Axl Signaling Research Articles

Abstract MP27: Human Hypertension And Endothelial Cell Activation Promote The Formation And Activation Of Axl + Siglec-6 + Dendritic Cells Via Endothelial Release Of Growth Arrest Specific 6

We have shown that dendritic cells (DCs) from hypertensive mice convey hypertension when adoptively transferred to recipients. Recently a novel subset of DCs in humans that express Axl and Sigelc-6 + (AS DCs) have been identified which drive T cell proliferation and produce IL-1β, IL-6 and IL-23, consistent with DCs we have observed in hypertension. We hypothesized that AS cells are increased in hypertension and contribute to immune activation in this disease. We quantified circulating AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a more than 2-fold increase in circulating AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml; p =0.0304). To investigate the mechanism by which AS DCs are formed in hypertension, we co-cultured human aortic endothelial cells (HAECs) undergoing either normotensive (5%) or hypertensive (10%) cyclical stretch for 48 hours with CD14 + monocytes from normotensive donors. Co-culture of monocytes with HAECs exposed to 10% stretch significantly increased AS DCs and AS DC IL-1β production when compared to 5% stretch alone as assessed by flow cytometry (21 ± 5 vs. 131 ± 32 IL-1β + AS DCs). Moreover, inhibition of Axl signaling with R248, completely abolished the production of IL-1β in AS DCs (34 ± 8 IL-1β + AS DCs). In additional experiments we found that 10% stretch caused a 50% increase in release of growth arrest 6 (GAS6), the ligand for Axl, from HAECs compared to 5% stretch. Treatment of human monocytes with GAS6 mimicked the effect of 10% stretch in promoting AS cell formation and IL-1β production. Based on the increased secretion of GAS6 from HAECs, we used a J-wire to harvest human endothelial cells from 23 additional volunteers to assess endothelial cell activation and GAS6 secretion in vivo. We found a positive association between pulse pressure and plasma GAS6 (R 2 =0.25, p =0.0079) and a striking positive association between GAS6 and ICAM-1 (R 2 =0.39, p =0.0012). These data show that secretion of GAS6 by an activated endothelial seems to promote the formation and activation of AS DCs. Thus, the interplay between endothelial-derived GAS6 and AS DCs seem to be an important mechanism in human hypertension and might be a novel therapeutic target for this disease.

AXL Mediates Cetuximab and Radiation Resistance Through Tyrosine 821 and the c-ABL Kinase Pathway in Head and Neck Cancer.

Radiation and cetuximab are therapeutics used in management of head and neck squamous cell carcinoma (HNSCC). Despite clinical success with these modalities, development of both intrinsic and acquired resistance is an emerging problem in the management of this disease. The purpose of this study was to investigate signaling of the receptor tyrosine kinase AXL in resistance to radiation and cetuximab treatment. To study AXL signaling in the context of treatment-resistant HNSCC, we used patient-derived xenografts (PDXs) implanted into mice and evaluated the tumor response to AXL inhibition in combination with cetuximab or radiation treatment. To identify molecular mechanisms of how AXL signaling leads to resistance, three tyrosine residues of AXL (Y779, Y821, Y866) were mutated and examined for their sensitivity to cetuximab and/or radiation. Furthermore, reverse phase protein array (RPPA) was employed to analyze the proteomic architecture of signaling pathways in these genetically altered cell lines. Treatment of cetuximab- and radiation-resistant PDXs with AXL inhibitor R428 was sufficient to overcome resistance. RPPA analysis revealed that such resistance emanates from signaling of tyrosine 821 of AXL via the tyrosine kinase c-ABL. In addition, inhibition of c-ABL signaling resensitized cells and tumors to cetuximab or radiotherapy even leading to complete tumor regression without recurrence in head and neck cancer models. Collectively, the studies presented herein suggest that tyrosine 821 of AXL mediates resistance to cetuximab by activation of c-ABL kinase in HNSCC and that targeting of both EGFR and c-ABL leads to a robust antitumor response.

