Tyro3, Axl, And Mer Research Articles

Dynamic conformational states of apo, ATP and cabozantinib bound TAM kinases to differentiate active-inactive kinetic models

The dynamically active and inactive conformations of kinases play a crucial role in the activation of intracellular downstream signaling pathways. The all-atom molecular dynamics (MD) simulations at microsecond (µs) timescale and longer provide robust insights into the structural details of conformational alterations in kinases that contribute to their cellular metabolic activities and signaling pathways. Tyro3, Axl and Mer (TAM) receptor tyrosine kinases (RTKs) are overexpressed in several types of human cancers. Cabozantinib, a small molecule inhibitor constrains the activity of TAM kinases at nanomolar concentrations. The apo, complexes of ATP (active state) and cabozantinib (active and inactive states) with TAM RTKs were studied by 1 µs MD simulations followed by trajectory analyses. The dynamic mechanistic pathways intrinsic to the kinase activity and protein conformational landscape in the cabozantinib bound TAM kinases are revealed due to the alterations in the P-loop, α-helix and activation loop that result in breaking the regulatory (R) and catalytic (C) spines, while the active states of ATP bound TAM kinases are retained. The co-existence of dynamical states when bound to cabozantinib was observed and the long-lived kinetic transition states of distinct active and inactive structural models were deciphered from MD simulation trajectories that have not been revealed so far. Communicated by Ramaswamy H. Sarma

STELLAR: A Phase II, Open-Label, Single-Arm, Prospective Clinical Study of Tislelizumab Combined with Sitravatinib as Consolidation Treatment after Chemoradiotherapy in Locally Advanced, Unresectable Non-Small-Cell Lung Cancer

<h3>Purpose/Objective(s)</h3> Consolidation durvalumab has become the standard of care in patients with unresectable, stage III non-small-cell lung cancer (NSCLC) without disease progression after concurrent chemoradiation therapy (cCRT) based on the PACIFIC study. However, there are still about 44% of patients experiencing disease progression or recurrence within one year. New regimens for consolidation treatment are worth exploring to further improve survival for this population. Tislelizumab, an anti-PD-1 antibody engineered to minimize binding to FcγR on macrophages to abrogate antibody-dependent phagocytosis (a potential mechanism of resistance), has shown improved efficacy in advanced NSCLC with a tolerable safety profile. Sitravatinib is a spectrum-selective tyrosine kinase inhibitor targeting TAM (Tyro3, Axl, and Mer) and split tyrosine-kinase domain-containing receptors (VEGFR2, KIT), which may potentially augment immune checkpoint blockade through reducing the number of myeloid-derived suppressor cells/regulatory T cells and increasing the ratio of M1/M2 polarized macrophages. An early trial of tislelizumab plus sitravatinib showed acceptable tolerability and promising antitumor activity in advanced NSCLC. STELLAR (NCT05176925) is evaluating tislelizumab plus sitravatinib as consolidation treatment in patients with unresectable, locally advanced, stage III NSCLC after cCRT. <h3>Materials/Methods</h3> This phase II, open-label, single-arm, prospective study is enrolling patients aged from 18-75 years with histologically/cytologically confirmed, locally advanced, unresectable, stage III NSCLC who have not progressed following definitive, platinum-based cCRT, and with ECOG PS of 0 or 1. Patients will be excluded if they have a mixed histological type of small cell lung cancer and NSCLC, or receive prior anti-VEGF mAb, VEGFR TKI or ICI therapy. Patients will receive tislelizumab (200 mg, IV, d1, Q3W) combined with sitravatinib (starting dose 70 mg, po, qd; escalating to 100 mg, qd, after 2 cycles) for up to 1 year until disease progression, intolerable toxicity, withdrawal of consent or death. The primary endpoint is 1-year progression-free survival (PFS) rate by investigator assessment per RECIST v1.1. Secondary end points are PFS, objective response rate, duration of response and disease control rate assessed by investigators per RECIST 1.1, overall survival, time to distant metastasis, and safety. AEs will be graded by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Enrollment begins in January 2022, and approximately 36 patients will be enrolled. <h3>Results</h3> N/A <h3>Conclusion</h3> N/A

Quantitative Tyrosine Phosphoproteome Profiling of AXL Receptor Tyrosine Kinase Signaling Network.

Simple SummaryAXL is a receptor tyrosine kinase belonging to the TAM (Tyro3, Axl and Mer) family. The AXL protein plays an important role in promoting cancer development, such as proliferation, migration, invasion and survival of cancer cells. In this study, we used mass spectrometry-based proteomics to quantify the cancer signaling regulated by AXL activation. Our study identified more than 1000 phosphotyrosine sites and discovered that activation of AXL can upregulate multiple cancer-promoting and cell migration/invasion-related signaling pathways. We also observed significant crosstalk as evidenced by rapid phosphorylation of multiple receptor tyrosine kinases and protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. These discoveries should serve as a potentially useful resource for studying AXL functions as well as for the development of effective therapeutic options to target AXL.Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.

AXL Receptor in Breast Cancer: Molecular Involvement and Therapeutic Limitations.

Breast cancer was one of the first malignancies to benefit from targeted therapy, i.e., treatments directed against specific markers. Inhibitors against HER2 are a significant example and they improved the life expectancy of a large cohort of patients. Research on new biomarkers, therefore, is always current and important. AXL, a member of the TYRO-3, AXL and MER (TAM) subfamily, is, today, considered a predictive and prognostic biomarker in many tumor contexts, primarily breast cancer. Its oncogenic implications make it an ideal target for the development of new pharmacological agents; moreover, its recent role as immune-modulator makes AXL particularly attractive to researchers involved in the study of interactions between cancer and the tumor microenvironment (TME). All these peculiarities characterize AXL as compared to other members of the TAM family. In this review, we will illustrate the biological role played by AXL in breast tumor cells, highlighting its molecular and biological features, its involvement in tumor progression and its implication as a target in ongoing clinical trials.

Red blood cell exchange for SARS-CoV-2: A Gemini of therapeutic opportunities

As of now, therapeutic strategies for the novel coronavirus (SARS-CoV-2) are limited and much focus has been placed on social distancing techniques to “flatten the curve”. Initial treatment efforts including ventilation and hydroxychloroquine garnered significant controversy and today, SARS-CoV-2 outbreaks are still occurring throughout the world. Needless to say, new therapeutic strategies are needed to combat this unprecedented pandemic. Nature Reviews Immunology recently published an article hypothesizing the pathogenesis of TAM (Tyro3, Axl, and Mer) receptor signaling in COVID-19. In it they expressed that hypercoagulation and immune hyper-reaction could occur secondary to decreased Protein S (PROS1). And hypoxia has been recently discovered to significantly decrease expression of PROS1. Regarding the cause of hypoxia in COVID-19; NIH funded research utilizing state-of-the-art topologies has recently demonstrated significant metabolomic, proteomic, and lipidomic structural aberrations in hemoglobin (Hb) secondary to infection with SARS-CoV-2. In this setting, Hb may be incapacitated and unable to respond to environmental variations, compromising RBCs and oxygen delivery to tissues. The use of red blood cell exchange would target hypoxia at its source; representing a Gemini of therapeutic opportunities.

Modulation of protein S and growth arrest specific 6 protein signaling inhibits pancreatic cancer cell survival and proliferation.

Thrombotic complications and hypercoagulopathies are commonly associated with the progression of pancreatic ductal adenocarcinoma (PDAC). Although the mechanistic link between the two phenomena is uncertain, there is evidently an increase in procoagulant proteins and a decrease in anticoagulants in PDAC patients. For example, the anticoagulant protein S (PS) is decreased during the progression of PDAC, a condition that possibly contributes to the hypercoagulopathies. PS is also an important signaling molecule that binds a family of tyrosine kinase receptors known as TAM (Tyro3, Axl and Mer) receptors; TAM receptors are often upregulated in different cancers. Growth Arrest Specific 6 or GAS6 protein, a homolog of PS, is also a TAM receptor family ligand. The downstream signaling pathways triggered by this ligand-receptor interaction perform diverse functions, such as cell survival, proliferation, efferocytosis, and apoptosis. Targeting the TAM receptors to treat cancer has had limited success; side effects are a significant obstacle due to the widespread numerous functions of TAM receptors. In the present study, it was revealed that PS-TAM interaction was pro-apoptotic, whereas GAS6-mediated TAM signaling promoted proliferation and survival in select PDAC cell lines. Furthermore, by regulating the balance between these two signaling pathways (by overexpressing PS or knocking down GAS6), the proliferative potential of the cells was decreased. Both long-term and short-term effects of natural PS overexpression were comparable to the treatment of the cells with the drug UNC2025, which inhibits the Mer-receptor. The present study lays the foundation for investigation of PS as a therapeutic agent to control cancer progression and to concurrently arrest thrombotic events.

