AXL signaling in cancer: from molecular insights to targeted therapies

Mechanisms of AXL activation

AXL regulation

AXL signaling is tightly regulated through a range of complex mechanisms, including epigenetic alterations, transcriptional, translational, and posttranslational modifications.²⁶ Several studies have demonstrated that AXL gene expression can be modulated by promoter methylation,⁵⁴ as well as by various transcription factors (HIF-��, AP-1, YAP1/TEAD, CREB, MZF1, and SP-1)^55–59. In addition, interactions with miRNAs also play a role in the regulation of AXL expression.⁶⁰

Posttranslational modifications, including glycosylation, phosphorylation, proteolytic cleavage, and ubiquitination, further influence the functionality of AXL in cancer progression.^61–63 Beyond these mechanisms, studies have identified the role of long non-coding RNA (LncRNAs) (DANCR, and XIST), which act as endogenous RNAs that compete with miRNAs (miR-33a-5p, and miR-93-5p) to regulate AXL signaling at the post-transcriptional level in various malignancies.^64–66 In addition to these regulatory mechanisms, transcriptomic sequencing of primary lung adenocarcinomas revealed the AXL-MBIP (MAP3K12 binding inhibitory protein 1) fusion gene as another potential mechanism for AXL activation.⁶⁷ These findings expand the understanding of AXL’s regulatory landscape and could provide new avenues for the development of targeted cancer therapies.

AXL in cell proliferation and survival

AXL signaling plays a pivotal role in promoting cell proliferation through various downstream pathways, including PI3K/Akt, JAK/STAT, NF-κβ, and RAS/RAF/MEK/ERK.^68,69 In addition, AXL enhances cell survival by regulating NF-κβ nuclear transport, which leads to increased expression of anti-apoptotic markers such as survivin and BCL-2, while simultaneously reducing the expression of pro-apoptotic markers like caspase 3 and BAD.⁷⁰ AXL inhibition has been shown to lower the levels of anti-apoptotic proteins, such as MCL-1 and BCL-2, while upregulating apoptosis-related proteins like Bim, particularly in chronic lymphocytic leukemia (CLL).⁷¹ Blocking the GAS6/AXL axis is critical for suppressing tumor growth in various cancers, including renal cell carcinoma (RCC), prostate cancer, lung adenocarcinoma, and colorectal cancer.⁷² For example, a study by Yu et al. identified AXL as a key survival factor for RCC cells, where it is overexpressed in human RCC tissues and cell lines. Targeting AXL using the monoclonal antibody hMAb173 induces apoptosis in both ex vivo and in vivo models.⁷³ These findings suggest that AXL plays a significant role in cancer cell proliferation and survival, making it a promising therapeutic target to inhibit cancer growth and enhance apoptosis in a range of malignancies.

AXL in cell migration and invasion

AXL overexpression is strongly linked to increased cell migration and invasion across various cancer types. AXL activity has been associated with the upregulation of GTP-binding proteins, such as Rho and Rac, which are key facilitators of cell migration.⁷⁴ Additionally, AXL activation promotes the expression of important factors such as Akt and MMP9, primarily through NF-κβ and Brg-1 signaling pathways.^70,75 In cancers, such as oral squamous cell carcinoma (OSCC) and ovarian tumors, AXL activation stimulates the PI3K/Akt pathway that mediates cancer cell invasion through increased expression of proteolytic enzymes like MMP2 and MMP9. In hepatocellular carcinoma, studies have shown that YAP1 knockdown results in reduced AXL activity, leading to decreased cell invasion. This emphasizes the significant role of AXL in invasion and migration processes.⁷⁶ Furthermore, AXL regulates genes associated with metastasis (e.g., somatostatin receptor 2 (SSTR2), fms-related receptor tyrosine kinase 4 (FLT4), matrix metalloproteinase 10 (MMP10), kisspeptin-1 (KISS1), collagen type IV alpha 2 chain (COL4A2), RAR-related orphan receptor B (RORB)) and is linked to the expression of stem cell markers.⁷⁷ It also facilitates the migration and invasion of breast cancer stem cells. Notably, AXL is involved in the production of cathepsin B, a facilitator of invasion, and the peripheral distribution of lysosomes in esophageal cancer cell lines.⁷⁸ These findings collectively suggest that AXL could be a promising target to inhibit cancer invasion and migration, thereby disrupting tumor progression.

AXL in epithelial-to-mesenchymal transition (EMT)

Recent studies have demonstrated that AXL plays a role in promoting EMT. Activation of AXL initiates a reversible transition from an epithelial to a mesenchymal phenotype, enhancing cell invasiveness and metastatic potential.⁷⁹ Several growth factors, including TGF-β, PDGF, and HGF as well as transcription factors such as Slug, Snail, ZEB1, and TWIST1, mediate EMT in various cancers.^77,80 AXL has been identified as a phenotypical marker in Non-small-cell lung cancer (NSCLC) cell lines and breast cancer stem cells.^79,81 Elevated levels of AXL are crucial for the downregulation of certain pro-epithelial factors such as E-cadherin, while simultaneously upregulating mesenchymal markers like N-cadherin, Slug, Snail, ��-catenin, α-SMA, and vimentin.^82,83

In addition to downregulating pro-epithelial factors and inducing mesenchymal markers, AXL enhances the transcriptional expression of EMT regulators Slug and N-cadherin by activating PI3K and NF-κβ pathways.^77,84 Overexpression of AXL in primary breast cancers is associated with reduced patient survival. Experimental breast cancer models have shown that knocking down AXL inhibits the metastasis of highly invasive breast carcinoma cells to lymph nodes and other organs, leading to improved overall survival.⁸¹ Similarly, TGF-β promotes AXL expression in HER2-positive breast cancer tissues, contributing to their invasive and metastatic properties.⁸⁵ In summary, AXL plays a dual role in promoting EMT across various cancers. It functions as an upstream signaling molecule that drives EMT and maintains cells in a mesenchymal state independently of GAS6. This mesenchymal state can be reversed by downregulating AXL, restoring an epithelial-like morphology.