Abstract 6114: Host and neoplastic cells utilize Axl for breast cancer bone metastasis

Abstract Breast cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related death for women in the United States. Approximately 90% of cancer-related death results from metastasis, and the bone is the most common site of metastasis for breast cancer. Upregulation of signaling pathways can promote the metastatic phenotypes of neoplastic cells, including cell migration, invasion, proliferation and angiogenesis. In addition, when breast cancer cells disseminate to the bone, neoplastic cells upregulate signaling pathways to activate osteoclast-induced bone resorption. A key pathway involved in metastatic spread of cancer cells is the receptor tyrosine kinase Axl. Genetic and pharmacologic Axl inhibition in both human and rodent breast cancer cell lines (MDA-MB-231 and 4T1) impaired tumor cell migration and invasion. Intracardiac inoculation of Axl knockdown (KD) MDA-MB-231 cells significantly decreased the number of bone metastases compared to the scrambled control MDA-MB-231 cells. Another mechanism by which Axl inhibition impaired bone metastasis is through the modulation of osteoclast maturation and activity. Co-culture of Axl KD breast cancer cells with murine osteoclast precursor cells (Raw264.7) significantly reduced the number of tumor cell-induced osteoclast resorption pits on bone slices. In addition, genetic and pharmacologic inhibition of Axl in Raw264.7 cells as well as bone marrow derived osteoclast precursor cells, significantly reduced osteoclast formation and activity. Scrambled control and Axl KD Raw264.7 cells were cultured in serum free medium for 24 hours, and their conditioned media were collected. The conditioned media of Axl KD Raw264.7 cells revealed significantly decreased levels of monocyte chemoattractant protein 1 (MCP-1), which known to promote osteoclastogenesis, compared to the scrambled control Raw264.7 cells. In summary, we have demonstrated that inhibition of Axl decreases breast cancer metastasis to the bone through modulation of the host and neoplastic cell functions. Collectively, these data indicate that Axl signaling may be a promising therapeutic target to impair tumor progression and metastasis. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cells utilize Axl for breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6114.

Abstract 6107: Stromal and immune changes in the aged lung microenvironment create a permissive niche for the metastatic outgrowth of melanoma

Abstract Our previous data has indicated that while tumors grow more slowly in aged mouse skin, they form more lung metastases. We queried whether the lung microenvironment undergoes age-related changes that provide a permissive niche for metastatic outgrowth. Our previous data has implicated dermal fibroblasts as the drivers of age-related changes in the skin, and so we analyzed young vs aged healthy human lung fibroblasts. The secreted changes in aged lung fibroblasts suggest that the lung microenvironment evolves during aging to promote proliferation of melanoma cells. Specifically, these changes involve the secretion of factors such as sFRP1, that we find suppresses Wnt5A and AXL signaling, which typically drives a metastatic but slow-growing/dormant cancer phenotype. Interestingly, this switch promotes activation of the MER tyrosine kinase pathway, increasing proliferation. This switch is reinforced by the MER ligand PROS1, which is upregulated in the aged lung microenvironment. Consistent with this, our in vivo studies indicate that tumor cells disseminate to the lungs at the same rate in young and aged mice. However, they persist as single cell populations in the young mouse lungs, whereas they grow out rapidly in the aged mouse lung. We next queried whether the immune microenvironment also played a role in the differential outgrowth of lung metastases in young and aged mice. We find that aged lung fibroblasts secrete higher levels of immunosuppressive factors ARG-1 and CXCL1, which are both necessary for efficient metastatic outgrowth in vivo. Analysis of the aged lung microenvironment reveals that immunosuppressive MDSCs and Treg populations are significantly increased compared with the young lung, while effector CD4 and CD8 T cells are decreased. Importantly, depletion of MDSCs or Tregs in the aged lung both significantly decrease metastasis. Overall, we find that aged-induced changes in fibroblast secretory phenotypes and immune subpopulations promote efficient metastatic melanoma progression in the lung. Citation Format: Mitchell Fane, Stephen Douglass, Gretchen Alicea, Marie Webster, Julio Aguirre-Ghiso, Ashani Weeraratna. Stromal and immune changes in the aged lung microenvironment create a permissive niche for the metastatic outgrowth of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6107.