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)- and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance involved a molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.

TAM Kinases Promote Necroptosis by Regulating Oligomerization of MLKL.

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members ofthe TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point ofinput from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressantTAM kinases are promoters ofpro-inflammatory necroptosis, shedding light on thebiological complexity of the regulation of inflammation.

1978-P: Possible Regulation between Estrogen and Gas6 Gene in Adipogenesis

Background: The physiological withdrawal of estrogen during menopause brings about changes in fat metabolism. Previous cell-based studies showed estrogen had direct effects on adipocytes. Gas6 is the ligand for the Tyro-3, Axl and Mer (TAM) receptors, and Gas6/TAM signaling stimulates cellular responses. Recent studies indicate that Gas6 gene was involved in adipocyte development, and human Gas6 levels were associated with obesity. Our clinical data showed Gas6 levels were positively correlated with estradiol (E2) levels in the postmenopausal women. However, the regulation mechanism between E2 and Gas6 gene in adipocyte functions is still unknown. In this study, we try to investigate the possible role of Gas6 gene that involved in estrogen effect on adipocyte development. Materials and Methods: Human and mouse adipocyte cell lines, SGBS and 3T3-L1, were used as in vitro cell model and ovariectomy rat was used as the postmenopausal animal model. Results: Our data showed increased estrogen receptor α (ERα) and Gas6 expression after E2 treatment in both SGBS and 3T3-L1 cell lines. Meanwhile, E2 treatment can decrease adipogenesis-related genes expression. Furthermore, we used estrogen antagonism in adipocyte that blocked the ERα and then reduced Gas6 expression and reversed the expression of adipogenesis-related genes. The luciferase assay confirmed that Gas6 gene expression was activated after E2 treatment through the genomic pathway. In addition, estrogen may also regulate Gas6 expression through non-genomic AMPK pathway. Finally, our animal model data showed significantly decreased Gas6 expression in adipose tissue from ovariectomy rats compared to the sham operation group. Interesting, the Gas6 expression in adipose tissue was increased in ovariectomy rats after E2 supplement. Conclusion: Our study first elucidates the E2 effect on adipogenesis-related genes expression and adipocyte function and may partially through genomic and non-genomic pathways to activate Gas6 gene expression. Disclosure C. Lee: None.

Phase II study of sitravatinib in combination with nivolumab in patients with advanced or metastatic urothelial carcinoma (UC) after checkpoint inhibitor therapy (CIT).

TPS498 Background: Combination therapy with agents targeting molecular and cellular mechanisms of CIT resistance is a rational approach to restoring CIT efficacy in patients (pts) with immunotherapy-resistant UC. Sitravatinib is a tyrosine kinase inhibitor (TKI) that targets multiple closely-related receptor tyrosine kinase (RTK) pathways, including the split RTKs VEGFR-2 and KIT, as well as the TAM (TYRO3, AXL, and MER) RTKs. Inhibition of the split RTKs enhances antitumor activity by reduction of immunosuppressive regulatory T cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME). Inhibition of TAM RTKs further restores a more immune-supportive TME by depletion of MDSCs and repolarization of tumor-associated macrophages to the M1 phenotype associated with secretion of pro-inflammatory cytokines. Given these pleiotropic immune-activating effects, the combination of sitravatinib with nivolumab is a rational approach to restoring or enhancing CIT clinical activity in pts with immunotherapy-resistant UC. This combination has been shown to be safe and tolerable in an ongoing Phase 2 study in pts with metastatic non-small cell lung cancer. Methods: This open-label Phase 2 study evaluates tolerability and clinical activity of sitravatinib in combination with nivolumab in pts with advanced or metastatic UC who experienced disease progression on or after CIT as the most recent systemic therapy. Enrollment is stratified by prior receipt of platinum-based chemotherapy. If the initial 2 cohorts are of high interest for efficacy, the protocol allows for addition of new cohorts to include pts previously treated with selected immunotherapies (anti-CTLA-4, anti-OX40 or anti–CD137 therapy) or who are CIT-naïve. The primary objective is Objective Response Rate. A Predictive Probability Design will be applied allowing for expansion to 40 pts per cohort. Sitravatinib is administered orally daily in continuous 28-day cycles at 120 mg; nivolumab intravenously at 240 mg every 2 weeks or 480 mg every 4 weeks. Status: The study is open for enrollment and recruitment is ongoing. Clinical trial information: NCT03606174.

