TAM Signaling Research Articles

AXL, along with PROS1, is overexpressed in papillary thyroid carcinoma and regulates its biological behaviour

BackgroundAXL, a TAM tyrosine kinase receptor, plays an essential role in the pathogenesis of various solid tumours. This study explores the role of AXL and its ligand PROS1 in the generation and biological behaviour of papillary thyroid cancer (PTC).MethodsThe expression levels of AXL in PTC cancer tissue were analysed using immunohistochemistry (IHC) staining. The expression levels of AXL in PTC and normal thyroid cell lines were analysed using real-time quantitative polymerase chain reaction (RT-qPCR). CCK-8 was used to assess the proliferation of the PTC cell line with and without the effect of the AXL inhibitor (R428). Scratching assays played a role in evaluating the cell migration rate.ResultsPROS1 and AXL were expressed in TPC-1, B-CPAP, and Nthy-Ori 3–1 cells at different levels. Expression was significantly higher in PTC cell lines (TPC-1 and B-CPAP) than in the normal thyroid cell line (Nthy-Ori 3–1) (p < 0.05). In addition, AXL expression in PTC tissues was significantly higher than in adjacent normal tissues (p < 0.05). CCK-8 experiments confirmed that R428 suppresses the proliferation of PTC cell lines in a dose-dependent manner, with an increase in concentration from 0.5 to 4 μM, decreasing the inhibitory effect (p < 0.01). In addition, R428 inhibited PTC cell line migration to different degrees in a range of concentrations from 0.5 to 2 μM compared to control cells (p < 0.01).ConclusionPROS1 and its downstream receptor AXL expression were significantly higher in PTC than in normal thyroid cells. AXL expression was also higher in human PTC tissues than in normal thyroid tissues. Inhibiting the PROS1-AXL-mediated TAM signaling pathway via the AXL blocker R428 suppressed the proliferation and migration of human PTC cells, highlighting the role of this cascade in human PTC development and progression.

GAS6/TAM signaling pathway controls MICA expression in multiple myeloma cells.

NKG2D ligands play a relevant role in Natural Killer (NK) cell -mediated immune surveillance of multiple myeloma (MM). Different levels of regulation control the expression of these molecules at cell surface. A number of oncogenic proteins and miRNAs act as negative regulators of NKG2D ligand transcription and translation, but the molecular mechanisms sustaining their basal expression in MM cells remain poorly understood. Here, we evaluated the role of the growth arrest specific 6 (GAS6)/TAM signaling pathway in the regulation of NKG2D ligand expression and MM recognition by NK cells. Our data showed that GAS6 as well as MERTK and AXL depletion in MM cells results in MICA downregulation and inhibition of NKG2D-mediated NK cell degranulation. Noteworthy, GAS6 derived from bone marrow stromal cells (BMSCs) also increases MICA expression at both protein and mRNA level in human MM cell lines and in primary malignant plasma cells. NF-kB activation is required for these regulatory mechanisms since deletion of a site responsive for this transcription factor compromises the induction of mica promoter by BMSCs. Accordingly, knockdown of GAS6 reduces the capability of BMSCs to activate NF-kB pathway as well as to enhance MICA expression in MM cells. Taken together, these results shed light on molecular mechanism underlying NKG2D ligand regulation and identify GAS6 protein as a novel autocrine and paracrine regulator of basal expression of MICA in human MM cells.

Up-regulated serum levels of TAM receptor tyrosine kinases in a group of Egyptian autistic children

TAM receptor family belongs to receptor tyrosine kinases (TAMRTKs). It includes three receptors; Tyro-3, Axl and Mer. TAMRTKs has a great role in resolution of inflammation due to their role in clearance of apoptotic cells by macrophages. Dysregulated TAM signaling pathways are associated with many autoimmune diseases and chronic inflammatory disorders. Autism may be an autoimmune disease in some patients. This work was the first study that investigated serum levels of the soluble ectodomain shed TAMRTKs in a group of autistic children. Serum levels of TAMRTKs were measured by ELISA in 30 autistic children aged between 3.5 and 11 years and 30 age and sex-matched healthy control children. Serum levels of TAMRTKs were significantly higher in autistic children than healthy control children (P < 0.001). Patients with severe autism had significantly higher serum levels of TAMRTKs than patients with mild to moderate autism (P < 0.01). In addition, there were significant positive correlations between scores of the Childhood Autism Rating Scale (CARS) and serum levels of TAMRTKs in autistic patients, (P < 0.01). In conclusions, serum levels of TAMRTKs were up-regulated in autistic children with significant positive correlations with the degree of the disease severity. This initial report requires further studies to investigate the relationship between TAMRTKs and autism.