Abstract 3051: A potent pyrimidinone Axl inhibitor DW17401

Abstract Aberrant signaling of Axl frequently occurs in many cancer types. Axl activation has been identified as a key trigger in epithelial to mesenchymal transition (EMT), which accounts for acquired drug resistance to many tyrosine kinase inhibitor therapies. It also has been revealed that Axl signaling is associated with immunosuppression. Therefore, Axl inhibitors may present a promising strategy for cancer therapeutics. We have designed and synthesized a series of pyrimidinone Axl inhibitors, and DW17401 was identified as a highly potent, orally available inhibitor against Axl, with IC50 values of 0.9 nM. DW17401 exhibited significant inhibitory effect against Axl driven cell proliferation, with an IC50 value of less than 0.02 nM for BaF3/TEL-AXL cell line. Oral dosing of DW17401 25 mg/kg (QD) resulted in complete tumor regressions in subcutaneous transplanted model of BaF3/TEL-AXL in nude mice. In addition, DW17401 could also inhibited c-Met kinase and c-Met-mediated tumor cell proliferation and growth with high potency, indicated it therapeutic potential in MET aberrant cancer. Of note, DW17401 presented favorable pharmacokinetic parameters on SD rat with a half-life time of 13.8 h and a plasma exposure value of 32647 ng.h/mL (p.o. at 3 mg/kg). All these data indicated that DW17401 was an effective promising Axl inhibitor for cancer treatment. Citation Format: Hefeng Zhang, Xia Peng, Yinchun Ji, Yang Dai, Yanyan Shen, Jing Ai, Wenhu Duan. A potent pyrimidinone Axl inhibitor DW17401 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3051.

Abstract 3052: Targeting AXL in PIK3R2-amplified ovarian cancer

Abstract The p85 regulatory subunit and its associated p110 catalytic subunit are components of the class IA PI3K that mediates the downstream signal of receptor tyrosine kinases. PIK3R2 (encoding p85β, one of the p85 isoforms) is commonly amplified in ovarian cancer patients. Importantly, high PIK3R2 expression significantly correlated with worse patient survival. Here, we showed that p85β promoted tumorigenic properties of ovarian cancer cells. The protein expression of a receptor tyrosine kinase AXL was positively regulated by p85β. AXL is a member of the TAM receptor family. The regulation was specific to AXL because the other two members (Tyro3 and MERTK) were not altered by p85β. Drug sensitivity assays suggested that inhibition of AXL reduced oncogenic activities of p85β in vitro and in vivo. Together, we propose p85β as a potential therapeutic target in ovarian cancer and therapeutic blockade of AXL signaling may be an effective strategy for ovarian cancer with PIK3R2 amplification. Citation Format: Ling Rao, Victor C. Mak, Yuan Zhou, Dong Zhang, Xinran Li, Yiling Lu, Gordon B. Mills, Lydia W. Cheung. Targeting AXL in PIK3R2-amplified ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3052.

Abstract 4711: Novel mechanism involved in the regulation ofoncogenic Axl receptor tyrosine kinase in cancer

