TAM Receptors Research Articles

The TAM receptor TYRO3 is a critical regulator of myelin thickness in the central nervous system.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). Major deficits arise in MS patients due to an inability to repair damaged myelin sheaths following CNS insult, resulting in prolonged axonal exposure and neurodegeneration. The TAM receptors (Tyro3, Axl, and Mertk) have been implicated in MS susceptibility, demyelination and remyelination. Previously, we have shown that Tyro3 regulates developmental myelination and myelin thickness within the optic nerve and rostral region of the corpus callosum (CC) of adult mice. In this study we have verified and extended our previous findings via a comprehensive analysis of axonal ensheathment and myelin thickness in the CC of unchallenged mice, following demyelination and during myelin repair. We show that the loss of the Tyro3 receptor correlates with significantly thinner myelin sheaths in both unchallenged mice and during remyelination, particularly in larger caliber axons. The hypomyelinated phenotype observed in the absence of Tyro3 occurs independently of any influence upon oligodendrocyte precursor cell (OPC) maturation, or density of oligodendrocytes (OLs) or microglia. Rather, the primary effect of Tyro3 is upon the radial expansion of myelin. The loss of Tyro3 leads to a reduction in the number of myelin lamellae on axons, and is therefore most likely a key component of the regulatory mechanism by which oligodendrocytes match myelin production to axonal diameter.

Relevance of placental type I interferon beta regulation for pregnancy success.

Pregnancy is a unique immunologic and microbial condition that requires an adequate level of awareness to provide a fast and protective response against pathogens as well as to maintain a state of tolerance to paternal antigens. Dysregulation of inflammatory pathways in the placenta triggered by pathogens is one of the main factors responsible for pregnancy complications. Type I IFNs are key molecules modulating immune responses at the level of the placenta and are crucial for protection of the pregnancy via their antiviral and immune modulatory properties. In this study, we elucidate the mechanisms controlling the basal expression of IFNβ and its negative feedback. Using in vitro and in vivo animal models, we found that TLR signaling maintains basal IFNβ levels through the TLR4-MyD88-independent TBK/IRF3 signaling pathway. We describe the role of the TAM receptor Axl in the regulation of IFNβ function in human and mouse trophoblast cells. The absence of TAM receptors in vivo is associated with fetal demise due to dysregulation of IFNβ expression and its pro-apoptotic downstream effectors. Collectively, our data describe a feedback signaling pathway controlling the expression and function of IFNβ in the trophoblast that is essential for an effective response during viral and microbial infections.

Targeting of TAM Receptors Ameliorates Fibrotic Mechanisms in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling, which progressively abolishes lung function in an RTK (receptor tyrosine kinase)-dependent manner. Gas6 (growth arrest-specific 6) ligand, Tyro3 (TYRO3 protein tyrosine kinase 3), and Axl (anexelekto) RTK expression and activity are increased in IPF. To determine if targeting these RTK pathways would inhibit fibroblast activation and the development of pulmonary fibrosis. Quantitative genomic, proteomic, and functional analyses were used to determine Gas6/TAM (Tyro3, Axl, and Mertk [MER proto-oncogene, tyrosine kinase]) RTK expression and activation in tissues and fibroblasts from normal and IPF lungs. The profibrotic impact of these RTK pathways were also examined in bleomycin-induced pulmonary fibrosis and in SCID/Bg mice that developed pulmonary fibrosis after the intravenous administration of primary IPF fibroblasts. Gas6, Axl, and Tyro3 were increased in both rapidly and slowly progressive IPF compared with normal lung samples and fibroblasts. Targeting these pathways with either specific antibodies directed at Gas6 or Axl, or with small-molecule TAM inhibitors indicated that the small molecule-mediated targeting approach was more efficacious in both in vitro and in vivo studies. Specifically, the TAM receptor inhibitor R428 (also known as BGB324) significantly inhibited the synthetic, migratory, and proliferative properties of IPF fibroblasts compared with the other Gas6/TAM receptor targeting agents. Finally, loss of Gas6 expression decreased lung fibrotic responses to bleomycin and treatment with R428 inhibited pulmonary fibrosis in humanized SCID/Bg mice. Gas6/TAM receptor activity contributes to the activation of pulmonary fibroblasts in IPF, suggesting that targeting this RTK pathway might be an effective antifibrotic strategy in this disease.

