TAM Receptors Research Articles

Immunological role of TAM receptors in the cancer microenvironment.

TAM receptors belong to the family of receptor tyrosine kinases, comprising of Tyro3, Axl and Mertk receptors (TAMs) and are important homeostatic regulators of inflammation in higher eukaryotes. Along with their ligands, Gas6 and ProteinS, TAMs acts as receptors to phosphatidylserine (PtdSer), an anionic phospholipid that becomes externalized on the surface of apoptotic and stressed cells. TAM receptors, specially Mertk, have been well established to play a role in the process of efferocytosis, the engulfment of dying cells. Besides being efferocytic receptors, TAMs are pleiotropic immune modulators as the lack of TAM receptors in various mouse models lead to chronic inflammation and autoimmunity. Owing to their immune modulatory role, the PtdSer-TAM receptor signaling axis has been well characterized as a global immune-suppressive signal, and in cancers, and emerging literature implicates TAM receptors in cancer immunology and anti-tumor therapeutics. In the tumor microenvironment, immune-suppressive signals, such as ones that originate from TAM receptor signaling can be detrimental to anti-tumor therapy. In this chapter, we discuss immune modulatory functions of TAM receptors in the tumor microenvironment as well role of differentially expressed TAM receptors and their interactions with immune and tumor cells. Finally, we describe current strategies being utilized for targeting TAMs in several cancers and their implications in immunotherapy.

Tyrosine Kinase Receptors Axl and MerTK Mediate the Beneficial Effect of Electroacupuncture in a Cuprizone-Induced Demyelinating Model.

Electroacupuncture has been shown to promote remyelination in a demyelinating model of multiple sclerosis (MS) through enhanced microglial clearance of degraded myelin debris. However, the mechanisms involved in this process are yet to be clearly elucidated. It has been revealed that TAM receptor tyrosine kinases (Tyro3, Axl, and MerTK) play pivotal roles in regulating multiple features of microglia, including the phagocytic function and myelin clearance. Therefore, the aim of this study is to further confirm whether electroacupuncture improves functional recovery in this model and to characterise the involvement of the TAM receptor during this process. In addition to naive control mice, a cuprizone-induced demyelinating model was established, and long-term electroacupuncture treatment was administrated. To evaluate the efficiency of functional recovery following demyelination, we performed beam-walking test and rotarod performance test; to objectify the degree of remyelination, we performed transmission electron microscopy and protein quantification of mature oligodendrocyte markers. Oil Red O staining was used to evaluate the deposit of myelin debris. We confirmed that, in cuprizone-treated mice, electroacupuncture significantly ameliorates motor-coordinative dysfunction and counteracts demyelinating processes, with less deposit of myelin debris accumulating in the corpus callosum. Surprisingly, mRNA expression of TAM receptors was significantly upregulated after electroacupuncture treatment, and we further confirmed an increased protein expression of Axl and MerTK after electroacupuncture treatment, indicating their involvement during electroacupuncture treatment. Finally, LDC1267, a selective TAM kinase inhibitor, abolished the therapeutic effect of electroacupuncture on motor-coordinative dysfunction. Overall, our data demonstrate that electroacupuncture could mitigate the progression of demyelination by enhancing the TAM receptor expression to facilitate the clearance of myelin debris. Our results also suggest that electroacupuncture may be a potential curative treatment for MS patients.

TAM receptors and their ligand-mediated activation: Role in atherosclerosis.

TAM family tyrosine kinase receptors including Tyro3, Axl, and MerTK are the key efferocytosis receptors presenting on antigen-presenting cell that mediate the clearance of apoptotic cells. They are thought to regulate inflammatory diseases by modulating inflammatory response and efferocytosis. Recent studies have revealed novel roles of TAM receptors in the biosynthesis of specialized pro-resolving mediators (SPMs) and inflammation resolution. In this chapter, we discuss the central roles of TAM signaling in atherosclerosis focusing on their regulation in efferocytosis and inflammation resolution and highlight the unique therapeutic potential of SPMs in blocking the progression of atherosclerosis.

Autophagic cell death in viral infection: Do TAM receptors play a role?