Abstract Background: Oncogenic receptor tyrosine kinase Axl is overexpressed and plays an important role in multiple human cancers. However the mechanisms of Axl overexpression in cancer remain unclear. This study is to investigate the mechanisms that regulate Axl expression in pancreatic cancer. Experimental procedures: The interactions between HPK1, Axl and c-Cbl were examined using co-immunoprecipitation and immunoblotting. Inhibitors of endocytic pathway and immunofluorescence were used to examine the underlying mechanisms of HPK1-mediated Axl degradation. The functional significance of HPK1-mediated Axl degradation in the invasive capability of pancreatic ductal adenocarcinoma (PDAC) cells and it downstream signaling pathways were examined using HPK1 stable PDAC cells. Immunohistochemistry was used to examine the in vivo correlation between HPK1 and Axl in human pancreatic intraepithelial neoplasia. The RNA sequencing data of HPK1 and Axl expression and survival data of 176 pancreatic cancer patients in the Cancer Genome Atlas (TCGA) database were downloaded from the Human Protein Atlas (https://www.proteinatlas.org). The expression of HPK1 and Axl was categorized as low or high using the cutoff values set by TCGA (3.42 for HPK1 and 14.09 for Axl). Survival analysis were performed using the Kaplan-Meier method and the log-rank test was used to evaluate the statistical significance of differences. Results: We identified Axl as a novel HPK1-interacting protein and demonstrated for the first time that HPK1 down-regulated Axl and decreased the half-life of Axl protein. HPK1-mediated Axl degradation was inhibited by leupeptin, baflomycin A1 and monensin which inhibit endocytic pathway. HPK1 accelerates the movement of Axl from the plasma membrane onto endosomes in pancreatic cancer cells after treated with Gas6. HPK1 increased the binding of Axl to c-Cbl, promoted Axl ubiquitination, decreased Axl signaling including phospho-Akt and phospho-Erk, and decreased the invasion capability of pancreatic cancer. More importantly, we showed that Axl expression inversely correlated with HPK1 expression in human pancreatic intraepithelial neoplasia (P=0.005). Low expression of HPK1 and high expression of Axl correlated significantly with poor survival in patients with PDAC (P<0.001). Conclusions: Our results suggest a novel tumor suppressor mechanism of HPK1 in which it targets Axl for degradation via the endocytic pathway. Loss of HPK1 may contribute to Axl overexpression and enhance its downstream signaling and tumor invasion in pancreatic cancer. Citation Format: Hua Wang, Xianzhou Song, Hironari Akasaka, Reza Abbasgholizadeh, Ji-Hyun Shin, Craig D. Logsdon, Anirban Maitra, Andrew J. Bean, Huamin Wang. Novel mechanism involved in the regulation ofoncogenic Axl receptor tyrosine kinase in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4711.

Abstract B29: AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells

Abstract Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer (TAM) family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in NSCLC and its expression correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogated the effects of the AXL inhibitor, TP-0903, on EMT in NSCLC cells using transcriptomic and proteomic profiling. Methods: Atomic force microscopy, Western blot analysis, RNA sequencing, and mass cytometry (CyTOF) were used to evaluate the phenotypic, transcriptomic, and proteomic profiles of A549 cells treated with 40 nM TP-0903 or shAXL knockdown. A549 and H1650 NSCLC xenograft models were used to explore the consequences of AXL inhibition in vivo. Results: As expected, TP-0903 treatment attenuated AXL signaling and downstream phosphorylation in the A549 cells. Interestingly, the treatment also reduced gene expression responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1, and TAZ. Consistent with AXL inhibition, TP-0903 reversed the mesenchymal phenotype in A549 and H2009 cell lines and decreased their migratory potential in culture. The CyTOF analysis on TP-0903-treated cells identified resistant clones overexpressing TGFβ receptor II (TGFBR2) and TAZ proteins and displaying hybrid EMT phenotypes. TP-0903 was also active in suppressing A549 or H1650 tumor growth in vivo. Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 has excellent therapeutic promise in NSCLC and we speculate that TP-0903 can target mesenchymal transitional states in NSCLC, possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis. Citation Format: Josephine A. Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel A. Osmulski, Maria E. Gaczynska, Chiou-Miin Wang, Nicholas D. Lucio, Meizhen Chen, Chih-Wei Chou, Alia Nazarullah, Shellye R. Lampkin, Lianquin Qiu, David J. Bearss, Steve Warner, Lars Mouritsen, Mark Wade, Daniel DeArmond, Ruben Mesa, Nameer Kirma, Tim H.-M. Huang. AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B29.

AXL confers cell migration and invasion by hijacking a PEAK1-regulated focal adhesion protein network

Aberrant expression of receptor tyrosine kinase AXL is linked to metastasis. AXL can be activated by its ligand GAS6 or by other kinases, but the signaling pathways conferring its metastatic activity are unknown. Here, we define the AXL-regulated phosphoproteome in breast cancer cells. We reveal that AXL stimulates the phosphorylation of a network of focal adhesion (FA) proteins, culminating in faster FA disassembly. Mechanistically, AXL phosphorylates NEDD9, leading to its binding to CRKII which in turn associates with and orchestrates the phosphorylation of the pseudo-kinase PEAK1. We find that PEAK1 is in complex with the tyrosine kinase CSK to mediate the phosphorylation of PAXILLIN. Uncoupling of PEAK1 from AXL signaling decreases metastasis in vivo, but not tumor growth. Our results uncover a contribution of AXL signaling to FA dynamics, reveal a long sought-after mechanism underlying AXL metastatic activity, and identify PEAK1 as a therapeutic target in AXL positive tumors.