TAM Signaling in the Nervous System.

Tyro3, Axl and Mertk are members of the TAM family of tyrosine kinase receptors. TAMs are activated by two structurally homologous ligands GAS6 and PROS1. TAM receptors and ligands are widely distributed and often co-expressed in the same cells allowing diverse functions across many systems including the immune, reproductive, vascular, and the developing as well as adult nervous systems. This review will focus specifically on TAM signaling in the nervous system, highlighting the essential roles this pathway fulfills in maintaining cell survival and homeostasis, cellular functions such as phagocytosis, immunity and tissue repair. Dysfunctional TAM signaling can cause complications in development, disruptions in homeostasis which can rouse autoimmunity, neuroinflammation and neurodegeneration. The development of therapeutics modulating TAM activities in the nervous system has great prospects, however, foremost we need a complete understanding of TAM signaling pathways.

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.

Anti-Inflammatory Role of TAM Family of Receptor Tyrosine Kinases Via Modulating Macrophage Function.

Macrophage is an important innate immune cell that not only initiates inflammatory responses, but also functions in tissue repair and anti-inflammatory responses. Regulating macrophage activity is thus critical to maintain immune homeostasis. Tyro3, Axl, and Mer are integral membrane proteins that constitute TAM family of receptor tyrosine kinases (RTKs). Growing evidence indicates that TAM family receptors play an important role in anti-inflammatory responses through modulating the function of macrophages. First, macrophages can recognize apoptotic bodies through interaction between TAM family receptors expressed on macrophages and their ligands attached to apoptotic bodies. Without TAM signaling, macrophages cannot clear up apoptotic cells, leading to broad inflammation due to over-activation of immune cells. Second, TAM signaling can prevent chronic activation of macrophages by attenuating inflammatory pathways through particular pattern recognition receptors and cytokine receptors. Third, TAM signaling can induce autophagy which is an important mechanism to inhibit NLRP3 inflammasome activation in macrophages. Fourth, TAM signaling can inhibit polarization of M1 macrophages. In this review, we will focus on mechanisms involved in how TAM family of RTKs can modulate function of macrophage associated with anti-inflammatory responses described above. We will also discuss several human diseases related to TAM signaling and potential therapeutic strategies of targeting TAM signaling.

The Dual Role of TAM Receptors in Autoimmune Diseases and Cancer: An Overview.

Receptor tyrosine kinases (RTKs) regulate cellular processes by converting signals from the extracellular environment to the cytoplasm and nucleus. Tyro3, Axl, and Mer (TAM) receptors form an RTK family that plays an intricate role in tissue maintenance, phagocytosis, and inflammation as well as cell proliferation, survival, migration, and development. Defects in TAM signaling are associated with numerous autoimmune diseases and different types of cancers. Here, we review the structure of TAM receptors, their ligands, and their biological functions. We discuss the role of TAM receptors and soluble circulating TAM receptors in the autoimmune diseases systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Lastly, we discuss the effect of TAM receptor deregulation in cancer and explore the therapeutic potential of TAM receptors in the treatment of diseases.

Cell-intrinsic regulation of murine epidermal Langerhans cells by protein S.

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.

The role of TAM family receptors and ligands in the nervous system: From development to pathobiology.

Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K-dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K-dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6-/- and TAM mutant mice models.

Gas6 /TAM system: physiological insights and diseases

Growth arrest specific 6 (Gas6) is a multimodular circulating protein, the actions are mediated by the interaction with three transmembrane tyrosine kinase receptors: Tyro3, Axl, and MerTK, named TAM. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. Gas6 /TAM system plays a role in the regulation of inflammation, coagulation, cell growth, and clearance of apoptotic bodies. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, and metastasis. We review the function of the Gas6/TAM system and the current evidence supporting its potential role in the pathogenesis of different diseases.

