Targeting of the AXL receptor tyrosine kinase by small molecule inhibitor leads to AXL cell surface accumulation by impairing the ubiquitin-dependent receptor degradation
Overexpression of AXL receptor tyrosine kinase (AXL) in various human cancers correlates with reduced patients overall survival and resistance to first line therapies. Therefore, several AXL tyrosine kinase inhibitors (TKIs) are currently under clinical evaluation.
AXL TKI BMS777607 treatment increased AXL protein levels after 24 h as observed by Western blot and flow cytometry analysis. Mechanistically, this inhibition-induced AXL cell surface accumulation was neither associated with epigenetic modifications, nor altered transcriptional and translational regulation. Further, we saw no impact on glycosylation and receptor shedding by α-secretases. However, we observed that BMS777607 increased the glycosylated 140 kDa AXL protein abundance, which was impaired in the kinase dead mutant AXL (K567R). We demonstrated that AXL kinase activity and subsequent kinase phosphorylation is necessary for GAS6-dependent receptor internalization and degradation. Blocking of kinase function by BMS777607 resulted in ubiquitination prohibition, impaired internalization and subsequent cell surface accumulation. Subsequently, AXL cell surface accumulation was accompanied by increased proliferation of 3D-Speroids induced by low μM levels of BMS777607 treatment.
Our data suggest a re-evaluation of anti-AXL clinical protocols due to possible feedback loops and resistance formation to targeted AXL therapy. An alternative strategy to circumvent feedback loops for AXL targeting therapies may exist in linkage of AXL TKIs to a degradation machinery recruiting unit, as already demonstrated with PROTACs for EGFR, HER2, and c-Met. This might result in a sustained inhibition and depletion of the AXL from tumor cell surface and enhance the efficacy of targeted anti-AXL therapies in the clinic.
KeywordsRTK AXL TKI Ubiquitin Degradation 3D spheroid
A Disintegrin and Metalloproteinase Domain
RAC-α serine/threonine protein kinase
AP-1 transcription factor subunit
AXL receptor tyrosine kinase
- BMS (BMS777607)
Castias B-lineage lymphoma
MET proto-oncogene, receptor tyrosine kinase
N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester
Epidermal growth factor receptor
eukaryotic translation initiation factor 4B
eukaryotic translation initiation factor 4E
Extracellular signal-regulated kinase
Fetal calf serum
Growth arrest specific gene 6
Growth factor receptor bound protein 2
erb-b2 receptor tyrosine kinase
Hypoxia inducible factor 1 subunit alpha
the nuclear factor-kappa B
p38 mitogen-activated protein kinase
Proteolysis targeting chimera
Receptor tyrosine kinase
Reverse transcription-quantitative PCR
Standard error of the mean
Sp1 transcription factor
Signal transducer and activator of transcription signaling
Tyrosine kinase inhibitors
Deregulated oncogenic activity of the AXL receptor tyrosine kinase (AXL) and elevated levels of its ligand GAS6 (growth arrest specific gene 6) are found in numerous types of human cancer and are directly correlated with diverse aspects of cancer pathogenesis . AXL up-regulation has also been described in cisplatin-resistant ovarian cancer, doxorubicin-resistant acute myeloid leukemia, lapatinib-resistant breast cancer, imatinib-resistant gastrointestinal stromal tumors, and imatinib-resistant chronic myeloid leukemia. Recently, AXL activation has been reported as a cause of resistance to epidermal growth factor receptor (EGFR)-targeted therapy in non–small cell lung cancers [2, 3].