EMT is also involved in the formation of circulating tumor cells (CTCs), which drive metastatic relapse and are key components of liquid biopsies.⁸⁶ A study by de Miguel-Pérez et al. suggested that assessing both tissue AXL expression and CTCs could improve predictions of patient outcome and tailor post-surgical therapy for resected lung adenocarcinomas.⁸⁷ Currently, CellSearch is the only FDA-cleared platform for CTC enumeration, demonstrating clinical value across various malignancies.^88–90 However, the limited expression of epithelial cell adhesion molecule (EPCAM) in CTCs has restricted their utility in advanced tumors.^91–93 Microcavity array technology, utilizing mesenchymal markers, has proven effective in identifying AXL-expressing CTCs in advanced NSCLC patients.⁹⁴ Furthermore, Bardol et al. provided the first evidence of CellSearch® detecting AXL⁺ CTCs in metastatic breast cancer, paving the way for future studies to explore anti-AXL treatments.⁹⁵

AXL in angiogenesis

TAM receptors, including AXL, are crucial not only for cancer cell proliferation and survival but also for vessel integrity and angiogenesis. They modulate various growth factors such as VEGF, FGF, and PDGF, which are essential for these processes.^96,97 AXL is expressed in endothelial and vascular smooth muscle cells, where it supports the stabilization and survival of endothelial cells and contributes to tissue remodeling, wound healing, and vessel integrity.⁹⁸ During neoplasm formation, both AXL and its ligand GAS6 are often overexpressed. They regulate the expression of matrix proteins, promoting endothelial cell proliferation, migration, and survival.⁹⁹ AXL has been implicated in stimulating growth and tube formation in human umbilical vein endothelial cells (HUVEC), through the VEGF-A-mediated activation of the PI3K/Akt pathway, which is crucial for tumor angiogenesis.^100,101 Knockdown studies of AXL and GAS6 have demonstrated reduced endothelial tube formation, attributed to decreased expression of pro-angiogenic proteins such as Dickkopf-related protein 3 (DKK3) and angiopoietin-2 (Ang-2),²⁷ which are key mediators of tumor angiogenesis. Inhibitors of AXL, when used in conjunction with antiangiogenic agents, have shown a significant reduction in vessel or tube formation in renal carcinoma xenografts.¹⁰² In addition, the GAS6/AXL/S100A10 axis has been found to promote plasmin generation, endothelial cell migration, and angiogenesis in clear cell renal cell carcinoma (ccRCC) cells, with AXL expression being associated with resistance to antiangiogenic therapies.¹⁰²

AXL in stemness

AXL is a key driver of stemness in cancer cells, facilitating self-renewal and contributing to the long-term maintenance of tumors. Its expression corelates with various cancer stem cell markers, including cell division cycle 2a (Cdc2a), bone gamma-carboxyglutamate protein 1 (Bglap1), aldehyde dehydrogenase 1 (ALDH1), enhancer of zeste homolog 2 (EZH2) and cluster of differentiation 44 (CD44).^70,103,104 Increased levels of these stemness markers enhance resistance to chemotherapy in squamous cell carcinoma and promote self-renewal activities in human mammary epithelial cells.¹⁰⁵ AXL maintains breast cancer stem cells (BCSCs) and epithelial plasticity through a positive feedback loop. Inhibiting AXL with inhibitors such as MP-470 or amuvatinib can effectively reverse the EMT process in BCSCs by downregulating the NF-κβ pathway. AXL expression is linked to the regulation of stem cell genes, metastatic genes, and enhanced tumor progression through increased cellular invasion and migration.⁷⁷ Specifically, the AXL-PYK2 (proline-rich tyrosine kinase 2)-PKCα (protein kinase C alpha) signaling axis has been shown to induce stemness in triple-negative breast cancer (TNBC). In TNBC, the expression of AXL, PYK2, and PKCα correlates with stemness in breast cancer patients. These molecules activate signal transducer and activator of transcription (STAT3), SMAD family member 3 (SMAD3), transcriptional coactivator with PDZ-binding motif (TAZ), and Fos-related antigen-1 (FRA1), along with pluripotent stem cell transcription factors Nanog and Oct4.¹⁰⁶

In cutaneous squamous cell carcinoma (SCC), AXL contributes to cell invasiveness and stemness by modulating Wnt and TGF-β signaling pathways and disrupting cell-to-cell adhesion.¹⁰³ Furthermore, suppressing AXL through inhibitors or shRNA extends the survival of chronic myeloid leukemia (CML) mice and reduces the growth of leukemia stem cells (LSCs). The GAS6/AXL complex has been shown to stabilize β-catenin in human CML CD4+ cells, presenting a potential target for eliminating CML LSCs.¹⁰⁷ Therefore, targeting AXL or its signaling pathways may enhance clinical response to anticancer therapies by reducing stemness, recurrence and metastasis.