Containment and clearance of invading pathogens, such as viruses, by suppression of viral replication through antiviral mechanisms (e.g. CRISPR, interferon response or programmed cell death) provide examples of evolutionary developed responses by hosts to limit the establishment of infection. Degradation of the cytoplasm en masse provides an ideal cellular response against intruding pathogens. Degradation of such scale is achieved by a process called (macro)autophagy, where double membrane vacuoles, autophagosomes, engulf cytoplasm and organelles for lysosomal degradation. However, chronic and unrestrained autophagy poses catastrophic consequences to a cell especially when vital organelles (e.g. mitochondria or nucleus) are engulfed and destroyed. Recent findings in the field of autophagy and cell death regulation describe mechanisms that distinguish whether autophagy takes a moderate or excess route. This review aims to present new perspectives and re-examines current assumptions related to cell death regulation by autophagy. The emerging role of TAM receptors in the modulation of autophagy (i.e. both homeostatic and lethal) in the context of virus infections is also discussed in addition to chemical strategies for studying autophagy.

Expression of AXL receptor tyrosine kinase relates to monocyte dysfunction and severity of cirrhosis.

Infectious complications in patients with cirrhosis frequently initiate episodes of decompensation and substantially contribute to the high mortality. Mechanisms of the underlying immuneparesis remain underexplored. TAM receptors (TYRO3/AXL/MERTK) are important inhibitors of innate immune responses. To understand the pathophysiology of immuneparesis in cirrhosis, we detailed TAM receptor expression in relation to monocyte function and disease severity prior to the onset of acute decompensation. TNF-α/IL-6 responses to lipopolysaccharide were attenuated in monocytes from patients with cirrhosis (n = 96) compared with controls (n = 27) and decreased in parallel with disease severity. Concurrently, an AXL-expressing (AXL+) monocyte population expanded. AXL+ cells (CD14+CD16highHLA-DRhigh) were characterised by attenuated TNF-α/IL-6 responses and T cell activation but enhanced efferocytosis and preserved phagocytosis of Escherichia coli Their expansion correlated with disease severity, complications, infection, and 1-yr mortality. AXL+ monocytes were generated in response to microbial products and efferocytosis in vitro. AXL kinase inhibition and down-regulation reversed attenuated monocyte inflammatory responses in cirrhosis ex vivo. AXL may thus serve as prognostic marker and deserves evaluation as immunotherapeutic target in cirrhosis.

Oncogenic role of TYRO3 receptor tyrosine kinase in the progression of pancreatic cancer.

The expression and functions of TYRO3, a member of the TAM receptor tyrosine kinase family, in pancreatic cancer (PC) have not been specifically elucidated. In this study, we confirmed TYRO3 expression in five human PC cell lines (PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and PK-9) using Western blotting. TYRO3 silencing and overexpression studies have revealed that TYRO3 promotes cell proliferation and invasion in PC via phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). Using a mouse xenograft model, we showed that tumor growth was significantly suppressed in mice subcutaneously inoculated with TYRO3-knockdown PC cells compared with mice inoculated with control PC cells. Furthermore, TYRO3 expression was examined in PC tissues obtained from 106 patients who underwent pancreatic resection for invasive ductal carcinoma through immunohistochemical staining. TYRO3-positive patients had poor prognoses for overall survival and disease-specific survival compared with TYRO3-negative patients. Multivariate analysis revealed that TYRO3 expression is an independent prognostic factor for overall survival. Our study demonstrates the critical role of TYRO3 in PC progression through Akt and ERK activation and suggests TYRO3 as a novel promising target for therapeutic strategies against PC.

Tumour-Secreted Protein S (ProS1) Activates a Tyro3-Erk Signalling Axis and Protects Cancer Cells from Apoptosis.

The TAM subfamily (Tyro3, Axl, MerTK) of receptor tyrosine kinases are implicated in several cancers, where they have been shown to support primary tumorigenesis as well as secondary resistance to cancer therapies. Relatively little is known about the oncogenic role of Tyro3, including its ligand selectivity and signalling in cancer cells. Tyro3 showed widespread protein and mRNA expression in a variety of human cancer cell lines. In SCC-25 head and neck cancer cells expressing both Tyro3 and Axl, Western blotting showed that both natural TAM ligands ProS1 and Gas6 rapidly stimulated Tyro3 and Erk kinase phosphorylation, with ProS1 eliciting a greater effect. In contrast, Gas6 was the sole stimulator of Axl and Akt kinase phosphorylation. In MGH-U3 bladder cancer cells, which express Tyro3 alone, ProS1 was again the stronger stimulator of Tyro3 and Erk stimulation but additionally stimulated Akt phosphorylation. Conditioned medium from ProS1-secreting 786-0 kidney cancer cells replicated the kinase activation effects of recombinant ProS1 in SCC-25 cells, with specificity confirmed by ProS1 ligand traps and warfarin. In addition, ProS1 protected cancer cells from acute apoptosis induced by staurosporine, as well as additionally, long-term serum starvation-induced apoptosis in MGH-U3 cells (Tyro3 only), which reflects its additional coupling to Akt signalling in these cells. In conclusion, we have shown that ProS1 is a tumour-derived functional ligand for Tyro3 that supports cancer cell survival. Furthermore, the ProS1-Tyro3 interaction is primarily coupled to Erk signalling although it displays signalling diversity dependent upon its representative expression as a TAM receptor in tumour cells.

Homeostatic Regulation of Apoptosis Governs Thymus Regeneration

Although the thymus has a remarkable capacity for repair following acute injury, such as that caused by the conditioning required for successful hematopoietic cell transplant (HCT), the mechanisms underlying this endogenous regeneration remain poorly understood. Delayed T cell reconstitution occurs following thymus insult and can exceed more than a year post-transplant due to a delay in full recovery of thymic output, function and T cell repertoire. Therefore, strategies to enhance T cell reconstitution post-transplant represents a rational approach to significantly improve the overall outcome of allo-HCT. We propose that enhancing thymic function will boost T cell reconstitution and substantially increase immune responses following allo-HCT. Our recent studies have identified two critical pathways that govern thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors (RAFs) after damage remain unclear. Given that our prior work revealed that the presence of DP thymocytes suppresses the production of RAFs like IL-23, a key downstream mediator of IL-22; and the high basal rate of thymocyte apoptosis, as apoptotic thymocytes form the bulk of developing T cells, we hypothesized that apoptotic DP thymocytes were mediating this suppression of RAFs under homeostatic conditions. Upon injury, loss of DP thymocytes leads to reduced apoptotic signaling and reduced suppression of RAFs, triggering thymic recovery (Fig 1A). Consistent with this hypothesis, our preliminary data shows a significantly reduced number of apoptotic thymocytes after total body irradiation (TBI, 550 cGy), as measured by cleaved caspase 3 levels (Fig 1B). Additionally, co-culture of apoptotic thymocytes results in reduced Bmp4 expression in ECs, which is rescued by inhibition of thymocyte apoptosis using the pan-caspase inhibitor zVAD-FMK (Fig 1C). One way in which apoptotic thymocytes could induce this suppression of RAFs is via TAM receptor activation, which is supported by our data demonstrating increased Bmp4 expression in ECs treated with a pan-TAM receptor antagonist and subsequently co-cultured with apoptotic thymocytes (Fig 1D). Interestingly, TAM receptors can activate Rac1, a Rho GTPases involved in actin cytoskeletal rearrangement; converging neatly on our previous data showing that inhibition of Rac1 with small molecule inhibitors led to robust induction of Bmp4 and Il23 expression. Therefore, we propose that in steady-state, apoptotic thymocytes activate TAM receptors on ECs and DCs and induce intracellular activation of Rac1, which ultimately suppresses the production of BMP4 and IL-23; but after damage, when the number of apoptotic thymocytes drops precipitously, this suppression is abrogated, allowing for thymic regeneration (Fig 1E). Importantly, we demonstrate here that this pathway can be therapeutically targeted, as inhibition of Rac1 in vivo with EHT1864 enhances thymus cellularity in models of acute injury (Fig. 1F), and age (Fig. 1G). As post-transplant T cell deficiency is associated with an increased risk of infections, relapse of malignancy, and the development of secondary malignancies, identifying molecular targets to enhance thymic recovery will aid in the development of therapeutics with imminent clinical need. These findings not only reveal a novel molecular mechanism governing tissue regeneration, but also offer a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by allo-HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