O2: Role of TAM receptors on decidual macrophage polarization and capacity for tissue repair

American Journal of Reproductive ImmunologyVolume 80, Issue S1 p. 49-49 ABSTRACTS O2: Role of TAM receptors on decidual macrophage polarization and capacity for tissue repair First published: 26 June 2018 https://doi.org/10.1111/aji.01_12983Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume80, IssueS1Special Issue: American-Sino Joint Meeting of Reproductive Immunology (ASRI-CSI 2018), June 28-1 July, 2018 Shanghai, ChinaJune 2018Pages 49-49 RelatedInformation

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.

Macrophage-Derived Protein S Facilitates Apoptotic Polymorphonuclear Cell Clearance by Resolution Phase Macrophages and Supports Their Reprogramming.

The complete resolution of inflammation requires the uptake of apoptotic polymorphonuclear cells (PMN) by local macrophages (efferocytosis) and the consequent reprogramming of the engulfing phagocytes to reparative and pro-resolving phenotypes. The tyrosine kinase receptors TYRO3, AXL, and MERTK (collectively named TAM) are fundamental mediators in regulating inflammatory responses and efferocytosis. Protein S (PROS1) is a ligand for all TAM receptors that mediates various aspects of their activity. However, the involvement of PROS1 in the resolution of inflammation is incompletely understood. Here, we report the upregulation of Pros1 in macrophages during the resolution of inflammation. Selective knockout of Pros1 in the myeloid lineage significantly downregulated macrophage pro-resolving properties. Hence, Pros1-deficient macrophages engulfed fewer apoptotic PMN remnants in vivo, and exogenous PROS1 rescued impaired efferocytosis ex vivo. Moreover, Pros1-deficient peritoneal macrophages secreted higher levels of the pro-inflammatory mediators TNFα and CCL3, while they secreted lower levels of the reparative/anti-inflammatory IL-10 following exposure to lipopolysaccharide in comparison to their WT counterparts. Moreover, Pros1-deficient macrophages expressed less of the anti-inflammatory/pro-resolving enzymes arginase-1 and 12/15-lipoxygenase and produced less of the specialized pro-resolving mediator resolvin D1. Altogether, our results suggest that macrophage-derived PROS1 is an important effector molecule in regulating the efferocytosis, maturation, and reprogramming of resolution phase macrophages, and imply that PROS1 could provide a new therapeutic target for inflammatory and fibrotic disorders.

Age-related changes in expression and signaling of TAM receptor inflammatory regulators in monocytes.

The multifactorial immune deterioration in aging--termed “inflamm-aging”--is comprised of a state of low-grade, chronic inflammation and complex dysregulation of responses to immune stimulation. The TAM family (Tyro 3, Axl, and Mer) of receptor tyrosine kinases are negative regulators of Toll like receptor-mediated immune responses that broadly inhibit cytokine receptor cascades to inhibit inflammation. Here we demonstrate elevated expression of TAM receptors in monocytes of older adults, and an age-dependent difference in signaling mediator AKT resulting in dysregulated responses to signaling though Mer. Our results may be especially significant in tissue, where levels of Mer are highest, and may present avenues for modulation of chronic tissue inflammation noted in aging.

Abstract A206: Small-molecule Cbl-b inhibitors as novel intracellular checkpoint inhibitors for cancer immunotherapy

