Tyro3 Receptor Tyrosine Kinases Research Articles

De Novo Multi-Omics Pathway Analysis Designed for Prior Data Independent Inference of Cell Signaling Pathways

New tools for cell signaling pathway inference from multi-omics data that are independent of previous knowledge are needed. Here, we propose a new de novo method, the de novo multi-omics pathway analysis (DMPA), to model and combine omics data into network modules and pathways. DMPA was validated with published omics data and was found accurate in discovering reported molecular associations in transcriptome, interactome, phosphoproteome, methylome, and metabolomics data, and signaling pathways in multi-omics data. DMPA was benchmarked against module discovery and multi-omics integration methods and outperformed previous methods in module and pathway discovery especially when applied to datasets of relatively low sample sizes. Transcription factor, kinase, subcellular location, and function prediction algorithms were devised for transcriptome, phosphoproteome, and interactome modules and pathways, respectively. To apply DMPA in a biologically relevant context, interactome, phosphoproteome, transcriptome, and proteome data were collected from analyses carried out using melanoma cells to address gamma-secretase cleavage-dependent signaling characteristics of the receptor tyrosine kinase TYRO3. The pathways modeled with DMPA reflected the predicted function and its direction in validation experiments.

Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS<3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2= 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Abstract 5783: Receptor tyrosine kinase Tyro3 interaction with ligands and inhibitors

Abstract TAM receptors (Tyro3, Axl, Mer) are a sub-family of receptor tyrosine kinases that regulate cell survival and homeostasis primarily through their role in clearance of apoptotic cells. While several receptor tyrosine kinases (RTKs) are targeted in various types of cancer therapies, interest in Tyro3 as a potential target in breast cancers has only recently grown. Determining the structure of Tyro3 with and without tyrosine kinase inhibitors (TKIs) bound will provide important missing information to aid in the design of treatments that can be combined with existing cancer treatments, as dual treatment is often necessary. I have designed constructs that will be used for crystallization of the Tyro3 kinase domains. I mapped the full portion of the kinase (called "Small"), an extended kinase ("Big"), and a smaller catalytic domain ("Cat"). The kinases were made through a molecular cloning method (Gibson Assembly), sequence verified, and expressed in insect cells for large scale purification. Ni-NTA and gel filtration will be used, followed by crystallization and soaking with inhibitors. Citation Format: Keisha Nicole Hardeman, Chrystal Starbird. Receptor tyrosine kinase Tyro3 interaction with ligands and inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5783.

Tyro3 promotes the maturation of glutamatergic synapses.

The receptor tyrosine kinase Tyro3 is abundantly expressed in neurons of the neocortex, hippocampus, and striatum, but its role in these cells is unknown. We found that neuronal expression of this receptor was markedly up-regulated in the postnatal mouse neocortex immediately prior to the final development of glutamatergic synapses. In the absence of Tyro3, cortical and hippocampal synapses never completed end-stage differentiation and remained electrophysiologically and ultrastructurally immature. Tyro3-/- cortical neurons also exhibited diminished plasma membrane expression of the GluA2 subunits of AMPA-type glutamate receptors, which are essential to mature synaptic function. Correspondingly, GluA2 membrane insertion in wild-type neurons was stimulated by Gas6, a Tyro3 ligand widely expressed in the postnatal brain. Behaviorally, Tyro3-/- mice displayed learning enhancements in spatial recognition and fear-conditioning assays. Together, these results demonstrate that Tyro3 promotes the functional maturation of glutamatergic synapses by driving plasma membrane translocation of GluA2 AMPA receptor subunits.

Tyro3 receptor tyrosine kinase contributes to pathogenic phenotypes of fibroblast-like synoviocytes in rheumatoid arthritis and disturbs immune cell balance in experimental arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by synovitis and joint damage, the underlying causes of which remain unclear. Our prior investigations revealed a notable correlation between the expression of Tyro3 Protein Tyrosine Kinase (Tyro3TK) and the progression of RA. To further elucidate the pathogenic role of Tyro3TK in RA, we analyzed the influence of Tyro3TK on pathogenic phenotypes of RA fibroblast like synoviocyte (FLS) in vitro and compared disease severity, joint damages and immunological parameters of K/BxN serum transfer arthritis (STA) in Tyro3TK−/− deficient mice and wild type controls. Our findings underscored the remarkable effectiveness of Tyro3TK blockade, as evidenced by diminished secretion of inflammatory cytokines and matrix metalloproteinases (MMPs), curtailed migration and invasiveness of RAFLS, and attenuated differentiation of pathogenic helper T cell subsets mediated by RAFLS. Correspondingly, our in vivo investigations illuminated the more favorable outcomes in Tyro3TK-deficient mice, characterized by reduced joint pathology, tempered synovial inflammation, and restored immune cell equilibrium. These data suggested that Tyro3TK might contribute to aggravated autoimmune arthritis and immunological pathology and act as a potential therapeutic target for RA.

Receptor tyrosine kinases Tyro3, Axl, and Mertk differentially contribute to antibody-induced arthritis

Tyro3, Axl, and Mertk (abbreviated TAMs) comprise a family of homologous type 1 receptor tyrosine kinases (RTKs) that have been implicated as inhibitory receptors that dampen inflammation, but their roles in the pathogenesis of rheumatoid arthritis remains understudied. Here, to investigate TAMs in an inflammatory arthritis model, antibody-induced arthritis in single TAM-deficient mice (Tyro3- KO, Axl-KO, Mertk-KO) was induced by K/BxN serum injection. Subsequently, joint inflammation and cytokine levels, as well as the expression of Fcγ Rs and complement receptors were assessed in WT and TAM-deficient mice. Compared with littermate control mice, Axl−/− and Mertk−/− mice developed more severe antibody-induced arthritis, while in contrast, Tyro3−/− mice showed diminished joint inflammation. Concomitantly, the levels of cytokines in joints of Axl−/− and Mertk−/− mice were also significantly increased, while cytokines in the Tyro3−/− joint tissues were decreased. At the molecular and cellular level, TAMs showed distinct expression patterns, whereby monocytes expressed Axl and Mertk, but no Tyro3, while neutrophils expressed Axl and Tyro3 but little Mertk. Moreover, expression of Fcγ receptors and C5aR showed different patterns with TAMs expression, whereby FcγRIV was higher in monocytes of Axl−/− and Mertk−/− mice compared to wild-type mice, while Tyro3−/− neutrophils showed lower expression levels of FcγRI, FcγRIII and FcγRIV. Finally, expression of C5aR was increased in Mertk−/− monocytes, and was decreased in Tyro3−/− neutrophils. These data indicate that Axl, Mertk and Tyro3 have distinct functions in antibody-induced arthritis, due in part to the differential regulation of cytokines production, as well as expression of FcγRs and C5aR.1eHRq_qjGV2iQGKkb-YYk-Video

Targeting BRD3 eradicates nuclear TYRO3-induced colorectal cancer metastasis.

Metastasis is the main cause of death in many cancers including colorectal cancer (CRC); however, the underlying mechanisms responsible for metastatic progression remain largely unknown. We found that nuclear TYRO3 receptor tyrosine kinase is a strong predictor of poor overall survival in patients with CRC. The metastasis-promoting function of nuclear TYRO3 requires its kinase activity and matrix metalloproteinase-2 (MMP-2)-mediated cleavage but is independent of ligand binding. Using proteomic analysis, we identified bromodomain-containing protein 3 (BRD3), an acetyl-lysine reading epigenetic regulator, as one of nuclear TYRO3's substrates. Chromatin immunoprecipitation-sequencing data reveal that TYRO3-phosphorylated BRD3 regulates genes involved in anti-apoptosis and epithelial-mesenchymal transition. Inhibition of MMP-2 or BRD3 activity by selective inhibitors abrogates nuclear TYRO3-induced drug resistance and metastasis in organoid culture and in orthotopic mouse models. These data demonstrate that MMP-2/TYRO3/BRD3 axis promotes the metastasis of CRC, and blocking this signaling cascade is a promising approach to ameliorate CRC malignancy.