AXL in immunosuppression

In recent years, significant progress has been made in cancer immunotherapy; however, acquired resistance remains a major clinical challenge. AXL has emerged as a key player in the development of tumor immune tolerance.²⁶ AXL induces immune evasion by upregulating BCL-2 and Twist, suppressing inflammatory signaling, and reducing the production of pro-inflammatory cytokines and chemokines such as interleukin 1 alpha (IL-1 α), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-X-C motif chemokine ligand 1, 2 and 5 (CXCL1, CXCL2, CXCL5), colony-stimulating factor 1-3 (CSF1-3) and C-C motif chemokine ligand 2-5 (CCL2-5).¹⁰⁸ It also contributes to radio-resistant and checkpoint immune checkpoint resistance by blocking MHC-I antigen presentation and promoting the production of myeloid-supporting chemokines and cytokines, which trigger an adverse immune response.^109,110 AXL confers therapeutic resistance by activating the PI3K/Akt pathway and increasing the transcription of programmed cell death ligand 1 (PD-L1) in head and neck cancer cells.^111,112 Overexpression of AXL has been linked to both intrinsic and acquired resistance to immune checkpoint blockade (ICB) in real cell carcinoma (RCC). A strong correlation exists between AXL and PD-L1 expression, with high levels of AXL in tumor cells associated with lower response rates and shorter progression-free survival following anti-PD-1 treatment. Furthermore, patients with tumors that display both high PD-L1 and AXL expression have the poorest overall survival and a reduced antitumor immune response.¹¹³

Knockout studies of AXL have demonstrated reduced tumor growth and increased sensitivity to immunotherapy due to the infiltration of CD8 + T cells in a murine model of breast cancer.^114,115 This indicates that AXL plays a multifaceted role in immunosuppression by regulating the secretion of immunosuppressive cytokines and mediators.¹¹⁶ AXL-mediated remodeling of the tumor microenvironment (TME) leads to the downregulation of immune effector cells, such as natural killer (NK) cells, T cells, and dendritic cells, while promoting the recruitment of immunosuppressive cells such as regulatory T cells (Tregs), tumor-related macrophages, and myeloid-derived suppressor cells (MDSCs).¹¹⁷

In addition, AXL signaling results in a reduction of cytotoxic T cells at effector sites and induces the transformation of macrophages from M1 to M2 polarization, ultimately facilitating tumor evasion and a poor prognosis.¹¹⁸ Therefore, inhibiting AXL can remodel the TME, enhance immune response and improve the therapeutic potential of immune checkpoint blockade inhibitors.

AXL in viral infections and inflammatory responses

Beyond its role in immune regulation, AXL has been implicated in the progression and severity of various viral infections, including Ebola virus (EBOV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Zika virus (ZIKV).^119–121 Its interactions with these viruses highlight its dual role in facilitating viral entry and modifying inflammatory responses, establishing a connection between infectious diseases and cancer progression.

Using a series of AXL mutants, Shimojima et al. demonstrated that EBOV particles activate AXL through cell surface adhesion and interaction with its immunoglobulin domains, enabling viral entry by endocytosis.¹²⁰ Similarly, AXL has been identified as a potential receptor for SARS-CoV-2, interacting directly with the spike protein’s N-terminal to facilitate viral attachment and entry into pulmonary epithelial cells.¹²² During Zika virus infection, AXL enhances viral entry in glial cells through GAS6 bridging and impacts innate immune responses.¹¹⁹ Studies involving Zika virus strain MR-766 in human glioblastoma cell lines revealed that AXL is essential for viral entry, though the exact mechanisms remain unclear and needs further investigations.¹²³ Moreover, AXL was recently identified as a co-receptor for parvovirus B19 in human erythroid progenitors, further emphasizing its role in viral pathogenesis.¹²⁴ These findings suggest that targeting AXL could be a promising strategy for future antiviral treatments.

In addition to facilitating viral entry, AXL plays a crucial role in regulating inflammatory responses. Activation of AXL by its ligand, GAS6, triggers a signaling cascade that suppresses innate immune defenses, allowing viruses to evade detection. This occurs through the upregulation of suppressors of cytokine signaling (SOCS), which in turn inhibits toll-like receptor (TLR) pathways essential for a robust immune response.¹²⁵ While this immunomodulatory function can reduce tissue damage during infections, it also creates a vulnerability to malignancy. Tumors often exploit these inflammatory pathways to escape immune surveillance, and elevated AXL expression can impair the function of natural killer (NK) cells and macrophages, weakening the antitumor immune response. For instance, the human papillomavirus type 16E6 (HPV16E6) oncoprotein has been shown to upregulate AXL expression in cervical cancer via the MAGI-2/PTEN/Akt/MZF1 network, increasing tumor invasiveness and promoting immune evasion by reducing responsiveness to NK cells-mediated lysis.¹²⁶

Thus, AXL serves as a critical link between viral infections, inflammatory responses, and cancer progression.¹²⁷ By facilitating viral entry and modulating the host’s immune response, AXL contributes to the pathogenesis of both infectious diseases and cancer metastasis. These insights position AXL as a potential therapeutic target in both virology and oncology, where its inhibition could enhance antitumor immunity and improve clinical outcomes.

However, studies suggest that AXL inhibition may also create a tumor-promoting environment by enhancing pro-inflammatory cytokine production and impairing the clearance of apoptotic neutrophils.¹²⁸ This is particularly relevant in comorbid conditions like inflammatory arthritis, where AXL, regulated by miR-34a, is essential for controlling dendritic cell (CD1c+ DC) activation and the generation of pro-inflammatory cytokines such as TNF, IL-6, IL-1β, and IL-23.¹²⁹ Moreover, AXL deficiency has been observed to exacerbate pulmonary arterial hypertension (PAH) via the bone morphogenetic protein receptor 2 (BMPR2) pathway, suggesting a protective role for the GAS6/AXL axis in PAH.¹³⁰ Therefore, AXL’s paradoxical roles as both a proto-oncogene and an immunological protector highlight its complexity in cancer and related comorbidities. Blocking AXL could interfere with its beneficial immunoregulatory effects, potentially exacerbating inflammation or immune dysregulation in comorbid diseases. Thus, a thorough investigation of AXL’s context-dependent effects is necessary to develop effective therapies.

Future research should focus on clarifying AXL’s roles in viral pathogenesis, tumor biology, and related comorbidities, paving the way for innovative treatment strategies. Nonetheless, the unpredictable mutations in viral genomes present a significant challenge for therapeutic development, necessitating further investigation.

AXL-TYRO3

TYRO3, a member of the TAM RTK family, shares the ligands GAS6 and Protein S with AXL. Although the signal transduction pathways of TYRO3 are not fully explored, its overexpression and activation have been linked to enhanced cell survival, proliferation, metastasis, and therapy resistance in various cancers.²⁰⁷

A study by Brown et al. identified a functional connection between TYRO3 and AXL, demonstrating that overexpressing kinase-active TYRO3 in Rat2 cells leads to increased GAS6-mediated AXL phosphorylation, independent of AXL expression levels. Moreover, the co-expression of TYRO3 and AXL in these cells significantly enhances proliferation, up to fivefold, upon GAS6 treatment. AXL overexpression also promotes transphosphorylation of both kinase-active and kinase-dead forms of TYRO3. This cross-phosphorylation modulates downstream of MAPK and PI3K signaling pathways, highlighting how RTK crosstalk alters cellular signaling.²⁰²

Both AXL and TYRO3 are overexpressed in thyroid cancer cells and leiomyosarcoma cells, contributing to cell growth and resistance to apoptosis.^208,209 Investigating the interplay between AXL and TYRO3 in cancer progression could offer insights into the varying responses of different cancer cells to AXL inhibitors.

AXL-MERTK

MERTK, another member of the TAM family of RTKs, plays a physiological role in regulating innate immunity, platelet aggregation, tissue homeostasis and repair. However, its aberrant expression in various cancers has been implicated in promoting neoplasia processes such as growth factor independence, increased proliferation, resistance to apoptosis, and metastasis.²¹⁰

While AXL and MERTK do not directly interact, their downstream signaling pathways significantly overlap. MERTK is primarily expressed in cells of the hematopoietic lineage, including monocytes, macrophages, dendritic cells, NK cells, and platelets, whereas AXL expression is mainly confined to epithelial and endothelial tissue and is linked to EMT.^99,189 Consequently, MERTK is more closely associated with the tumor microenvironment, while AXL is linked to both tumor cells and the tumor microenvironment. This cooperation between AXL and MERTK has been shown to accelerate the growth of breast cancer cells by combining oncogenic signaling and evading host antitumor immunity.²¹¹

AXL and MERTK are frequently overexpressed in multiple cancers including head and neck, lung, colon, lung, skin, sarcoma, and glioblastoma multiforme.^212–216 McDaniel et al. identified MERTK upregulation as an inherent and adaptive feedback mechanism that leads to resistance against anti-AXL agents. However, co-targeting both MERTK and AXL significantly inhibited tumor growth by affecting several signaling pathways, including Akt, STAT6, P70S6K, S6RP, and C-RAF.²¹⁵ Interestingly, the dual inhibitor INCB081776 demonstrated immunomodulatory and antitumor effects primarily by inhibiting MERTK activation in macrophages. This effect partially counteracted M2 macrophage-mediated inhibition of T-cell proliferation and suppression of phospho-Akt.²¹⁶

These findings suggest that co-targeting the TAM RTKs could confer dual anticancer effects. First in the tumor cells, where AXL inhibition would disrupt developmental, proliferative, and migratory pathways, and second, in the tumor microenvironment, where MERTK suppression would modulate the innate immune response.

AXL-c-MET

The c-MET RTK is a proto-oncogene known to derive the invasion and metastasis of various cancers.^215,217 In clear cell renal cell carcinoma (ccRCC), GAS6/AXL signaling enhances cellular invasion through lateral stimulation of c-MET, mediated by another proto-oncogene, SRC.⁵⁷ AXL and c-MET crosstalk has been observed in triple-negative breast cancer (TNBC), where exposure to hepatocyte growth factor (HGF) leads to phosphorylation of both c-MET and AXL. Interestingly, AXL knockdown does not affect the HGF-mediated phosphorylation of downstream proteins, indicating a complex interplay between c-MET and AXL. Conversely, GAS6 exposure results only in AXL phosphorylation, c-MET knockdown alters the timing and intensity of GAS6-mediated signaling. Despite these complexities, Reverse Phase Protein Arrays (RPPAs or RPLAs) analyses suggest that AXL and c-MET receptors exhibit crosstalk and may physically interact in breast cancer cells.²¹⁸

In addition, Li et al. identified that the clustering of c-MET, AXL, ELMO2, and DOCK180 proteins on the plasma membrane constitutes a crucial mechanism through which HGF-dependent signaling initiates RAC1-mediated migration and invasion of glioblastoma cells.²¹⁹ Notably, in the HGF/c-MET pathway, AXL (in its higher-molecular-weight form, p140) undergoes transphosphorylation at Tyr-779, potentially playing a significant role in tumorigenesis. This contrasts with the phosphorylation of the lower-molecular-weight AXL (p120) by GAS6 at Y702.²¹⁹

Overall, these findings suggest that using inhibitors targeting both AXL and c-MET may be a promising strategy for preventing the progression and metastasis of cancers driven by AXL/c-MET signaling.

AXL-EGFR

Epidermal growth factor receptor (EGFR) is primarily involved in maintaining epithelial tissue. However, aberrant EGFR signaling can lead to epithelial transformation, making it one of the most studied tyrosine kinase receptors in cancer.²²⁰ Despite the clinical efficacy demonstrated by various EGFR-targeted therapies, acquired resistance often limits long-term effectiveness in cancer treatment.

Interestingly, AXL has been found to influence acquired resistance to EGFR-targeted therapies in both breast and lung cancers. In triple-negative breast cancer (TNBC), EGFR-mediated transactivation of AXL reduces responsiveness to EGFR-targeted therapies by diversifying EGFR signaling, including its interactions with c-MET and PDGFR, but not with IGF1R (insulin-like growth factor-1 receptor) or INSR (insulin receptor).²²¹ In addition, co-expression of AXL and EGFR is frequently observed in lung adenocarcinomas, regardless of EGFR mutation status.^206,222 Furthermore, crosstalk between AXL and EGFR allows squamous cell carcinomas to bypass the anti-proliferative effects of PI3Kα inhibitors by activating the PLCγ-PKC/mTOR signaling pathway.¹³³ In glioblastoma multiforme, this hetero-interaction between AXL and EGFR facilitates EGFR-mediated pro-invasive signaling.⁵¹

Examining the co-expression patterns of EGFR and AXL in various cancers could provide valuable insights into potential biomarkers. Additionally, the concurrent targeting EGFR and AXL may enhance the therapeutic efficacy of EGFR-targeted therapies and help mitigate therapeutic resistance.

AXL-HER2

Another mechanism of RTK cross-communication involves the stabilization of one receptor by another, as seen with AXL and human epidermal growth factor receptor 2 (HER2). HER2, a member of the epidermal growth factor receptor family, plays a significant role in the etiology of various human cancer by regulating cellular proliferation and differentiation. Approximately 20% of breast cancer cases exhibit HER2 gene amplification and overexpression.²²³

In HER2-positive breast cancer cells, AXL physically interact with HER2, facilitating AXL’s stability and recruitment to the cell surface. This interaction leads to AXL transphosphorylation by HER2, promoting metastatic processes such as intravasation, growth of metastatic cells, and extravasation.⁸⁵ In addition, AXL stabilizes HER2 and regulate Hypoxia-inducible factor-1 alpha (HIF-1α) levels under hypoxic conditions, potentially through activation of the PI3K/Akt pathway, which drives metastasis and modulates the tumor microenvironment in HER2-positive breast cancer cells.²²⁴ The crosstalk between AXL and HER2 also activates the PI3K/Akt and ERK pathways, enabling HER2-positive breast cancer cells to develop resistance against lapatinib, an inhibitor of HER2/neu and EGFR pathways.²²⁵

Interestingly, AXL promotes EMT and therapeutic resistance in breast cancer by heterodimerizing with HER2, thereby inducing PI3K/Akt and MAPK signaling independent of its ligands. This suggests that targeting HER2-AXL heterodimerization could be crucial for preventing HER2 inhibitor-mediated therapeutic resistance, especially in breast cancer.¹³⁷

AXL-HER3

Human epidermal growth factor receptor 3 (HER3) is a member of the HER-RTK family, with neuregulins 1 (NRG1) and 2 (NRG2) as its primary ligands. Although HER3 has low intrinsic tyrosine kinase activity, it can interact with other RTK or non-RTK members to increase transphosphorylation and activate downstream pathways, ultimately promoting metastasis and drug resistance.²²⁶

AXL and HER3 collaborate to counteract the effects of AXL inhibitors (such as BMS777607 or R428) in MDA-MB231 and Ovcar8 cells. when AXL activity is lost, HER3 transcription is upregulated, leading to NRG1-dependent phosphorylation (Y1289) as a compensatory mechanism. Notably, the activation of phospho-Akt (S473) at low basal levels is typically required for HER3 activation upon AXL inhibition.²²⁷

In addition, crosstalk between AXL and HER3 promotes invasion and metastatic progression in melanoma cells by regulating invadopodia formation.²⁰⁵ In NSCLC, AXL interacts with HER3 with EGFR within a negative feedback system involving SPRY4, resulting in resistance to the EGFR inhibitor osimertinib.^222,228 In head and neck cancer, AXL mediates cetuximab resistance through two mechanisms: activation of c-ABL kinase via AXL’s Y821 phosphorylation²²⁹ and HER3 activation due to upregulated NRG1.³¹

This predictive relevance of the AXL and HER3 association highlights the importance of developing dual-targeted inhibitors or combination therapies to enhance treatment efficacy against acquired resistance.