Abstract Cancer cells evade destruction by co-opting checkpoint signaling in the immune system. Therapeutics aimed at preventing this, such as anti-PD1 and anti-CTLA4 antibodies, promote cytotoxic T lymphocyte (CTL)-mediated antitumor immune responses against advanced solid tumors. Harnessing the antitumor activity of CTLs, and that of their innate counterpart natural killer (NK) cells, is a promising approach. Activation of CTLs and NK cells is tightly regulated. CTLs require costimulatory signals, such as CD28, for robust activation. NK cell activation can be kept in check by a variety of inhibitory receptors, such as those of the TAM tyrosine kinase receptor family. Ultimately, these signaling events are orchestrated by the ubiquitin signaling system. One ubiquitin ligase in particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), is a negative regulator of both CTL and NK cell activation. In T cells, Cbl-b attenuates TCR signaling by negatively regulating several downstream signaling components, enforcing the requirement for costimulation. In NK cells, Cbl-b regulates TAM receptor internalization at the plasma membrane via ubiquitylation, and this process is important in enabling inhibitory signaling via the TAM receptors. Accordingly, mice deficient in active Cbl-b exhibit hyper-responsive immunity and reject a variety of implanted metastatic and nonmetastatic tumors. Outgrowth of spontaneous tumors in these mice is also significantly delayed. This tumor resistance is mediated by activated CD8+ T cells and NK cells. Thus, Cbl-b is a promising target for developing small-molecule cancer immunotherapy agents. Using proprietary HTS technologies, Progenra has discovered novel and selective Cbl-b inhibitors that decrease ubiquitylation of TAM receptor family members (Tyro3). These inhibitors also promote CD3-mediated activation of T cells in a CD28-independent manner, markedly increasing proliferation, IL-2 secretion, and TNFα production in T cells. Additionally, these Cbl-b inhibitors increase NK cell activation (as measured by IFNγ and degranulation) in ex vivo assays. ADME/DMPK as well as in vivo efficacy data will be discussed. These molecules are expected to synergize with other approved immunotherapy agents. Citation Format: Christopher Riling, Ivan Sokirniy, Brigid Cunnion, Emily Todd, Michael Mattern, Jian Wu, Taku Kambayashi, Suresh Kumar. Small-molecule Cbl-b inhibitors as novel intracellular checkpoint inhibitors for cancer immunotherapy [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 A206.

Circulating Levels of ADAM-10 and Soluble Standard CD44 in Patients with Hepatocellular Carcinoma: Relationship with Soluble TAM Receptors and their Ligands

Objective: The aim of the study was to examine correlations of the plasma levels of soluble (s) CD44 standard (std), and a disintegrin and metalloproteinase (ADAM) 10, with those of the soluble Tyro3, Axl, and Mer receptors (sTAMRs) and their ligands (GAS6 and PROS1) in patients with hepatocellular carcinoma (HCC), and other liver diseases.Method: The subjects were 55 patients with HCC, 4 with fulminant hepatitis (FH), 18 with acute hepatitis (AH), 10 with chronic hepatitis (CH), 20 with liver cirrhosis (LC), and 20 healthy normal controls (NCs). Plasma levels of sCD44std, ADAM10, sTAMRs, GAS6, PROS1, des-γ-carboxy prothrombin (DCP), and des-γ-carboxy GAS6 were measured by enzyme-linked immunosorbent assay.Results: The levels of sCD44std, ADAM10, sTAMRs, and Gas6 were significantly higher in HCC and other liver diseases (except CH) compared to those in NCs, but PROS1 levels in these diseases were significantly lower than those in NCs. These were significant positive correlations of sCD44std levels with ADAM10, and sMer levels in HCC and other liver diseases. SAxl was lower in stage HCC than in stage HCC, but GAS6 increased with progression of HCC stages. There was a significant positive correlation between the levels of DCP and des-γ-carboxy GAS6 in HCC. The subjects were roughly classified into three groups of HCC, inflammatory diseases, and normal controls using ratios of TAMRs and their ligands.Conclusion: The measurement of these blood factors facilitates a unified view of HCC therapy