Abstract 2548: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity

Abstract Casitas B-lineage lymphoma (CBL) is a family of RING-type E3 ligases that target proteins for degradation via the proteasomal pathway. CBL-b specifically functions as a negative effector of T cell activation by down regulating the T cell receptor, whereas c-CBL interacts with various receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation. Therefore, the CBL family regulates the biology of immune cells and targeting CBL-b or c-CBL is a potential therapeutic strategy for the treatment of cancer, infection, or autoimmune diseases. In this study, we used TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) to uncover new mechanistic aspects of CBL activation. Our assays were designed and optimized using purified components (E1/E2/c-CBL or CBL-b E3 ligase and kinase substrate Tyro3 or Axl) to recreate the polyubiquitination cascade in vitro. The assays use a Europium cryptate-labeled Ubiquitin (donor) and a Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the technique measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay is homogeneous, making it especially suitable for screening applications and real-time kinetics. Validation of the assay indicated that the presence of a kinase substrate (Tyro3 or Axl) activates CBL-b and c-CBL, which are known to display a similar mechanism of activation. Importantly, our assay design allowed us to show that known CBL-B inhibitor CBL-B-IN-1 is not strictly selective toward CBL-B but also affects c-CBL E3 ligase activity to similar extend since the IC50 of the compound for c-CBL and CBL-B differ by only 2-fold. Finally, it has been proposed that CBL-b phosphorylation at Y106, Y133, and Y363 by Tyro3 kinase is required for its activation. Indeed, in our assay, we observed that CBL-b mutation at Y363F decreases TYRO3 ubiquitination drastically. Surprisingly, phospho-mimetic mutation Y363E did not restore CBL-b-mediated ubiquitination of Tyro3, indicating that substrate binding close to Y363 is the predominant requirement for CBL activation. The weak E3 ligase activity of CBL-b Y363E mutant was insensitive to CBL-B-IN-1 treatment.In conclusion, our data indicates that no significant difference in the parameters of substrate ubiquitination exists between CBL-B and c-CBL, and that ubiquitination of receptor tyrosine kinase Tyro3 by either c-CBL or CBL-B depends on the kinase binding to CBL but not on the phosphorylation of CBL. Citation Format: Kasia Zientara-Rytter, Veronique Baron, Pavel Shashkin, Henry Zhu. Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2548.

Chicken-derived MERTK protein inhibits Newcastle disease virus replication by increasing STAT1 phosphorylation in DF-1 cells

The receptor tyrosine kinases TYRO3, AXL, and MERTK (TAM) are transmembrane proteins associated with the regulation of the innate immune response. In this study, the role of the chicken-derived MERTK protein (chMertk) in the regulation of the type I interferon (IFN) signaling pathway and its antiviral effect were investigated in vitro. Newcastle disease (ND) caused by the Newcastle disease virus (NDV) is able to widely spread in chickens and give rise to massive losses in the poultry industry around the world. We found that the overexpression of the exogenous chMertk upregulated the STAT1 phosphorylation and the expression of IFN-stimulated gene IFITM3 and significantly reduced the NDV titer (p < 0.05). A mutation assay showed that three tyrosine residues (Y739, Y743, and Y744) in chMertk promoted STAT1 phosphorylation and inhibited NDV replication. However, the chicken-derived E3 ubiquitin ligase CBL significantly negatively regulated chMertk expression, thus attenuating STAT1 phosphorylation. chMertk function was restored by the ubiquitin-proteasome inhibitor MG132, demonstrating that chMertk was controlled by Casitas B-lineage proto-oncogene (CBL) ubiquitination and degradation. Together, these results suggested that chMertk participated in regulating the immune responses to NDV infection, and that CBL significantly downregulated the expression of chMertk through its ubiquitination and degradation, to maintain cellular homeostasis. Overall, our study provided new insights into the role of chMertk in regulating the innate immune response and its anti-NDV activity.

Abstract 240: MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism

Abstract The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In previous studies, inhibition of MERTK decreased PD-1 checkpoint proteins in the leukemia microenvironment and prolonged survival in a syngeneic BCR-ABL+/Arf-/- B-cell acute leukemia model, implicating MERTK as a promising immune-oncology target in leukemia. Strikingly, Mertk-/- mice were largely protected from leukemia. In our current studies, Tyro3-/- almost completely prevented development of leukemia, comparable to Mertk-/-, while Axl-/- mice died with similar timing to wild type (WT) mice (20-40 days). These data demonstrate differential roles for TAM kinases in the anti-leukemia immune response. Depletion studies were conducted to evaluate potential roles for T cells and dendritic cells (DCs) in anti-leukemia immunity in Mertk-/- mice. Selective depletion of CD8+ T cells abrogated protection from leukemia in Mertk-/- mice, but survival was still prolonged relative to WT. Thus, while CD8+ T cells were required for complete protection from leukemia, the anti-leukemia response remained partially intact even in the absence of CD8+ T cells, implicating an innate immune mechanism. Indeed, combined depletion of CD8+ T cell and CD8α+ DC subsets completely abrogated the anti-leukemic effects in Mertk-/- mice, revealing a critical immunosuppressive role for MERTK in DCs in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Similarly, single cell RNA sequencing revealed CD8+ DCs with a more mature and antigen-presenting phenotype in Mertk-/- mice compared to WT, while antigen-presenting DCs were not increased in Tyro3-/- mice. Single cell sequencing data also suggest induction of an anti-leukemic DC - T cell axis in WT leukemic mice treated with the MERTK-selective inhibitor MRX-2843. DCs were nearly absent in leukemic bone marrow from saline-treated mice and were dramatically increased in response to treatment with MRX-2843. Treatment with MRX-2843 also decreased the incidence of CD8+ T cells expressing high levels of Tox, which has been associated with T cell exhaustion. These changes coincided with decreased leukemic blasts, even in the context of established disease.Together, our findings support a model whereby MERTK inhibition promotes DC function and CD8+ T cell activity, leading to anti-leukemia immunity. In contrast, anti-leukemia immunity in response to TYRO3 inhibition is less dependent on DCs. Differential roles for the TAM kinases in the leukemia microenvironment provide rationale for development of MERTK and/or TYRO3 targeted immunotherapies to treat acute leukemia. Citation Format: Justus M. Huelse, Swati S. Bhasin, Beena E. Thomas, Madison L. Chimenti, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 240.

White matter tract conductivity is resistant to wide variations in paranodal structure and myelin thickness accompanying the loss of Tyro3: an experimental and simulated analysis.

Myelination within the central nervous system (CNS) is crucial for the conduction of action potentials by neurons. Variation in compact myelin morphology and the structure of the paranode are hypothesised to have significant impact on the speed of action potentials. There are, however, limited experimental data investigating the impact of changes in myelin structure upon conductivity in the central nervous system. We have used a genetic model in which myelin thickness is reduced to investigate the effect of myelin alterations upon action potential velocity. A detailed examination of the myelin ultrastructure of mice in which the receptor tyrosine kinase Tyro3 has been deleted showed that, in addition to thinner myelin, these mice have significantly disrupted paranodes. Despite these alterations to myelin and paranodal structure, we did not identify a reduction in conductivity in either the corpus callosum or the optic nerve. Exploration of these results using a mathematical model of neuronal conductivity predicts that the absence of Tyro3 would lead to reduced conductivity in single fibres, but would not affect the compound action potential of multiple myelinated neurons as seen in neuronal tracts. Our data highlight the importance of experimental assessment of conductivity and suggests that simple assessment of structural changes to myelin is a poor predictor of neural functional outcomes.

Intrinsic and Extrinsic Control of Hepatocellular Carcinoma by TAM Receptors.

Simple SummaryTyro3, Axl, and MerTK are receptor tyrosine kinases of the TAM family, which are activated by their ligands Gas6 and Protein S. TAM receptors have large physiological implications, including the removal of dead cells, activation of immune cells, and prevention of bleeding. In the last decade, TAM receptors have been suggested to play a relevant role in liver fibrogenesis and the development of hepatocellular carcinoma. The understanding of TAM receptor functions in tumor cells and their cellular microenvironment is of utmost importance to advances in novel therapeutic strategies that conquer chronic liver disease including hepatocellular carcinoma.Hepatocellular carcinoma (HCC) is the major subtype of liver cancer, showing high mortality of patients due to limited therapeutic options at advanced stages of disease. The receptor tyrosine kinases Tyro3, Axl and MerTK—belonging to the TAM family—exert a large impact on various aspects of cancer biology. Binding of the ligands Gas6 or Protein S activates TAM receptors causing homophilic dimerization and heterophilic interactions with other receptors to modulate effector functions. In this context, TAM receptors are major regulators of anti-inflammatory responses and vessel integrity, including platelet aggregation as well as resistance to chemotherapy. In this review, we discuss the relevance of TAM receptors in the intrinsic control of HCC progression by modulating epithelial cell plasticity and by promoting metastatic traits of neoplastic hepatocytes. Depending on different etiologies of HCC, we further describe the overt role of TAM receptors in the extrinsic control of HCC progression by focusing on immune cell infiltration and fibrogenesis. Additionally, we assess TAM receptor functions in the chemoresistance against clinically used tyrosine kinase inhibitors and immune checkpoint blockade in HCC progression. We finally address the question of whether inhibition of TAM receptors can be envisaged for novel therapeutic strategies in HCC.

G-749 Promotes Receptor Tyrosine Kinase TYRO3 Degradation and Induces Apoptosis in Both Colon Cancer Cell Lines and Xenograft Mouse Models.

G-749 is an FLT3 kinase inhibitor that was originally developed as a treatment for acute myeloid leukemia. Some FLT3 kinase inhibitors are dual kinase inhibitors that inhibit the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and are used to treat solid cancers such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). AXL promotes metastasis, suppression of immune response, and drug resistance in NSCLC and TNBC. G-749, a potential TAM receptor tyrosine kinase inhibitor, and its derivative SKI-G-801, effectively inhibits the phosphorylation of AXL at nanomolar concentration (IC50 = 20 nM). This study aimed to investigate the anticancer effects of G-749 targeting the TAM receptor tyrosine kinase in colon cancer. Here, we demonstrate the potential of G-749 to effectively inhibit tumorigenesis by degrading TYRO3 via regulated intramembrane proteolysis both in vitro and in vivo. In addition, we demonstrated that G-749 inhibits the signaling pathway associated with cell proliferation in colon cancer cell lines HCT15 and SW620, as well as tumor xenograft mouse models. We propose G-749 as a new therapeutic agent for the treatment of colon cancer caused by abnormal TYRO3 expression or activity.

The Akt-mTORC1 pathway mediates Axl receptor tyrosine kinase-induced mesangial cell proliferation.

Glomerulonephritis (GN), an important pathologic feature of many renal diseases, is frequently characterized by mesangial cell proliferation. We and others have previously shown that the TAM family receptor tyrosine kinases Axl, Mer, and Tyro-3 contribute to cell survival, proliferation, migration, and clearance of apoptotic cells (ACs); that Axl contributes to GN by promoting mesangial cell proliferation; and that small molecule inhibition of Axl ameliorates nephrotoxic serum-induced GN in mice. We now show that stimulation of renal mesangial cell Axl causes a modest increase in intracellular Ca2+ and activates NF-κB, mTOR, and the mTOR-containing mTORC1 complex, which phosphorylates the ribosomal protein S6. Axl-induction of Akt activation is upstream of NF-κB and mTOR activation, which are mutually codependent. Axl-induced NF-κB activation leads to Bcl-xl up-regulation. Axl is more important than Mer at mediating AC phagocytosis by mesangial cells, but less important than Mer at mediating phagocytosis of ACs by peritoneal macrophages. Taken together, our data suggest the possibility that Axl mediates mesangial cell phagocytosis of ACs and promotes mesangial cell proliferation by activating NF-κB and mTORC1.

GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection.

Multiple sclerosis (MS) is a highly disabling neurodegenerative autoimmune condition in which an unbalanced immune response plays a critical role. Although the mechanisms remain poorly defined, helminth infections are known to modulate the severity and progression of chronic inflammatory diseases. The tyrosine kinase receptors TYRO3, AXL, and MERTK (TAM) have been described as inhibitors of the immune response in various inflammatory settings. We show here that patients with concurrent natural helminth infections and MS condition (HIMS) had an increased expression of the negative regulatory TAM receptors in antigen-presenting cells and their agonist GAS6 in circulating CD11bhigh and CD4+ T cells compared to patients with only MS. The Th17 subset was reduced in patients with HIMS with a subsequent downregulation of its pathogenic genetic program. Moreover, these CD4+ T cells promoted lower levels of the co-stimulatory molecules CD80, CD86, and CD40 on dendritic cells compared with CD4+ T cells from patients with MS, an effect that was GAS6-dependent. IL-10+ cells from patients with HIMS showed higher GAS6 expression levels than Th17 cells, and inhibition of phosphatidylserine/GAS6 binding led to an expansion of Th17 effector genes. The addition of GAS6 on activated CD4+ T cells from patients with MS restrains the Th17 gene expression signature. This cohort of patients with HIMS unravels a promising regulatory mechanism to dampen the Th17 inflammatory response in autoimmunity.

TAM family receptors in conjunction with MAPK signalling are involved in acquired resistance to PI3K\u03b1 inhibition in head and neck squamous cell carcinoma

BackgroundAberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3Kα inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs).MethodsFive unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance.ResultsProlonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3Kα inhibition. In vivo, resistance to alpelisib emerged following 20–35 days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib.ConclusionsWe have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3Kα inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3Kα inhibition and suggest a means to prevent, or at least delay, resistance to PI3Kα inhibition in order to improve outcomes for HNSCC patients.

Role of Vitamin K-Dependent Factors Protein S and GAS6 and TAM Receptors in SARS-CoV-2 Infection and COVID-19-Associated Immunothrombosis.

The vitamin K-dependent factors protein S (PROS1) and growth-arrest-specific gene 6 (GAS6) and their tyrosine kinase receptors TYRO3, AXL, and MERTK, the TAM subfamily of receptor tyrosine kinases (RTK), are key regulators of inflammation and vascular response to damage. TAM signaling, which has largely studied in the immune system and in cancer, has been involved in coagulation-related pathologies. Because of these established biological functions, the GAS6-PROS1/TAM system is postulated to play an important role in SARS-CoV-2 infection and progression complications. The participation of the TAM system in vascular function and pathology has been previously reported. However, in the context of COVID-19, the role of TAMs could provide new clues in virus-host interplay with important consequences in the way that we understand this pathology. From the viral mimicry used by SARS-CoV-2 to infect cells, to the immunothrombosis that is associated with respiratory failure in COVID-19 patients, TAM signaling seems to be involved at different stages of the disease. TAM targeting is becoming an interesting biomedical strategy, which is useful for COVID-19 treatment now, but also for other viral and inflammatory diseases in the future.

Galectin-3 Stimulates Tyro3 Receptor Tyrosine Kinase and Erk Signalling, Cell Survival and Migration in Human Cancer Cells.

The TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and protein S (ProS1), are implicated in tumorigenesis and chemoresistance in various cancers. The β-galactoside binding protein galectin-3 (Gal-3), which is also implicated in oncogenesis, has previously been shown to be a ligand for MerTK. However, the selectivity of Gal-3 for the other TAM receptors, and its TAM-mediated signalling and functional properties in cancer cells, remain to be explored. The present study was aimed at determining these, including through direct comparison of Gal-3 with the two canonical TAM ligands. Exogenous Gal-3 rapidly stimulated Tyro3 receptor phosphorylation to the same extent as the Tyro3 ligand ProS1, but not Axl, in the cultured human cancer cell lines SCC-25 (express both Tyro3 and Axl) and MGH-U3 (express Tyro3 only). Gal-3 also activated intracellular Erk and Akt kinases in both cell lines and furthermore protected cells from acute apoptosis induced by staurosporine but not from serum-starvation induced apoptosis. In addition, Gal-3 significantly stimulated cancer cell migration rate in the presence of the Axl blocker BGB324. Therefore, these results have shown Gal-3 to be a novel agonist for Tyro3 RTK, activating a Tyro3-Erk signalling axis, as well as Akt signalling, in cancer cells that promotes cell survival, cell cycle progression and cell migration. These data therefore reveal a novel mechanism of Tyro3 RTK activation through the action of Gal-3 that contrasts with those of the known TAM ligands Gas6 and ProS1.

Gas6 expression is reduced in advanced breast cancers

Growth arrest-specific gene 6 (Gas6) is a cytokine that binds to receptor tyrosine kinases Tyro3, Axl, and Mer. Numerous studies have suggested that macrophage-derived Gas6 interacts with Axl to promote cancer progression, and Axl has been associated with poor clinical outcome. However, the expression and relevance of Gas6 in human breast cancer patients has not been studied. Analysis of tissue microarrays showed that Gas6 was highly expressed in ductal carcinoma in situ (DCIS) but markedly decreased in invasive breast cancer. Gas6 and Axl were weakly correlated, suggesting that their functions may not exclusively rely on each other. Analyses of publicly available databases showed significantly improved overall and relapse-free survival in patients with high Gas6 mRNA, particularly in luminal A breast cancers. These findings indicate that tumor-derived Gas6 is not overexpressed in invasive breast cancer, and may not be a negative prognostic factor in human breast cancer.

New Insights into the Role of Tyro3, Axl, and Mer Receptors in Rheumatoid Arthritis.

Rheumatoid Arthritis (RA) is the most common chronic inflammatory autoimmune disease involving joints. Among several pathogenic mechanisms, the impairment of homeostatic regulators of inflammation seems to be critically important to sustain persistent infiltration and activation of immune and stromal cells within the diseased synovium. Tyrosine kinase receptors Tyro3, Axl, and Mer are members of the TAM family. Upon binding their ligands Growth Arrest-Specific gene 6 (Gas6) and Protein S (ProS1), TAM receptors (TAMs) exert numerous and diverse biologic functions. Activated Axl and Mer, for instance, can negatively regulate the inflammatory cascade and mediate phagocytosis of apoptotic cells, contributing to prevent the development of autoimmunity. Thus, a role for TAMs has been hypothesized in RA. In this review, we will summarise unmet clinical needs in RA, depict the biology of TAMs and TAM ligands, focussing on their ability to regulate the immune system and inflammation cascade, and finally offer an overview of the state-of-the-art literature about the putative role of TAM axis in RA.