Abstract B159: Exploring the functions of TAM kinases: assay development and characterization of potential therapeutics

Abstract TAM kinases, a relatively recent group of RTKs, have gained interest as therapeutic targets in cancer, chronic inflammatory, and autoimmune diseases. Comprising Tyro-3, Axl, and MerTK, TAMs need an extracellular lipid-protein complex for their activation, which is unique to this family of RTKs. For example, they bind to phosphatidyl serine bound gamma-carboxylated proteins as Gas-6 on apoptotic cells as ligands. Normally, TAMs function as regulators of the immune system such as controlling innate immunity or differentiation of natural killer cells, and lately a significant role in hematopoiesis has been deciphered. Though not potent oncogenic drivers, deregulated TAM signaling is often strongly associated with cancer progression, metastasis, and resistance to targeted therapies. They promote tumor growth by activation of prosurvival signals in tumor cells and reduction of antitumor immune responses. Several TAM kinase inhibitors are under (pre-)clinical investigation, e.g., BGB324 (R428), an Axl inhibitor is currently in Phase 1 trials as mono- or combination therapy. While for dual MerTK-Flt3 inhibitors MRX-2843 has received FDA Investigational New Drug (IND) status, UNC2025 is restricted to certain cancers like acute myeloid leukemia since inhibition of Flt3 has been related to severe hematopoietic toxicity. With the rapid increase in TAM-selective targeting strategies, there is an unmet need of assay systems to identify and categorize specific inhibitors. Due to their unique activation mechanism, set-up of cellular assay systems especially poses a challenge. Here, we report on the progress of set-up of various cell-based assays for all TAM kinases. To this end, we successfully developed a MefToff system for Axl and full-length expression in one cell background (e.g. Rat1) for Axl and Tyro-3 to measure cellular kinase activity. Additionally, we compared the data and feasibility of our cellular systems with biochemical tests using recently published commercially available inhibitors including UNC2025. To identify off-targets, we also tested 320 kinases in our ProQinase biochemical wild-type profiler assays. Furthermore, phenotypic screens were run to substantiate these outcomes. Currently, we try to set up assays for MerTK using various transmembrane domain mutants and chimeric systems. Assays with primary as well as tumor cells are in progress that would allow us to validate results from the cell-based artificial systems in to a more physiologic context. We believe such assays will allow a detailed molecular characterization of TAM kinases and will open opportunities for development of more advanced molecules as well for many new areas of interest. Citation Format: Alokta Chakrabarti, Daniel Feger, Frank Totzke, Sarah Umber, Orysya Stus, Marianne Birkle, Oliver Siedentopf, Marcel Pathe, Thomas Weber, Michael H.G. Kubbutat, Jan E. Ehlert. Exploring the functions of TAM kinases: assay development and characterization of potential therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B159.

TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy.

Cancer immunotherapy utilizing T-cell checkpoint inhibitors has shown tremendous clinical success. Yet, this mode of treatment is effective in only a subset of patients. Unresponsive patients tend to have non-T-cell-inflamed tumors that lack markers associated with the activation of adaptive anti-tumor immune responses. Notably, elimination of cancer cells by T cells is critically dependent on the optimal activity of innate immune cells. Therefore, identifying new targets that regulate innate immune cell function and promote the engagement of adaptive tumoricidal responses is likely to lead to the development of improved therapies against cancer. Here, we review the TAM receptor tyrosine kinases-TYRO3, AXL, and MERTK-as an emerging class of innate immune checkpoints that participate in key steps of anti-tumoral immunity. Namely, TAM-mediated efferocytosis, negative regulation of dendritic cell activity, and dysregulated production of chemokines collectively favor the escape of malignant cells. Hence, disabling TAM signaling may promote engagement of adaptive immunity and complement T-cell checkpoint blockade.

Porphyromonas gingivalis Promotes Unrestrained Type I Interferon Production by Dysregulating TAM Signaling via MYD88 Degradation

Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ Tcells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.