Abstract A140: MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents

Abstract The TAM family of receptor tyrosine kinases (RTKs) has been discovered to play a predominant role in promoting the growth, survival, and metastatic spread of several tumor types. AXL and MERTK are two TAM family RTKs that are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. The AXL receptor is the most well-characterized TAM receptor and has been found to mediate resistance to both conventional and targeted cancer therapies. Since AXL is overexpressed in aggressive tumor types, cancer patients are currently being enrolled in clinical trials testing AXL inhibitors. In the current study, we analyzed the efficacy of AXL inhibitors—both small molecule and monoclonal antibody therapy—in HNSCC, TNBC, and NSCLC preclinical models. We observed limited efficacy of anti-AXL targeting strategies across these different models, which was attributed to the upregulation of MERTK. MERTK was robustly overexpressed in cell lines and patient-derived xenografts treated with AXL inhibitors. Inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibitors, leading to a more potent blockade of downstream signaling, decreased expansion of tumor cells in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These results suggest that cotargeting both AXL and MERTK may be highly beneficial in a variety of tumor types where both receptors are expressed and may therefore prolong antitumor effects and improve the survival of patients with lethal malignancies. Citation Format: Nellie K. McDaniel, Christopher T. Cummings, Toni M. Brand, Mari Iida, Justus Hulse, Hannah E. Pearson, Rachel A. Orbuch, Olivia J. Ondracek, Kurtis D. Davies, Parkash Gill, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Randall J. Kimple, Paul M. Harari, Deborah DeRyckere, Douglas K. Graham, Deric L. Wheeler. MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents [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 A140.

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction.

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme-linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In-vitro osteoclast differentiation assay showed further that tartrate-resistant acid phosphatase (TRAP)+ osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis.

Requirement of Gamma-Carboxyglutamic Acid Modification and Phosphatidylserine Binding for the Activation of Tyro3, Axl, and Mertk Receptors by Growth Arrest-Specific 6.

The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.

Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation.

Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1β production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1β response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1β processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.

Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma

Background:Leiomyosarcoma (LMS) are 15% of adult sarcomas and remain seldom curable in metastatic phase. The TAM receptors and their ligands are overexpressed or activated in multiple malignancies, including LMS.Methods:The TAM receptor and ligand expression was evaluated in LMS cell lines and 358 sarcoma samples by either gene expression or immunohistochemistry. TYRO3 and AXL were knocked down. Crizotinib and foretinib were investigated in vitro.Results:High expression of TYRO3 and AXL was detected in LMS cell lines. TYRO3 or AXL gene knockdown reduced cell proliferation/colony formation. Crizotinib and foretinib decreased TYRO3 and AXL phosphorylation, apoptosis, G2/arrest and reduced colony formation. Immunohistochemistry performed in 107 sarcomas showed higher expression of TYRO3 and GAS6 in LMS vs other sarcomas and nuclear TYRO3 only in LMS. Microarray gene expression performed in 251 sarcomas revealed significantly higher expression of TYRO3 and GAS6 in LMS than other sarcomas. Leiomyosarcoma patients with high expression of GAS6 or PROS1 present a significantly worse PFS.Conclusions:Leiomyosarcoma patients, especially those whom develop metastasis, express higher levels of TYRO3 and GAS6. Crizotinib and foretinib showed effective antitumour activity in LMS through TYRO3 and AXL deactivation indicating that clinical trials using TYRO3 and AXL inhibitors are warranted in advanced LMS.

Studies of Lassa Virus Cell Entry.

Host cell entry is the first and most fundamental step of every virus infection and represents a major barrier for zoonotic transmission and viral emergence. Targeting viral entry appears further as a promising strategy for therapeutic intervention. Several cellular receptors have been identified for Lassa virus, including dystroglycan, TAM receptor tyrosine kinases, and C-type lectins. Upon receptor binding, LASV enters the host cell via a largely unknown clathrin- and dynamin-independent endocytotic pathway that delivers the virus to late endosomes, where fusion occurs after engagement of a second, intracellular receptor, the late endosomal/lysosomal resident protein LAMP1. Here, we describe a series of experimental approaches to investigate LASV cell entry and to test candidate inhibitors for their action at this early and decisive step of infection.

Vitamin K-Dependent Protein S: Beyond the Protein C Pathway.

Protein S is a vitamin K-dependent plasma glycoprotein circulating in plasma at a concentration of around 350 nM. Approximately 60% of protein S in human plasma is bound to the complement regulatory protein C4b-binding protein (C4BP) in a high-affinity, high-molecular-weight complex. Protein S in plasma has multiple anticoagulant properties and heterozygous protein S deficiency is associated with increased risk of venous thrombosis. Homozygous deficiency in man and mice is associated with severe thrombosis in fetal life, defects in the vascular system development, and not compatible with life. Protein S has additional functions beyond being an anticoagulant. It affects the complement regulatory properties of C4BP, and moreover, protein S interacts with tyrosine kinase receptors of the TAM family, which comprises Tyro3, Axl, and Mer. The TAM receptor interaction is important for the ability of protein S to stimulate phagocytosis of apoptotic cells. This review will discuss the multiple functions of protein S, describing its role as cofactor to activated protein C with a subsequent focus on the other functions of protein S.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.