AXL expression is regulated via various mechanisms. It is subject to epigenetic modifications, i.e. DNA methylation and histone acetylation . Hypomethylation of the AXL gene has been associated with high expression in different cancer entities. Multiple transcription factors, including SP1, AP-1 and HIFα, can induce AXL transcription [5, 6]. Expression of splice variants is regulated post-transcriptionally and by translation initiation factors, e.g. eIF4E and eIF4B . AXL undergoes either N-linked or O-linked glycosylation in the Golgi apparatus before it is properly recruited to the membrane [8, 9]. AXL kinase activity is increased after GAS6 binding leading to activation of downstream signaling cascades . AXL signaling stimulates phosphatidylinositide 3-kinase/RAC-α serine/threonine protein kinase (PI3K/AKT), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase cascades (MAPK), the nuclear factor-kappa B (NF-κB) pathway as well as signal transducer and activator of transcription signaling (STAT) . Therefore, biological processes including invasion, angiogenesis, resistance to chemotherapeutics and targeted drugs, survival, and proliferation, as well as receptor downregulation are tightly regulated. Phosphorylated AXL is internalized for subsequent degradation or recycling via proteasomal, lysosomal or endosomal pathways [11, 12, 13]. Since deregulated AXL expression is associated with cancer and other pathological conditions, a better understanding of AXL regulation is critical for AXL-targeted treatment approaches. A complex of γ-carboxylated GAS6 Ca2+-dependently bound to phospholipid PtdSer causes homo-dimerization of the receptor, leading to the optimal activation of AXL kinase by phosphorylation of tyrosine residues at the receptor C-terminal kinase domain. This phosphorylation event creates docking sites for AXL downstream signaling molecules [14, 15]. In parallel, AXL phosphorylation leads to recruitment of the GRB2 adaptor protein, which recruits the E3 ubiquitin-protein ligase CBL in similar fashion as shown for c-MET [16, 17]. An additional mechanism is the direct recruitment and activation of CBL through the CBL tyrosine kinase binding (TKB) domain. Upon binding and activation of CBL, AXL is ubiquitinated . This posttranslational modification is required for efficient AXL degradation in the lysosome . An alternative pathway of AXL degradation is the proteolytic shedding by ADAM10 and ADAM17, as described by Miller et al., releasing a soluble 85 kDa N-terminal fragment (sAXL) and a short intracellular 55 kDa C-terminal fragment . This soluble sAXL receptor can be used as a biomarker and companion diagnostic tool as shown for hepatocellular carcinoma (HCC) and malignant peripheral nerve sheath tumors (MPNST) [20, 21]. Because AXL is considered an important molecular target in cancer therapy, various strategies, like sAXL decoy receptor (GL2I.T and MYD1–72) and antagonistic monoclonal antibody targeting AXL (YW327.6S2 and 20G7-D9) are currently in preclinical development. Other applications have already proceeded to clinical trials . Among the most promising AXL-inhibitory approaches are small molecule tyrosine kinase inhibitors (TKIs). In the present study we used the preclinical AXL/MET small molecule inhibitor BMS777607 (BMS) . Here we show that AXL inhibition by BMS causes accumulation of the AXL receptor on the cellular surface due to impaired receptor downregulation. We propose that inhibition of AXL kinase phosphorylation by the small molecule inhibitors causes reduced ubiquitination of AXL leading to reduced lysosomal degradation. This might be linked to therapy resistance displaying a major problem of targeted cancer therapies in the clinic. This study elucidates the complex regulation of AXL expression.
Cells were cultured in a humidified incubator with 5% CO2 at 37 °C. MDA-MB231 was maintained in DMEM supplemented with 1% sodium pyruvate and 10% FCS. MDA-MB231-D3H2LN (Caliper)  was grown in MEM supplemented with 1% sodium pyruvate, 1% GlutaMAX, 1% nonessential amino acids, and 10% FCS. Hs578T and NCI-H292 (H292) and NCI -1792 (H1792) were cultured in RPMI 1640 medium with 1% GlutaMAX and 10% FCS. Cell culture media and supplements were purchased from Gibco (Thermo Fisher Scientific, Darmstadt, Germany).
Three-dimensional (3D) spheroid culture and cell viability
Matrigel basement membrane matrix (BD Biosciences—Corning, No. 354234; Kaiserslautern, Germany) was diluted to a concentration of 3 mg·mL− 1 in cell line-corresponding serum-free medium. 7000 cells were seeded on top of the solid Matrigel in each well (96-well plate). Viability of cells was determined by ATP quantification using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mannheim, Germany). The luminescence signal was measured by Clariostar multimode microplate reader (BMG Labtech, Ortenberg, Germany).
Tyrosine kinase inhibitors and cell treatment
BMS777607 was purchased from ShangHai Biochempartner Co., Limited, Wuhan, China (Cas No.:1196681–44-3) with a purity > 98%. Chloroquine Phosphate (CQ, No.S4157 in H2O 10 mg/ml) and DAPT were purchased from Seleckchem (No. S2215, BIOZOL GmbH, Eching, Germany). BB94/batimastat (BB94, No. 2961) and human recombinant GAS6 (#885-GSB-050) were purchased from R&D Systems GmbH. Cycloheximide (No. C1988) was purchased from Sigma Aldrich. All inhibitors were dissolved in DMSO (Sigma Aldrich, Taufkirchen, Germany), except CQ, and stored at room temperature in 10 mM stock solutions.
Reverse transcription-quantitative PCR (RT-qPCR)
Gene expression was analyzed following the isolation of total RNA using the RNeasy minikit (Qiagen, Hilden, Germany) and cDNA using random hexamers and Verso cDNA synthesis kit (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s instructions. RT-qPCR was performed with DyNAmo ColorFlash SYBR green qPCR kit (Thermo Fisher Scientific, Darmstadt, Germany) using a LightCycler 480 II (Roche, Mannheim, Germany). Relative gene expression levels were calculated according to the 1.9 -Δ(CT(housekeeping)-CT(gene of interest). Primer sequences: ALAS1, forward: 5′-CTGCAAAGATCTGACCCCTC-3′, reverse: 5′-CCTCATCCACGAAGGTGATT-3′, Human GAPDH, forward: 5′-ACCCAGAAGACTGTGGATGG-3′, reverse: 5′-TTCTAGACGGCAGGTCAGGT-3′, AXL P3. forward: 5′-GAGGGAGAGTTTGGAGCTGT-3′, reverse: 5′-TCATGACGTTGGGATGGTCA-3′, AXL PB, forward: 5′-CAGCTTCGGCTAGGCAG-3′, reverse: 5′-TCCGCGTAGCACTAATGTTCT-3′.
Western blot analysis
Standard mini gel electrophoresis and blotting system was used (Bio-Rad Laboratories GmbH, München, Germany). Proteins were transferred to PVDF membranes and incubated overnight at 4 °C with the primary antibody. Anti- HA-Tag (No.3724), GAPDH (No.5174), AXL (No.8661) were purchased from Cell Signalling Technonoly (New England Biolabs GmbH, Frankfurt am Main, Germany), and AXL H-3 (No.sc-166,269) from Santa Cruz Biotechnology as well as β-ACTIN from Sigma. Species corresponding fluorophore-labeled secondary antibodies (Li-Cor, Lincoln, NE) were incubated for 1 h at room temperature. The fluorescence signals were detected with an Odyssey CLx system and quantified by Image Studio software (Li-Cor). All bands were normalized as described to ACTIN or GAPDH as loading control.
Immunoprecipitation was performed as previously described . The FK-2 antibody (No. BML-PW8810–0100, Enzo Life Sciences GmbH, Lörrach, Germany) was used for ubiquitin precipitation in the concentration of 1 μg per 750 μg lysate. Subsequently, the AXL antibody (No.8661, Cell Signaling, New England Biolabs GmbH, Frankfurt am Main, Germany) was used for western blot analysis.
Cells were detached with Accutase (No. A1110501, Thermo Fisher Scientific, Darmstadt, Germany). Centrifugation was performed at 300 g for 3 min. Cells were fixed with 3.7% PFA at 37 °C for 10 min and permeabilized with − 20 °C methanol for 5 min. Primary antibody Ab 154 (MAB154, R&D Systems GmbH) or Ab 259/2 homemade antibody (clone 259/2, IgG1 isotype) was incubated for 1 h and subsequently incubated for 30 min at 37 °C with Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK). Fluorescence intensity was measured using a BD Acccuri Flow Cytometer (BD, Heidelberg, Germany).
SOMA (single oligonucleotide mutagenesis and cloning approach)
Plasmid for AXL wildtype overexpression with HA-Tag was purchased at Sino Biological (Cat: HG10279-CY). Primer for K567R mutation (GTGGCCCTGGGGAGGACTCTGGGAGAG). SOMA was performed according to Pfirrmann et al. with the following modifications: Q5-polymerase was used in combination with standard PCR protocol [30 cycles 1 min per kb elongation]. DNA was isolated using Qiagen PCR purification kit (Qiagen, Hilden, Germany) . DH5α competent E. coli were used for plasmid amplification.