AXL-VEGFR

Vascular endothelial growth factor receptor (VEGFR), a member of the RTK family, plays a pivotal role in angiogenesis, with its three primary subtypes identified as VEGFR1, VEGFR2, and VEGFR3. RTKs can communicate indirectly through various signaling intermediates. One such example is how VEGFR2 activates Src family kinases via TSAd (T-cell-specific adaptor protein), promoting ligand-independent autophosphorylation of AXL at YXXM motifs. This activation facilitates interaction with PI3K, stimulating Akt signaling. Consequently, activated AXL contributes to VEGF-A/VEGFR2-dependent cancer cell migration, tube formation, vascular permeability, and corneal neovascularization.¹⁰¹

Conversely, GAS6 impedes ligand-dependent VEGFR2 activation and angiogenic signaling in vascular endothelial cells by inducing the phosphorylation of SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2) and facilitating its interaction with AXL.²³⁰ This intricate regulatory mechanism underscores the complexity of crosstalk between AXL and VEGFR in modulating angiogenic programs.

In addition, the localization and expression of AXL and VEGFR exhibit significant variation between monolayer and spheroid ovarian cancer models. For instance, OVCAR8 spheroids show a tenfold higher concentration of VEGFR1 on the plasma membrane and greater heterogeneity compared to monolayers, characterized by a bimodal distribution of low and high-AXL subpopulations. This variation, including a 100-fold difference in plasma membrane AXL concentration between chemo-sensitive and chemoresistance cells,²³¹ highlights the importance of selecting appropriate cancer models for therapeutic screening.

In conclusion, comprehensive studies across various cancers are needed to unravel the precise molecular mechanisms underlying GAS6/AXL crosstalk with VEGFR and its regulation of angiogenic programs.

AXL-FLT3

The Fms-related receptor tyrosine kinase 3 (FLT3) gene, a class III RTK, plays a crucial role in hematopoiesis regulation. Upon binding to its ligand, FLT3 undergoes homodimerization on the plasma membrane, leading to autophosphorylation and initiation of downstream signaling pathways involved in cell proliferation, apoptosis, and differentiation in bone marrow. Mutations that result in constitutive activation of FLT3 are notably implicated in myeloid and lymphoblastic leukemia.²³²

AXL significantly influences the progression of FLT3 internal tandem duplication (ITD)-positive acute myeloid leukemia (AML) by promoting constitutive FLT3 phosphorylation.²³³ In FLT3-ITD positive AML cells, elevated AXL expression promotes cell growth, increases therapy resistance, and suppresses apoptosis and differentiation through regulation of downstream molecules such as Akt, STAT5, and ERK.^151,234 Notably, inhibition of AXL activity has shown a marked reduction in the development of resistance in AML against FLT3 inhibitors, including PKC412 and AC220.²³⁵ Furthermore, dual inhibition of FLT3 and AXL using Gilteritinib in FLT3-ITD AML has demonstrated effectiveness in overcoming resistance mechanisms driven by the hematopoietic niche.²³⁶

The potential of FLT3-ITD inhibitors to induce apoptosis is influenced by the GSK-3β/Mcl-1 axis, which is regulated by both FLT3-ITD and AXL signaling pathways.²³⁷ Overall, combined inhibition of FLT3 and AXL presents a promising therapeutic strategy for leukemia, offering new alternatives to overcome resistance and improve patient outcomes.

AXL-PDGFR

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) are expressed in various cancers, where PDGF/PDGFR signaling activation influences cell proliferation, migration, invasion, and angiogenesis through the PI3K-PKB/Akt and MAPK/ERK pathways.²³⁸ AXL and PDGFR demonstrate crosstalk, as observed in gastrointestinal stromal tumors (GISTs) treated with imatinib mesylate. In this context, inhibition of α-PDGFR (KIT) by imatinib mesylate leads to AXL overexpression, resulting in resistance to therapy.²³⁹

In human bladder cancer, c-MET has been shown to promote disease progression by transactivating the expression of AXL and PDGFR-α via MEK/ERK1/2 signaling, independent of Ras and Src.²⁰² Similarly, in triple-negative breast cancer cells, AXL interacts with other ErbB receptor family members, c-MET, and PDGFR contributing to resistance against EGFR TKIs.²²¹

Further exploration of the mechanisms underlying receptor switching from PDGFR to AXL in different cancers could provide valuable insights into the signaling networks and help overcome acquired resistance.

Overall, the crosstalk between AXL with other RTKs contributes to resistance against both conventional and targeted therapies across diverse cancers. Therefore, co-targeting AXL along with other RTKs presents a promising strategy to address the challenges associated with RTK signaling rewiring and improve cancer management.

Bemcentinib combinations

Bemcentinib, the first selective small-molecule AXL inhibitor to receive US FDA fast-track designation, has shown promising results in combination studies across various cancer types. Its potent inhibition of both ligand-dependent and ligand-independent AXL signaling makes it an attractive candidate for combination therapies aimed at overcoming resistance and improving treatment outcomes.