Mouse macrophages show different requirements for phosphatidylserine receptor Tim4 in efferocytosis

Protein S (ProS) and growth arrest-specific 6 (Gas6) bind to phosphatidylserine (PtdSer) and induce efferocytosis upon binding TAM-family receptors (Tyro3, Axl, and Mer). Here, we produced mouse ProS, Gas6, and TAM-receptor extracellular region fused to IgG fragment crystallizable region in HEK293T cells. ProS and Gas6 bound Ca2+ dependently to PtdSer (Kd 20-40 nM), Mer, and Tyro3 (Kd 15-50 nM). Gas6 bound Axl strongly (Kd < 1.0 nM), but ProS did not bind Axl. Using NIH 3T3-based cell lines expressing a single TAM receptor, we showed that TAM-mediated efferocytosis was determined by the receptor-binding ability of ProS and Gas6. Tim4 is a membrane protein that strongly binds PtdSer. Tim4 alone did not support efferocytosis, but enhanced TAM-dependent efferocytosis. Resident peritoneal macrophages, Kupffer cells, and CD169+ skin macrophages required Tim4 for TAM-stimulated efferocytosis, whereas efferocytosis by thioglycollate-elicited peritoneal macrophages or primary cultured microglia was TAM dependent, but not Tim4 dependent. These results indicate that TAM and Tim4 collaborate for efficient efferocytosis in certain macrophage populations.

Abstract 4176: Targeting TAM family members with antibody or small molecule inhibitors enhances therapeutic modalities of HNSCC

Abstract Radiation, cisplatin, and cetuximab are clinical therapeutics used in the treatment of head and neck squamous cell carcinoma (HNSCC). Despite clinical success with these modalities, development of both intrinsic and acquired resistance is an emerging problem in the management of HNSCC. Thus, identifying and targeting molecules driving this drug resistance is essential for improving efficacy of treatment approaches. Recent studies have identified a role for the TAM family of receptor tyrosine kinases (Tyro, Axl, and Mer) in tumor biology, especially the Axl receptor in promoting tumor growth and metastasis. Previously, we identified Axl as a logical molecular target in HNSCC and indicated that it may play a pivotal role in resistance to radiation, cisplatin, and cetuximab. In the current study, we advanced these early findings into pre-clinical models using patient-derived xenografts (PDXs). We have shown that small molecules targeting Axl can enhance therapy in PDXs that express high levels of Axl and have been identified as resistant to radiation, cisplatin, and cetuximab. Current studies are focusing on antibody-based targeting of Axl using PDXs and genetically-modified models of resistance. Furthermore, studies focused on co-targeting of Axl and Mer in vitro and in vivo have shown striking results and denote the importance of establishing logical approaches to target TAMs in the management of HNSCC. Finally, investigations into the molecular mechanisms of how Axl signaling can lead to resistance have underscored the importance of tyrosine 821 (Y821) of Axl. Overexpression of Axl rendered cetuximab sensitive lines resistant, but cell lines overexpressing Axl-Y821F retained their sensitivity to cetuximab. Advancing this line of study in vivo indicated that tumors expressing Axl were resistant to cetuximab whereas tumors harboring the Y821 mutation were sensitive demonstrating that signals emanating from Y821 may be critical for cetuximab resistant pathways. Collectively, the studies presented herein identify the TAM family of receptors as key players in radiation, cisplatin, and cetuximab resistance. These results provide rationale for the clinical targeting of TAM receptors to enhance the therapeutic modalities used in treating HNSCC. Citation Format: Nellie K. Black, Mari Iida, Tamara S. Rodems, Toni M. Brand, Randall J. Kimple, Deric L. Wheeler. Targeting TAM family members with antibody or small molecule inhibitors enhances therapeutic modalities of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4176. doi:10.1158/1538-7445.AM2017-4176

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy

TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion <30 mg/24 hours and >300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n=9) and controls (n=6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an invitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During invitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM.