas6 protein expression or serum levels, as a potential biomarker for patient response and outcome, is also warranted. Since small molecule tyrosine kinase inhibitors often have multiple targets, results from clinical trials utilizing a monoclonal antibody against Axl (i.e., Axl-107-MMAE and BA301), a chimeric antigen receptor T (CAR-T) cell against Axl (CCT301 CAR-T), and a soluble receptor against Gas6 (ABV-500) are highly anticipated and will offer efficacy and toxicity profiles and guidance for the future development of treatment strategies. Still, preclinical and clinical findings for various Gas6 and Axl inhibitors are favorable and make targeting this axis an attractive and promising approach to impair tumor progression and dissemination.