In uterine serous carcinoma, bemcentinib enhanced sensitivity to paclitaxel chemotherapy by inhibiting AXL activation.²⁴⁰ Similarly, in diffuse intrinsic pontine glioma, and aggressive colorectal adenocarcinoma, combining bemcentinib and agents like panobinostat (an HDAC inhibitor) and galunisertib (a TGF-β inhibitor) showed synergistic effects in reversing mesenchymal transition and reducing the mesenchymal phenotype, respectively.^241,242

In NSCLC, bemcentinib combined with VX-970 (an ATR inhibitor) enhanced DNA damage and replication stress, increasing susceptibility to ATR inhibitors.¹⁷² In addition, in metastatic melanoma, bemcentinib improved the therapeutic efficacy of the BRAF inhibitor vemurafenib by triggering apoptosis, accelerating ferroptosis, and reducing autophagy.²⁴³ In older AML patients who cannot tolerate intense chemotherapy, bemcentinib showed an anti-leukemic effect when combined with low-dose cytarabine or decitabine (NCT02488408). Parallel studies have shown that combining bemcentinib with erlotinib (NCT02424617), docetaxel (NCT02922777), and pembrolizumab (a humanized antibody that targets PD-1; NCT03184571) is viable and tolerable in advanced NSCLC.

A phase Ib/II trial is currently studying the combination of bemcentinib with pembrolizumab or dabrafenib/trametinib in advanced non-resectable or metastatic myeloma (NCT02872259). Similarly, the combination of bemcentinib and pembrolizumab is being evaluated in a phase II study for mesothelioma compared to other targeted treatments (NCT03654833).

While bemcentinib/pembrolizumab has demonstrated a favorable clinical response in several trials, its investigation in refractory TNBC was halted due to the lack of complete or partial response in patients (NCT03184558). The encouraging results from these combination studies collectively support further exploration of bemcentinib in conjunction with other small-molecule inhibitors or immunotherapeutic agents for the effective treatment of various human malignancies.

TP-0903 combinations

In small-cell lung cancer, combining WEE1 inhibitors (such as AZD1775) and AXL inhibitors (like TP-0903, an oral selective AXL inhibitor) or with mTOR inhibitors (such as RAD001) can overcome resistance to WEE1 inhibition driven by downstream activation of mTOR and CHK1 which are involved in DNA damage repair pathways.¹⁷¹ Additionally, the combination of AXL (TP-0903) and JAK1 (ruxolitinib) inhibition has been identified as a novel therapeutic strategy based on single-cell proteomic profiling in lung cancer.²⁴⁴ Our lab has demonstrated that silencing AXL (either through gene knockdown or using TP-0903), effectively inhibits therapeutic resistance against cabozantinib in renal cell carcinoma both in vitro and in vivo.^245–247 Moreover, targeting the heterodimerization of AXL-HER2 with TP-0903 and trastuzumab (a HER2 inhibitor) in breast cancer has been observed to overcome resistance to HER2 blockade.¹³⁷

The preliminary clinical activity of TP-0903 in combination with decitabine has also been reported in the phase 1b/2 trial (NCT03013998) involving patients aged ≥60 years with prognostically poor TP53 mutant/complex karyotype AML.²⁴⁸ Together, these studies support the use of TP-0903 in combinations with various therapeutics to overcome the emergence of therapeutic resistance.

Antibody–drug conjugate combinations (CAB-AXL-ADC & Enapotamab vedotin)

Several clinical trials are investigating the safety and efficacy of antibody–drug conjugates (ADCs), such as CAB-AXL-ADC. These trials are examining its effectiveness in combination with PD-(L)1 inhibitor against various cancers, including sarcomas (phase I, NCT03425279), NSCLC (phase II, NCT04681131) and ovarian cancers (phase II, NCT04918186).

Recently, Boshuizen et al. also demonstrated the role of another AXL-targeting antibody–drug conjugate (Enapotamab vedotin) in potentiating anti-PD-1 (pembrolizumab) therapy in melanoma and lung cancer models resistant to immunotherapy. This combination results in a significant benefit from de novo immune checkpoint inhibition.²⁴⁹

ONO-7475 combinations

ONO-7475, a dual inhibitor targeting AXL and MERTK, has shown potential in combination therapies for overcoming resistance in various cancers. In FLT3-ITD-dependent AML, ONO-7475 can synergize with sorafenib (a multikinase inhibitor) and venetoclax (a BCL-2 inhibitor) to address resistance to FLT3 and BCL-2 inhibitors, respectively.^250,251 In addition, combining ONO-7475 with Osimertinib (an EGFR inhibitor) has demonstrated efficacy in suppressing resistance in EGFR-mutated NSCLC cells that overexpress AXL.²⁵²

Despite these promising results, some clinical trials have faced challenges. The phase I/II trial (NCT03176277) exploring ONO-7475 alone or with venetoclax in AML was terminated due to protocol-defined futility criteria. Similarly, the dose-escalation study of ONO-7475 monotherapy, and in combination with anti-PD-1 therapy (ONO-4538/Nivolumab) in advanced solid tumors (NCT03730337) was also suspended following temporary completion of enrollment.

PF-07265807 combinations

PF-07265807, an inhibitor of AXL and MERTK, has demonstrated promising antitumor activity in preclinical models, particularly when combined with PD-1 inhibitors.²⁵³ A clinical trial (NCT04458259) is currently assessing the safety, tolerability, pharmacokinetics, and early antitumor efficacy of PF-07265807 as a single agent and in combination with sasanlimab (an anti-PD-1 antibody), with or without axitinib (a VEGFR inhibitor), in patients with advanced or metastatic renal cell carcinoma.²⁵⁴

In addition, Q702, a selective inhibitor targeting AXL, MERTK, and CSF1R, has shown favorable pharmacologic activity and an acceptable safety profile in patients with advanced solid tumors.²⁵⁵ This inhibitor is being evaluated in a clinical trial (NCT04648254).

Recently studies have also explored the use of AXL, TAM, and non-TAM RTK inhibitors in combination therapies to reverse acquired resistance to EGFR TKIs and improve the outcomes of immunotherapy. These combinations often show greater efficacy compared to TAM receptor-specific inhibitors alone.

Cabozantinib combinations

Cabozantinib, a multikinase inhibitor targeting VEGFR2, c-MET, RET, and AXL, has shown efficacy in overcoming resistance to other therapies across various cancers. In renal cancer, prolonged sunitinib treatment increases c-MET and AXL expression, leading to acquired resistance against sunitinib, which can be countered with cabozantinib.²⁵⁶ Similarly, cabozantinib combined with gefitinib has been effective in countering resistance to crizotinib and ROS1-TKIs in lung cancers with ROS1 fusions, where active HB-EGF/EGFR and AXL signaling contribute to resistance.²⁵⁷

Cabozantinib combined with osimertinib has also shown potential in overcoming AXL-mediated resistance in EGFR-mutated NSCLC.¹⁶⁸ Multiple trials (NCT00596648, NCT01866410, and NCT01708954) are exploring the combination of cabozantinib with erlotinib to address EGFR-TKI resistance in NSCLC, while cabozantinib combined with panitumumab has demonstrated a favorable safety profile and encouraging activity in RAS wild-type colorectal cancer (NCT02008383).²⁵⁸

Cabozantinib is also being investigated in combination with immunotherapies. The COSMIC-021 phase 1b trial is evaluating cabozantinib alone or with atezolizumab in various solid tumors. In metastatic castration-resistant prostate cancer, cabozantinib and atezolizumab have shown a tolerable safety profile and clinically relevant activity (NCT03170960).²⁵⁹ The CONTACT-2 phase III trial reported that the combination significantly improved progression-free survival compared to abiraterone, enzalutamide, and prednisone (NCT04446117).²⁶⁰

In renal cell carcinoma, the combination of cabozantinib and nivolumab has improved overall survival compared to sunitinib and showed durable responses in advanced cases (NCT03141177).²⁶¹ The COSMIC-313 trial showed adding cabozantinib to nivolumab and ipilimumab significantly prolonged progression-free survival in advanced renal cell carcinoma (NCT03937219).²⁶² Ongoing trials, including the PDIGREE study (NCT03793166) and a phase I/II trial (NCT01658878), continue to explore these combinations, highlighting Cabozantinib’s versatility in treating various cancers and improving outcomes.

Sitravatinib combinations

Sitravatinib, a receptor kinase inhibitor targeting TYRO3, AXL, MERTK, and VEGFR2, is being studied in combination with nivolumab for various cancers. Ongoing clinical trials focus on its efficacy in locally advanced clear cell renal cell carcinoma (ccRCC) (NCT03015740; NCT04904302; NCT03680521), advanced NSCLC (NCT02954991; NCT03906071) and oral cancer (NCT03575598). A phase I study is also exploring the combination of sitravatinib with nivolumab and ipilimumab in ccRCC and other solid malignancies (NCT04518046). Sitravatinib combined with the anti-PD-1 antibody tislelizumab has demonstrated tolerance and early anticancer activity in patients with unresectable advanced hepatocellular carcinoma and gastric cancer (NCT03941873) and promising results in TNBC²⁶³ (NCT04734262), advanced melanoma²⁶⁴ and NSCLC²⁶⁵ (NCT03666143). A phase III trial is testing sitravatinib and nivolumab in advanced NSCLC (NCT04921358), while a phase II trial investigates this combination in advanced biliary tract cancer (NCT04727996). However, a phase II study in urothelial carcinoma found a tolerable safety profile but no significant objective response (NCT03606174), with 51.2% of patients experienced grade 3 treatment-related adverse events (TRAEs) 3.3% encountered grade 4 TRAEs, and one patient died from cardiac failure.²⁶⁶ The phase III SAPPHIRE trial also failed to show improved survival for sitravatinib plus nivolumab compared to docetaxel in advanced NSCLC (NCT03906071).²⁶⁷ Additional multitargeted AXL inhibitors are in development. NPS-1034, a dual inhibitor of AXL/MET, enhances gefitinib or erlotinib sensitivity in EGFR-mutant NSCLC.²⁶⁸ Ningetinib, an inhibitor of c-MET, AXL and VEGFR2 showed promise when combined with gefitinib in EGFR-TKI-resistant T790M-negative NSCLC (NCT03758287). CB469, another AXL/MET inhibitor, demonstrated efficacy with EGFR TKIs in NSCLC with acquired resistance from AXL and c-MET activation.²⁶⁹

Anlotinib combined with osimertinib has also been found to sensitize osimertinib-resistant NSCLC by targeting c-MET/MYC/AXL signaling.²⁷⁰

In addition, BMS777607, a selective MET kinase inhibitor, combined with lapatinib, overcame HER3-induced resistance to AXL TKIs.²²⁷ S49076, an inhibitor of MET, AXL, and FGFR, enhanced radiotherapy efficacy in MET-dependent and independent models.²⁷¹

These studies support the ongoing exploration of AXL inhibitors in combination therapies to improve safety and overcome resistance in various cancers.