Statistical data analysis
Mean values and SEM are shown. The statistical analysis was performed by the application of an two-way ANOVA in combination with Bonferroni multiple comparison post-test using GraphPad prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). Differences with *P < 0.05 were considered as statistically significant.
Increased AXL protein level after 24 h of low μM treatment with selective AXL TKI BMS777607
Increased viability of tumor spheroids upon low μM treatment with BMS777607
We performed ATP measurements after 72 h of treatment with increasing concentrations of BMS (0.197 μM up to 12.5 μM), to evaluate the impact of increased AXL protein levels on cell viability of 3D-spheroids. Hs578T, MDA-MB231, Caliper, H1792 and H292 were used for spheroid formation and subsequent ATP measurement. Three different cell viability phenotypes were observed. Hs578T cells displayed a concentration-dependent decrease of cell viability. In contrast, H1792 cells showed a concentration-dependent increase of cell viability. The third phenotype was observed in MDA-MB231, Caliper and H292 cell lines, where BMS concentrations higher than 5 μM caused reduced cell viability, whereas concentrations lower than 1 μM led to increased cell viability compared to DMSO- controls, as shown in Fig. 1d. The elevated AXL protein levels correlated with increased cell viability in MDA-MB231, Caliper, H1792 and H292 cell line which might indicate that AXL protein upregulation is part of AXL TKI resistance or feedback mechanism.
Transcription of AXL mRNA was not affected by low μM treatment with BMS777607
The major mechanism that increases protein concentrations is the transcription of coding mRNA. Therefore, we evaluated the transcription of AXL mRNA in Hs578T, H292 and H1792 cells by RT-qPCR after 0.25, 0.5, 1, 2 and 4 h of treatment with 0.5 μM BMS compared to DMSO control. We observed no significant changes of mRNA levels within 4 h of treatment with BMS (Fig. 1e-g), although a 1.3-fold increase of AXL protein levels was evident already after 3 hours of BMS treatment in H292 cells (Fig. 3e). Based on these results we conclude that low μM BMS treatment does not influence AXL mRNA level. AXL mRNA quantity did not change after 24 h as well (data not shown).
The significant increase of AXL protein levels by 0.5 μM BMS777607 treatment is depending on culture conditions
GAS6 mRNA levels were significantly different in Hs578T, H292 and H1792 cell lines. H1792 cells showed significant induction by serum deprivation
GAS6-dependent degradation of AXL was prevented by BMS777607 treatment
BMS777607 impaired GAS6-dependent internalization of AXL leading to cell surface accumulation of AXL protein
BMS777607 prevented ubiquitination of AXL after GAS6 stimulation
K567R gate keeper mutation prohibited the phosphorylation-dependent ubiquitination and subsequent internalization of AXL
BMS is an efficient AXL TKI, prohibiting AXL kinase phosphorylation [23, 25]. Finally, we investigated, if ubiquitin-dependent AXL internalization and subsequent lysosomal degradation depends on AXL kinase activity. Therefore, we introduced HA-tagged AXL gatekeeper mutants into Hs578T (Fig. 6c) and H292 cells (Fig. 6d). The K567R gate keeper mutation prohibits the binding of ATP to the hinge region of the kinase domain, which completely abolished the AXL phosphorylation. Analogous to previous experiments we analyzed the effect of 0.5 μM BMS on AXL abundance after 24 h of treatment . The fully glycosylated 140 kDa AXL is localized at the cell surface and thereby accessible to GAS6. Consequently, only this 140 kDa AXL protein can be protected from GAS6-dependent degradation by BMS (Fig. 6e). We used the 120 kDa AXL protein as a normalization control for transfection efficacy in Hs587T and H292 cells (Fig. 6c-d). We observed that BMS increased the fully glycosylated 140 kDa AXL protein abundance in wildtype AXL transfected cells in similar fashion as the K567R gate keeper mutation. BMS treatment displayed no additional impact on the 140/120 kDa ratio in K567R gate keeper mutants. Consequently, we assume that AXL kinase activity and subsequent RTK phosphorylation is necessary for ligand-dependent receptor internalization and degradation. Blocking of kinase function by BMS resulted in phosphorylation prohibition, impaired internalization and subsequent cell surface accumulation, as observed in Fig. 1a-c. By using a plasmid based overexpression system without 3’and 5’UTR regions, we additionally excluded the impact of BMS on posttranscriptional regulation by miRNAs potentially binding to AXL mRNA untranslated regions.
In summary, we postulate that phosphorylation of AXL is a prerequisite of ubiquitin-dependent internalization and lysosomal degradation, which is completely abolished by BMS. The inhibition of AXL phosphorylation subsequently prevents ubiquitination and results in the accumulation of cell surface AXL via impaired internalization after GAS6 binding.
Our study identified elevated AXL cell surface expression after 24 h of BMS777607 AXL TKI treatment. The complex AXL RTK biology requires a better understanding of the underling mechanism for successful implementation of AXL targeting therapeutics, as therapy resistance still remains the major problem for targeted therapies. Therefore, we focused on mechanisms, by which AXL protein quantity is regulated, starting with transcriptional regulation [28, 29]. AXL expression is controlled by DNA methylation, histone acetylation and transcription factors, including SP1, AP-1 and HIFα, as reviewed by Gay et al. . We excluded the impact of these transcriptional regulators on AXL protein abundance caused by BMS treatment as this would necessarily be caused by increased mRNA levels which could not be observed in our study (Fig. 1e-g). AXL expression is further regulated by post-transcriptional modifications. In dendritic cells, AXL expression is abundant, and in bone marrow-derived macrophages, AXL expression is minimal; however, there is essentially no difference in AXL mRNA copy number in these cells, suggesting a significant role for post-transcriptional or post-translational regulatory mechanisms of AXL expression . Other studies have identified target sequences for microRNA (miRs) including miR-34 and miR-199a/b in the AXL 3′ untranslated region, with correlative findings confirming the effects of miRs on AXL expression [31, 32]. In our study we show that low μM BMS treatment does not influence AXL mRNA level. This was interesting, as we observed a minor increase of AXL protein levels already after 3 hours of BMS treatment in H292 cells (Fig. 3e-f). Using a plasmid based overexpression system without 3′ and 5′ UTR regions we exclude the impact of BMS on posttranscriptional regulation by miRNAs, potentially binding to AXL untranslated mRNA regions (Fig. 6c-d). Alternative splicing events have not been analyzed in this study, although three different splicing variants were described for the axl gene . We haven’t observed a dramatic shift in AXL protein in western blot analysis after BMS treatment, but we cannot exclude a splice variant shift on mRNA level. Translational regulation of oncogenes play an important role in carcinogenesis . Emerging evidence indicates that AXL expression may also be regulated at the translational level. A critical protein for translation initiation is eIF4E, which binds to the 5′ m7G cap of mRNA molecules and thus facilitates ribosomal recruitment . In preliminary experiments we saw a slight, but significant, increase of eIF4B S422 phosphorylation in MDA-MB231 cells and increased ribosome-bound nascent chain puromycinylation in Hs578T and H292 cells after 24 h of low μM BMS treatment (data not shown). In contrast to that result we failed to validate a significant impact on translation by polysome-fractionation and subsequent RT-qPCR of bound AXL mRNA. Therefore, we cannot draw a clear picture, whether the translational machinery is significantly affected by BMS treatment leading to enhanced AXL protein enrichment. To become a functionally mature protein, important posttranscriptional modifications, including glycosylation and signal peptide cleavage, need to occur. Another type of AXL cleavage is commonly referred to as ‘ectodomain shedding’, in which the extracellular domain is cleaved from the cell membrane through actions of various matrix metalloproteinases and A Disintegrin and Metalloproteinase Domain (ADAM) family members, e.g. ADAM10 and ADAM17 . We analyzed the impact of α-secretases and γ-secretases by combinatorial treatments of BMS or BB94 together with DAPT. BB94 blocks α-secretase activity and DAPT is a known inhibitor of γ –secretases. When taking reduction of receptor ectodomain shedding as a potential mechanism for 140 kDa AXL cell surface accumulation, then α-secretases have to be inhibited. Blocking of γ-secretases by DAPT treatment leads to the stabilization of the 55 kDa C-terminal fragment of AXL and causes no accumulation of the 140 kDa AXL protein. We