AXL Is a Potential Target for the Treatment of Intestinal Fibrosis.

Fibrosis is the final common pathway to intestinal failure in Crohn's disease, but no medical therapies exist to treat intestinal fibrosis. Activated myofibroblasts are key effector cells of fibrosis in multiple organ systems, including the intestine. AXL is a receptor tyrosine kinase that has been implicated in fibrogenic pathways involving myofibroblast activation. We aimed to investigate the AXL pathway as a potential target for the treatment of intestinal fibrosis. To establish proof of concept, we first analyzed AXL gene expression in 2 in vivo models of intestinal fibrosis and 3 in vitro models of intestinal fibrosis. We then tested whether pharmacological inhibition of AXL signaling could reduce fibrogenesis in 3 in vitro models of intestinal fibrosis. In vitro testing included 2 distinct cell culture models of intestinal fibrosis (matrix stiffness and TGF-β1 treatment) and a human intestinal organoid model using TGF-β1 cytokine stimulation. Our findings suggest that the AXL pathway is induced in models of intestinal fibrosis. We demonstrate that inhibition of AXL signaling with the small molecule inhibitor BGB324 abrogates both matrix-stiffness and transforming growth factor beta (TGF-β1)-induced fibrogenesis in human colonic myofibroblasts. AXL inhibition with BGB324 sensitizes myofibroblasts to apoptosis. Finally, AXL inhibition with BGB324 blocks TGF-β1-induced fibrogenic gene and protein expression in human intestinal organoids. The AXL pathway is active in multiple models of intestinal fibrosis. In vitro experiments suggest that inhibiting AXL signaling could represent a novel approach to antifibrotic therapy for intestinal fibrosis such as in Crohn's disease.

Synthetic Lethal and Resistance Interactions with BET Bromodomain Inhibitors in Triple-Negative Breast Cancer

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

P85\u03b2 regulates autophagic degradation of AXL to activate oncogenic signaling

PIK3R2 encodes the p85β regulatory subunit of phosphatidylinositol 3-kinase and is frequently amplified in cancers. The signaling mechanism and therapeutic implication of p85β are poorly understood. Here we report that p85β upregulates the protein level of the receptor tyrosine kinase AXL to induce oncogenic signaling in ovarian cancer. p85β activates p110 activity and AKT-independent PDK1/SGK3 signaling to promote tumorigenic phenotypes, which are all abolished upon inhibition of AXL. At the molecular level, p85β alters the phosphorylation of TRIM2 (an E3 ligase) and optineurin (an autophagy receptor), which mediate the selective regulation of AXL by p85β, thereby disrupting the autophagic degradation of the AXL protein. Therapeutically, p85β expression renders ovarian cancer cells vulnerable to inhibitors of AXL, p110, or PDK1. Conversely, p85β-depleted cells are less sensitive to these inhibitors. Together, our findings provide a rationale for pharmacological blockade of the AXL signaling axis in PIK3R2-amplified ovarian cancer.

Targeting AXL-JAK1 in tumor and immune ecosystem of lung cancer

Abstract Intercellular crosstalk between lung adenocarcinoma cells (LACs) and tumor-associated macrophages (TAMs) has been implicated in promoting cancer metastasis; however, how oncogenic signaling networks corroborate in this tumorigenic process remains unexplored. Our findings suggest that coordinated activation of AXL and JAK1-pSTAT3 signaling engenders a vicious cycle of dependence between LACs and TAMs for tumor progression. AXL-overexpressing LACs polarize TAMs to an M2-like phenotype in a JAK1-STAT3-dependent manner, facilitating the development of a proinflammatory tumor microenvironment. Conversely, M2-like TAMs release Gas6 ligand to enhance AXL-mediated cancer stemness and epithelial-to-mesenchymal transition (EMT) in LACs. Cytometry by time-of-flight (CyTOF) is a single-cell detection technology that allows for measurement of multiple protein markers in diverse cell subpopulations. We designed a 34-antibody CyTOF panel to identify immune cells, stromal cells, epithelial cells, oncogenic signaling, stemness and EMT components for LAC tumor profiling. CyTOF profiling of ~642,241 single cells detected 55 subpopulations among LAC cell lines (n=4 with co-culture conditions) and lung tumors (n=12), which were further differentiated based on their cell types (LACs, TAMs, stromal cells, granulocytes, natural killer, B and T lymphocytes). High AXL expression was noticed in tumor cell subpopulation while high pSTAT3 expression was detected in the TAM subpopulation. Moreover, pSTAT3 expression levels were increased in U937 differentiated macrophage after co-culture with A549 cells. This novel platform may facilitate the optimal selection of lung cancer patients for dual targeting of AXL and JAK1-pSTAT3.

Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFβ, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Kinetic and Mechanistic Characterization of a Potent and Selective Inhibitor for Human AXL Receptor Tyrosine Kinase

The AXL receptor tyrosine kinase (AXL) is a member of the TAM RTK family comprising TYRO3, AXL and MER. AXL signaling is activated by binding of the growth arrest‐specific protein 6 (Gas6) or vitamin K‐dependent protein S (PROS1), which subsequently induces homodimerization and autophosphorylation, thereby initiating a conventional RTK signaling pathway. Maximal stimulation requires an extracellular lipid, phosphatidylserine (PtdSer).Activation of AXL regulates an array of signal transduction pathways, including those mediating cell survival, proliferation and migration, as well as promoting an immunosuppressive phenotype within the tumor microenvironment 1, 2. AXL has been shown to be highly expressed in sarcoma, renal cell carcinoma, pancreatic adenocarcinoma and other types of tumors, correlative with poor prognosis in cancer patients. Furthermore, AXL has been shown to facilitate both intrinsic and acquired resistance to multiple anti‐cancer drugs 3, suggesting therapeutic targeting of AXL may be an effective anti‐cancer strategy.We have developed a potent, reversible and competitive small molecule AXL inhibitor. This inhibitor (Compound 1) inhibits the activity of recombinant human AXL kinase domain with nanomolar potency, as determined by detection of phosphorylated substrate using homogeneous time‐resolved fluorescence (HTRF). Compound 1 is also highly selective against TYRO3, MERTK and MET kinase domains. The compound binds to the intracellular AXL kinase domain with a nanomolar dissociation rate constant measured by monitoring displacement of a competitive fluorescent tracer using bioluminescence resonance energy transfer (BRET). In order to assess the inhibitory effect of the compound on downstream signal transduction, AXL autophosphorylation‐induced SH2 translocation was determined using an AXL PathHunter® assay and phosphorylation of AKT Ser473 induced by AXL activation was also assessed by AlphaLISA.Support or Funding Information

LncRNA XIST modulates HIF-1A/AXL signaling pathway by inhibiting miR-93-5p in colorectal cancer.

BackgroundLong noncoding RNA (LncRNA) XIST is one of the genes that exists in different types of cancers. Earlier researches showed that XIST can advance the progression of colorectal cancer. Nevertheless, the potential molecular mechanism of XIST in combination with miR‐93‐5p has not been explored in colorectal cancer.MethodsWe performed qRT‐PCR to explore the level of XIST. And a serious experiments in vitro and in vivo were performed to explore the function of XIST. The relationship between XIST/HIF‐1A and miR‐93‐5p was confirmed by RIP and dual‐luciferase assays.ResultsIn the present research, our team demonstrated the upregulation of XIST expression, which was related to tumor progression, and the downregulation of miR‐93‐5p in cells and tissues of colorectal cancer. XIST is the competitive endogenous RNA of miR‐93‐5p to promote HIF‐1A, and then the upregulated AXL level facilitates the EMT process, migration, and proliferation of colorectal cancer. At last, we proved that XIST enhanced the in vivo and in vitro activities of colorectal cancer by regulating AXL signaling.ConclusionIn summary, the above results indicate that XIST promotes colorectal cancer tumorigenesis by regulating miR‐93‐5p/HIF‐1A/AXL signaling pathway, which will supply a novel perspective to diagnose and treat colorectal cancer disease.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

Introduction:Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC.Methods:We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC.Results:We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018).Conclusion:Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

Cabozantinib inhibits AXL- and MET-dependent cancer cell migration induced by growth-arrest-specific 6 and hepatocyte growth factor

Cabozantinib is known as an inhibitor of receptor tyrosine kinases mainly targeting AXL receptor tyrosine kinase (AXL), MET proto-oncogene-encoded receptor tyrosine kinase (MET), and vascular endothelial growth factor receptor 2. Growth arrest-specific 6 (GAS6) and hepatocyte growth factor (HGF), the natural ligands of AXL and MET, respectively, are associated with the induction of cancer cell proliferation or metastasis. Currently, it is still unclear how cabozantinib regulates cancer cell migration and invasion by inhibiting AXL and MET. This study was conducted to investigate the mechanism underlying the anti-cancer effects of cabozantinib through regulation of AXL and MET signaling.The results of Boyden chamber assays showed that cancer cell migration was induced by GAS6 and HGF in SKOV3 cells in serum-free medium. Combinatorial treatment with GAS6 and HGF exerted an additive effect on cell migration. Furthermore, we examined the role of AXL and MET signaling in cell migration. Short interfering RNA targeting AXL and MET inhibited GAS6- and HGF-induced migration, respectively. Double knockdown of AXL and MET completely suppressed cell migration induced by combination treatment with GAS6 and HGF compared to AXL or MET inhibition alone. Finally, we investigated the effects of cabozantinib on cell migration and invasion. Cabozantinib inhibited AXL and MET phosphorylation and downregulated the downstream mediators, phosphorylated SRC in the presence of both GAS6 and HGF in SKOV3 cells. The cell migration and invasion induced by combined GAS6 and HGF treatment were suppressed by cabozantinib, but not by capmatinib, a selective MET inhibitor.Our data indicate that the GAS6-AXL and HGF-MET signal pathways markedly contribute to cancer cell migration and invasion in an independent manner, suggesting that simultaneous inhibition of these two pathways contributes to the anti-cancer effects of cabozantinib.

AXL signaling in primary sensory neurons contributes to chronic compression of dorsal root ganglion-induced neuropathic pain in rats.

Low back pain is a chronic, highly prevalent, and hard-to-treat condition in the elderly. Clinical studies indicate that AXL, which belongs to the tyrosine kinase receptor subfamily, mediates pathological pain. However, it is not clear exactly how AXL regulates pain behaviors. In this study, we used a model of chronic compression of dorsal root ganglion-induced neuropathic pain to recreate clinical intervertebral foramen stenosis and related lumbocrural pain to explore whether AXL in primary sensory neurons contributes to this neuropathic pain in rats. Using double-labeling immunofluorescence, we observed that both phosphorylated AXL and AXL were localized primarily on isolectin B4-positive and calcitonin gene-related peptide-positive neurons, while AXL was also localized in neurofilament-200-positive neurons. Chronic compression of dorsal root ganglion-induced pain was associated with the upregulation of AXL mRNA and protein in injured dorsal root ganglia. Repeated intrathecal administration of the AXL inhibitor, TP0903, or the AXL small interfering RNA effectively alleviated chronic compression of dorsal root ganglion-induced pain hypersensitivities. Moreover, repeated intrathecal administration of either TP0903 or AXL small interfering RNA reduced the expression of mammalian target of rapamycin in injured dorsal root ganglia, suggesting that mammalian target of rapamycin may mediate AXL’s actions. These results indicate that the upregulation of dorsal root ganglion AXL may be part of a peripheral mechanism of neuropathic pain via an intracellular mammalian target of rapamycin-signaling pathway. Thus, while AXL inhibitors have so far primarily shown clinical efficacy in tumor treatment, AXL intervention could also serve as a potential target for the treatment of neuropathic pain.

Commentary Referring to Pericyte FAK Negatively Regulates Gas6/ Axl Signalling To Suppress Tumour Angiogenesis and Tumour Growth

Commentary Referring to Pericyte FAK Negatively Regulates Gas6/ Axl Signalling To Suppress Tumour Angiogenesis and Tumour Growth