TAM receptor signaling in development.

TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

The roles of TAM receptor tyrosine kinases in the mammalian testis and immunoprivileged sites.

Three members of a receptor tyrosine kinase family, including Tyro3, Axl, and Mer, are collectively called as TAM receptors. TAM receptors have two common ligands, namely, growth arrest specific gene 6 (Gas6) and protein S (ProS). The TAM-Gas6/ProS system is essential for phagocytic removal of apoptotic cells, and plays critical roles in regulating immune response. Genetic studies have shown that TAM receptors are essential regulators of the tissue homeostasis in immunoprivileged sites, including the testis, retina and brain. The mechanisms by which the TAM-Gas6/ProS system regulates the tissue homeostasis in immunoprivileged sites are emerging. The roles of the TAM-Gas6/ProS system in regulating the immune privilege were intensively investigated in the mouse testis, and several studies were performed in the eye and brain. This review summarizes our current understanding of TAM signaling in the testis and other immunoprivileged tissues, as well as highlights topics that are worthy of further investigation.

The association of Tyro3/Axl/Mer signaling with inflammatory response, disease activity in patients with primary Sjögren's syndrome

ObjectivesDefects in Tyro3/Axl/Mer signaling may lead to impaired phagocytosis of apoptotic cells, eventually contributing to the development of autoimmune disease. The association of TAM signaling with several autoimmune disease has been investigated, but it remains unclear in primary Sjögren's syndrome. Therefore, the aim of this study was to evaluate the level of TAM signaling in primary Sjögren's syndrome with its clinical significance. MethodsReal-Time Polymerase Chain Reaction was used to determine the mRNA expression of Mer, Tyro-3, Axl, Gas6, and Protein S in Peripheral Blood Mononuclear Cell from 43 pSS and 46 control. The Enzyme-Linked Immunosorbent Assay method was used to test plasma levels of soluble TAM signaling from those individuals, and the relationship of their levels with clinical characteristic was evaluated. ResultsThe mRNA expression levels of Tyro-3, Axl were decreased in pSS patients. When considering the plasma level, increased levels of soluble Mer was observed with statistically significant difference. Soluble Mer levels were positively correlated with IgG levels (r=0.53, P<0.01), Erythrocyte Sedimentation Rate levels (r=0.44, P<0.01) and Sjögren's Syndrome Disease Activity Index (r=0.48, P<0.01). And the levels of soluble Mer in patients with the presence of SSA/SSB were higher than those without SSA/SSB. ConclusionsThe plasma levels of sMer were increased in pSS patients, which was associated with inflammatory response and disease activity.

TAM Receptor Signaling in Immune Homeostasis

The TAM receptor tyrosine kinases (RTKs)-TYRO3, AXL, and MERTK-together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease.

Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

Tyro3, Axl, and Mertk receptor signaling in inflammatory bowel disease and colitis-associated cancer.

Three receptor tyrosine kinases, Tyro3, Axl, and Mertk (TAM) and their ligands Gas6 and Protein S, have emerged as potent negative regulators of innate immune responses. A number of studies using genetic ablation of TAM loci in mice have elucidated the mechanism of TAM engagement and function during the immune response and removal of apoptotic cells. Following phagocytosis of apoptotic cells or the induction of T-cell dependent adaptive immune responses, ligand-induced TAM signaling dampens proinflammatory cytokine production and thus prevents exaggerated or prolonged inflammation. It is believed that the TAM pathway may play an important role in the pathogenesis of inflammatory bowel disease. Suppression of inflammation and removal of apoptotic cells followed by tissue repair are essential processes for disease remission and the successful management of inflammatory bowel disease. In light of the key role of TAMs in controlling inflammatory responses, here, we review the recent advances on TAM research vis-à-vis the resolution of intestinal inflammation. Targeted activation of TAM receptor tyrosine kinases may represent a potent therapeutic opportunity in inflammatory bowel disease.

Biology of the TAM Receptors

The TAM receptors--Tyro3, Axl, and Mer--comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands--Gas6 and Protein S--are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.