could not prove an impact of BMS on the activity of α- secretases as shown in Fig. 4. Glycosylation is essential for maturation and function of membrane proteins regulating their routing, conformation and ligand binding. For example, inhibition of glycosylation sensitizes cancer cells that are resistant to EGFR targeted therapy to radiation. Tunicamycin inhibits N-acetylglucosamine (GlcNAc) transferase, which catalyzes the first step of protein N-glycosylation in the endoplasmic reticulum. Krishnamoorthy et al., 2013 could show that tunicamycin treatment of CAL62 cells led to AXL protein accumulation as a 100 Da protein in western blot, representing the core polypeptide, whereas the 140 and 120 kDa bands disappeared, indicating that both were N-glycosylated isoforms of AXL . We have not focused on this aspect of posttranslational modification in the present study, but it is unlikely that glycosylation is impaired by BMS treatment as we could even observe an increase of the fully glycosylated 140 kD. Only exogenous over-expression of Ha-tagged-AXL showed the appearance of a potential non-glycosylated AXL protein at 110 kDa in western blot. Neither the 110 kDa band nor the 120 kDa band was significantly regulated by BMS treatment in contrast to the fully glycosylated 140 kDa band (Fig. 6e). This glycosylation of the endogenous or exogenous 140 kDa AXL protein is necessary for ligand accessibility and subsequent full activation of the kinase function . Like for other RTKs, phosphorylation plays an essential role governing the activity and the fate of AXL. Upon GAS6 binding, AXL undergoes dimerization and Y698, Y702 and Y703 phosphorylation. Subsequently, three adjacent residues (Y779 and 821 and Y866) are phosphorylated, representing active docking sites for signal transduction. ATP binding is prohibited by a K567R gate keeper mutation resulting in an inactive kinase domain with loss of binding capability to adaptor proteins, like PI3K subunit p85 [16, 36]. Upon GAS6 binding AXL is activated, internalized and sorted to endosomes through endocytosis, which is clathrin- and dynamin-dependent. From endosomes, AXL can either be transported to the plasma membrane or delivered to lysosomes for degradation [37, 38]. We used chloroquine as a lysosomotropic agent that prevents endosomal acidification leading to inhibition of lysosomal enzymes. We could not show an additional impact by inhibition of lysosomal degradation with chloroquine on the abundance of AXL after BMS treatment (Fig. 4). AXL is also subject to ubiquitin-mediated proteasomal degradation. AXL degradation by proteasomes has been well demonstrated by Paolino et al.. Castias B-lineage lymphoma (CBL) is an E3 ubiquitin ligase, responsible for TAM family ubiquitination and degradation . We could show in preliminary experiments that inhibition of the proteasom by MG132 does not lead to an accumulation of the 140 kDa AXL protein (data not shown). Phosphorylation of AXL tyrosine residues creates a docking site for recruiting CBL . Alternatively, CBL may be recruited to AXL through its interaction with the adaptor protein GRB2 as shown for MET . GRB2 is also implicated in the clathrin-mediated endocytosis of activated EGFR  and Pao-Chun et al. showed that GRB2 is binding to AXL after GAS6 ligand stimulation . A similar GRB2/CBL-dependent internalization mechanism may exist for AXL as well, taking into account that Y821 is an essential tyrosine for binding of GRB2 . AXL small molecule inhibitors prohibit receptor phosphorylation and prevent the binding of adaptor proteins. Among these, CBL leads to receptor downregulation by ubiquitination and successive degradation in the lysosomes. The activation-dependent downregulation is important for keeping a steady-state between active and non-active AXL modes. Here we demonstrated that kinase inhibition hinders the AXL activation-dependent downregulation. As a consequence, the AXL accumulates on the cell surface, where it potentially could be stimulated, once the administration of the inhibitor is stopped. Based on our data, we propose that AXL TKIs inhibit the phosphorylation on the CBL binding site, disrupting the interaction between AXL and CBL and thus restricting ubiquitination of AXL, which subsequently cannot be internalized and degraded in the lysosomes. Proper RTK downregulation is a crucial step of activity regulation, and if not tightly controlled, may cause oncogenic events. ATP binding of AXL is prohibited by a K567R gate keeper mutation resulting in an inactive kinase domain and shows analogous, but not additional effects like BMS treatment on AXL abundance (Fig. 6c-e). Here we report that impairment of downregulation takes place upon inhibiting the activity of AXL by TKIs. Although in the current case the receptor accumulates initially in an inactive state, enhanced phosphorylation of AXL might take place as well within 24–36 h, as shown for BMS by Baumann et al. and for R428 by Chen et al. [38, 42]. This might be relevant in vivo, where concentration gradients of drugs are common in tumors. Our findings could possibly translate into appalling consequences once the inhibitor drops below the inhibitory concentration in some cells within the tumor mass. To our knowledge, this is the first report to describe that targeting of the AXL by small molecule inhibitors leads to its cell surface accumulation by potential interference with a downregulation-associated mechanism. This phenomenon might represent clinically relevant aspect to be considered when following AXL inhibition in clinical setups. Additionally, the release of the soluble 85 kDa N-terminal fragment (sAXL) might be used as biomarker and campaign diagnostic tool as shown for early hepatocellular carcinoma (HCC) and malignant peripheral nerve sheath tumors (MPNST) [20, 21]. We demonstrated that the 55 kDa C-terminal fragment, which is generated as the sAXL is shedded by ADAM10 and 17, was accumulating after BMS treatment in Hs578T (Fig. 4). Consequently, sAXL might be increased in serum of AXL TKI treated patents . Beside kinase inhibition with TKIs or decoy receptors also receptor downregulation is a common mode of action for AXL targeting therapies. This was shown for antagonistic monoclonal antibody targeting AXL , 17-Allylamino-17-demethoxygeldanamycin  and yuanhuadine . As a linkage of kinase inhibition and receptor degradation, proteolysis targeting chimera (PROTAC) technology could be used as a powerful tool for AXL targeting therapy. Burselm et al. could demonstrate that PROTACs are capable of inducing the degradation of active EGFR, HER2, and c-Met. In most cases, PROTACs capable of degradation inhibit downstream signaling and cell proliferation at lower concentrations than similar TKIs without linked degradation machinery recruiting unit. Furthermore, degradation provides a more sustained reduction in signaling, as evidenced by the reduction in kinome rewiring, as observed previously with EGFR, HER2, and c-Met inhibitors, as well as the sustained duration of response even after washout . Based on our study, we would suggest a PROTAC-based strategy for AXL inhibition to achieve a sustained inhibition and depletion of AXL. This might enhance the efficacy of targeted AXL therapies in the clinics.
AXL tyrosine kinase inhibitors (TKIs) are currently under clinical evaluation. We observed by Western blot and flow cytometry analysis that AXL TKI BMS increases AXL protein levels after 24 h of treatment. We demonstrate that AXL kinase activity and subsequent RTK phosphorylation is necessary for GAS6-dependent receptor internalization and degradation. Blocking of kinase function by BMS results in phosphorylation prohibition, impaired internalization and subsequent cell surface accumulation. Our data suggest careful consideration of anti-AXL clinical protocols because feedback loops and resistance formation might countervail targeted AXL therapy. An alternative strategy to circumvent feedback loops for AXL targeting therapies may exist in linkage of AXL TKIs to a degradation machinery recruiting unit (PROTACs). This might result in a sustained inhibition and depletion of the AXL from tumor cell surface and enhance the efficacy of targeted anti-AXL therapies in the clinics.
We thank Dres. Singh, AK., Neuhaus, H. and Holstein, I. for critical reading of the manuscript.
This study was designed by T.R. W.A and L.M performed the experiments; T.R and L.M analyzed and interpreted the data; T.R and L.M wrote the manuscript. All authors read and approved the final manuscript.
HaPKoM, University Halle-Wittenberg, Halle (Saale), Germany.
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Consent for publication
The authors declare that they have no competing interests.
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