TYRO3: A potential therapeutic target in cancer.

Cancer is among the leading causes of death worldwide. In 2016, 8.9 million people are estimated to have died from various forms of cancer. The current treatments, including surgery with chemotherapy and/or radiation therapy, are not effective enough to provide full protection from cancer, which highlights the need for developing novel therapy strategies. In this review, we summarize the molecular biology of a unique member of a subfamily of receptor tyrosine kinase, TYRO3 and discuss the new insights in TYRO3-targeted treatment for cancer therapy.

Plasma Growth Arrest-Specific 6 Protein and Genetic Variations in the GAS6 Gene in Patients with Metabolic Syndrome.

Growth arrest-specific 6 (Gas6) is a vitamin K-dependent protein secreted by immune cells, endothelial cells, vascular smooth muscle cells, and adipocytes. Recent studies indicate that Gas6 and receptors of the TAM (Tyro3, Axl, and Mer) family may be involved in the pathogenesis of obesity, systemic inflammation, and insulin resistance. The aim of this study was to investigate the association between plasma Gas6 protein and the c.843 + 7G>A Gas6 polymorphism in metabolic syndrome (MetS). Two hundred five adults (88 men and 117 women) were recruited in this study. Plasma Gas6 concentration, general, and biochemical data were measured. All subjects were genotyped for the c.843 + 7G>A Gas6 polymorphism. Plasma Gas6 concentrations decreased in parallel with various MetS components in all groups (P = 0.017 for trend). Patients in the second and third tertiles of Gas6 level had higher high-density lipoprotein cholesterol (HDL-C) levels than those in the first tertile overall and in the female group. Plasma Gas6 levels were significantly positively correlated with HDL-C level and negatively with fasting glucose level in the female patients. The A allele and genotype AA in single nucleotide polymorphism c.843 + 7G>A were less frequent in the subjects with MetS compared to those without MetS. Our results demonstrated a positive correlation between Gas6 protein values and HDL-C and reinforce the association with fasting glucose. In addition, the presence of c.843 + 7G>A Gas6 polymorphisms, especially the AA genotype, had an association with MetS. The potential role of the Gas6/TAM system in MetS deserves further investigation.

Abstract 2488: Functional characterization of nuclear TYRO3 in colorectal cancer

Abstract Colorectal cancer is one of most common death-related cancers worldwide. Aberrant expression of oncogenes is regarded as a predominant factor in colorectal cancer development. TYRO3 belongs to TAM (TYRO3, AXL, and MER) family, which is a subfamily of receptor tyrosine kinases. Our previous study discovered TYRO3, an oncogene overexpressed in colorectal cancer, promotes cell proliferation, metastasis, and tumor progression. Herein, we further identified that TYRO3 is present in nucleus of colon cancer cells using immunofluorescence staining in conjunction with confocal microscopy. By staining with three different antibodies recognizing N-terminal, central region, and C-terminal of TYRO3, respectively, we identified that central and C-terminal of TYRO3 is the fragment to be translocated to the nucleus. Transfection of exogenous Myc-TYRO3-GFP fusion protein further confirmed this finding. By bioinformatic tool prediction, we found there is a nuclear localization signal (NLS) and a nuclear export signal (NES) in TYRO3. To evaluate the function of nuclear TYRO3, we mutated NLS of TYRO3 and performed several functional studies. Results of immunofluorescent staining showed that after NLS mutation, TYRO3 cannot be translocated to nucleus, which was accompanied by distorted morphology. Furthermore, cancer cells transfected with NLS-mutated TYRO3 increased activated caspase-3 and number of apoptotic cells, suggesting nuclear localization of TYRO3 is essential for cell survival. Clinical data further support that levels of nuclear TYRO3 are a poor prognostic marker that is positively associated with colon cancer treatment failure and malignancy. These data support that nuclear TYRO3 is important for colon cancer cell survival and is a valuable biomarker for prognosis. Citation Format: Shaw-Jenq Tsai, Chun-Wei Chien, Sih-Yu Chen, Shao-Chieh Lin, Bo-Wen Lin, Jenq-Chang Lee. Functional characterization of nuclear TYRO3 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2488.

Activation of the Receptor Tyrosine Kinase AXL Regulates the Immune Microenvironment in Glioblastoma.

Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors.Significance: These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. Cancer Res; 78(11); 3002-13. ©2018 AACR.

Antiphospholipid Antibodies Inhibit Trophoblast Toll-Like Receptor and Inflammasome Negative Regulators.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti-β2 -glycoprotein I (anti-β2 GPI) antibodies can activate Toll-like receptor 4 (TLR-4) and NLRP3 in human first-trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation. Human trophoblasts were not treated or were treated with anti-β2 GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest-specific protein 6 (GAS6) or the autophagy-inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Antiphospholipid antibody-induced trophoblast inflammation was measured by qRT-PCR, activity assays, and ELISA. Anti-β2 GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR-4-mediated interleukin-8 (IL-8) response. However, the NLRP3 inflammasome-mediated IL-1β response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti-β2 GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL-induced inflammasome function and IL-1β secretion. Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti-β2 GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response.

Elevated Plasma Growth Arrest-Specific 6 Protein Levels Are Associated with the Severity of Disease During Hantaan Virus Infection in Humans.

Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaan virus (HTNV) and Seoul virus infection in Asia. The clinical manifestation of HFRS is characterized by the rapid loss of renal function (acute kidney injury) and thrombocytopenia. The specific immune mechanisms that cause thrombocytopenia in HFRS are not well described. The growth arrest-specific 6 (Gas6) protein and TAM (Tyro3, Axl and Mer) receptors have been recently shown to play prominent roles in immune regulation, and high plasma levels of Gas6 may predict the severity of diseases. The association of TAM receptors with several autoimmune diseases has been investigated, although the relationship between TAM receptors and these diseases remains unclear in HFRS. Therefore, the aim of this study was to evaluate the clinical significance of Gas6 and TAM receptor expression in HFRS. The concentrations of Gas6 in the plasma from 144 patients and the expression of TAM receptors on monocytes from 117 patients were quantified. The relationship between Gas6 levels and disease course, severity, and clinical parameters was analyzed. We first found that the plasma Gas6 levels were significantly higher in HFRS patients, whereby they were positively correlated with white blood cell counts and negatively correlated with platelet counts. The expression of Tyro3 was increased on monocytes in HFRS patients compared with that in controls. Taken together, our data indicate that elevated plasma Gas6 levels is associated with the severity of disease during HTNV infection in humans, suggesting that Gas6 may play an important role by binding with Tyro3 on monocytes, which will be assessed in future studies.

Role of Tyro3, Axl, and Mer Receptors and Their Ligands (Gas6, and Protein S) in Patients with Hepatocellular Carcinoma

The Tyro3, Axl and Mer (TAM) receptor tyrosine kinases are activated by endogenous ligands, protein S1 (PROS1) and growth arrest-specific gene 6 (Gas6), and those have important effects on cell biology. These receptors (Rs) can be shad from the cell membrane and their soluble(s) forms can be found in plasma. We investigated the fluctuation and interactive role of sTAMRs and its ligands in patients with hepatocellular carcinoma (HCC), hepatitis groups, and healthy normal adult controls (NC). The measurement cases were 45 patients with HCC group (stage 1 in 4, stage 2 in 8, stage 3 in 16, and stage 4 in 17), 4 patients with fulminant hepatitis (FH), 14 patients with acute hepatitis (AH), 10 patients with chronic hepatitis (CH), 16 patients with liver cirrhosis (LC), and 20 NCs matched by age. Plasma levels of three sTAMRs and their ligands were measured by ELISA. In comparison with NCs, Gas6, des-γ-carboxy Gas6, and sTAMRs levels were significantly higher in HCC patients, but free PROS1 levels were significantly lower. The sTyro3 and sAxl levels peaked HCC stages 2 and 3 respectively, and gradually decreased afterwards while maintaining high levels. sMer levels increased with the progression of HCC. Gas6 and des-γ-carboxy Gas6 levels gradually increased, and PROS1 levels decreased with the progression of HCC. Gas6 levels were positively correlated with sAxl levels, whereas sMer levels were negatively correlated with free PROS1 levels. sTAMRs and Gas6 levels increased in parallel to the progression of HCC fibrosis. Through the progression of HCC, Axl played the major role in TAMRs activation. However, sTYro3 continued increasing rapidly from the early stage, and that of Mer increased throughout the progression. Roles of Axl may be changed in Mer, because des-γ-carboxy Gas6 levels increasing with Gas6 in the advanced stage of HCC cannot send a signal to Axl.

The TAM receptors Axl and Mer play a joint-specific protective role in experimental arthritis

Abstract Introduction Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by an unrestrained inflammatory response in synovial joints. One family of tyrosine kinase receptors involved in anti-inflammatory feedback mechanism are Tyro3, Axl and Mer (TAM). The plasma level of the TAM receptor ligand Gas6 is significantly reduced in RA patients suggesting this feedback mechanism is impaired in RA. We investigated the individual role of each TAM receptor in experimental arthritis. Methods The KRN serum transfer model of arthritis was induced by two intraperitoneal injections of arthritic K/BxN serum in TAM receptor single gene knock-out mice (Tyro3−/−, Axl−/−, Mer−/−) and wild-type (WT) mice. Ankle joints were macroscopically scored for 7 days and knee and ankle joints were taken at the end of the experiment for histology and immunohistochemistry. Results Mer−/− mice had an increased macroscopic ankle score until day 4 whereas Axl−/− mice had an enhanced macroscopic score from day 4 until the end of the experiment. Histology of the ankle joints showed significantly increased arthritis pathology in Axl−/− mice compared to WT. In contrast, enhanced macroscopic score and arthritis pathology in the knee joints of Mer−/− mice was observed. Both in ankle and knee joints, no protective or aggravating role of Tyro3 was seen. To explain the discrepancy of Axl involvement between ankle and knee, we looked for Axl expression in synovium of naïve mice. The lining layer of ankle synovium was completely Axl-positive whereas only mild expression in the knee joints was observed. Conclusion These findings identify the TAM receptors Axl and Mer as important protective players in arthritis in addition to being expressed in a joint-specific manner.

Opposing roles of tyrosine kinase receptors Mer and Axl determine clinical outcome in a mouse model of nephritis

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology. Lupus nephritis remains one of the most severe manifestations of SLE, with considerable morbidity and mortality. There remains a major void in the successful management of lupus nephritis. TAM (Tyro-3, Axl and Mer) receptor tyrosine kinases play an important role in the maintenance of immune homeostasis. Mer is constitutively highly expressed on glomerular endothelial cells; Axl expression is inducible in kidney under inflammatory conditions. To investigate the functions of Axl and Mer in lupus nephritis, we induced glomerulonephritis in WT, Axl-KO, Mer-KO, and A/M-KO (Axl/Mer-double knockout) mice by injection of nephrotoxic serum (NTS). Mer-KO mice developed severe glomerulonephritis, with significantly decreased survival rate and increased blood urea nitrogen (BUN) levels as compared to WT mice given the same treatment. Two weeks after injection, necrotic cell death was evident in the glomeruli of Mer-KO mice. However, Axl-KO presented with significantly increased survival rate and reduced BUN as compared to the WT, Mer-KO and A/M-KO mice injected with the same serum. Interestingly, the A/M-KO mice developed kidney inflammation comparable to WT mice. Western blot analysis revealed significantly increased Stat3 phosphorylation and caspase-1 activation in the kidney of Mer-KO mice after NTS injection. In contrast, Axl deficient NTS-injected mice developed decreased Akt phosphorylation and Bcl-xl upregulation. The reciprocal activation of Axl and Mer receptor tyrosine kinases may have a major impact on renal inflammation. Results from these studies may lead to novel therapeutic drug development targeted to lupus nephritis.

Axl Receptor Axis: A New Therapeutic Target in Lung Cancer

Abstract The Axl receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of proteins and is upregulated in multiple types of cancers, including non-small cell lung cancer. In normal tissues, TAM receptor tyrosine kinases contribute to immune regulation. In cancer, Axl expression is associated with poor outcomes and is controlled through various transcriptional and epigenetic mechanisms. Preclinical studies have shown that Axl is involved in cancer cell migration, growth, survival and resistance to chemotherapy through activation of multiple downstream signaling pathways, such as Ras/MAPK, PI3K and Rac1. Axl is also expressed on endothelial cells and plays a role in angiogenesis. In preclinical models, Axl inhibition decreases cancer growth and impedes lung cancer metastases. Small molecule Axl inhibitors have been developed and are currently being used as monotherapy or in combination with cytotoxic chemotherapy or anti-EGFR therapy in early clinical trials. Here, we review Axl structure, functions, regulation, and preclinical and clinical studies in lung cancer.