ACTR-48. CLINICAL DEVELOPMENT TARGETING AXL IN RECURRENT GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a lethal disease without effective therapies. Previously we reported that the TAM receptor tyrosine kinase family member AXL is upregulated in mesenchymal GBM and its knockdown induces apoptosis of mesenchymal but not proneural glioma sphere cultures (GSCs). In this study, we report that BGB324, a novel small molecule inhibitor for AXL, prolongs the survival of immunocompromised mice bearing mesenchymal GSC-derived GBM-like intracranial tumors by inhibition of AXL activation in tumor cells. Mechanistically, we identified that protein S (PROS1), known as a ligand for other TAM receptors, is secreted by tumor-associated macrophages, thereby physically associates and activates AXL in mesenchymal GSCs. The PROS1-driven phosphorylation of AXL resulted in the NF-kB activation in mesenchymal GSCs and this activation was eliminated by BGB324 treatment. In addition, BGB324 downregulated the immune checkpoint protein PD-L1 both in vitro and in vivo. Combination of BGB324 with Nivolumab - the neutralizing antibody for PD-1 (a receptor for PD-L1) was effective to prolong the survival of immunocompetent mice bearing syngeneic GBM models. Clinically, both AXL and PROS1 expression indicated poorer prognosis of GBM patients. These data suggest that the PROS1/AXL pathway regulates intrinsic mesenchymal signaling as well as extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. To translate the pre-clinical data to human GBM patients, we designed a surgical PK/PD clinical trial with BGB324 for recurrent GBM. As of May 2019, this trial, termed ABTC1701, is in pending approval by the Brain Malignancy Steering Committee in the National Cancer Institute.

A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target

Background and AimsGAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.MethodsGAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients.ResultsIn primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.ConclusionAXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer.

Targeted therapies and immunotherapy have shown promise in patients with non-small cell lung cancer (NSCLC). However, the majority of patients fail or become resistant to treatment, emphasizing the need for novel treatments. In this study, we confirm the prognostic value of levels of AXL, a member of the TAM receptor tyrosine kinase family, in NSCLC and demonstrate potent antitumor activity of the AXL-targeting antibody-drug conjugate enapotamab vedotin across different NSCLC subtypes in a mouse clinical trial of human NSCLC. Tumor regression or stasis was observed in 17/61 (28%) of the patient-derived xenograft (PDX) models and was associated with AXL mRNA expression levels. Significant single-agent activity of enapotamab vedotin was validated in vivo in 9 of 10 AXL-expressing NSCLC xenograft models. In a panel of EGFR-mutant NSCLC cell lines rendered resistant to EGFR inhibitors in vitro, we observed de novo or increased AXL protein expression concomitant with enapotamab vedotin-mediated cytotoxicity. Enapotamab vedotin also showed antitumor activity in vivo in 3 EGFR-mutant, EGFR inhibitor-resistant PDX models, including an osimertinib-resistant NSCLC PDX model. In summary, enapotamab vedotin has promising therapeutic potential in NSCLC. The safety and preliminary efficacy of enapotamab vedotin are currently being evaluated in the clinic across multiple solid tumor types, including NSCLC.

TAM receptors attenuate murine NK-cell responses via E3 ubiquitin ligase Cbl-b.

TAM receptors (Tyro3, Axl, and Mer) are receptor tyrosine kinases (RTKs) that are expressed by multiple immune cells including NK cells. Although RTKs typically enhance cellular functions, TAM receptor ligation blocks NK-cell activation. The mechanisms by which RTKs block NK-cell signaling downstream of activating receptors are unknown. In this report, we demonstrate that TAM receptors attenuate NK cell responses via the activity of E3 ubiquitin ligase Casitas B lineage lymphoma b (Cbl-b). Specifically, we show that Tyro3, Axl, and Mer phosphorylate Cbl-b, and Tyro3 ligation activates Cbl-b by phosphorylating tyrosine residues 133 and 363. Ligation of TAM receptors by their ligand Gas6 suppresses activating receptor-stimulated NK-cell functions such as IFN-γ production and degranulation, in a TAM receptor kinase- and Cbl-b-dependent manner. Moreover, Gas6 ligation induces the degradation of LAT1, a transmembrane adaptor protein required for NK cell activating receptor signaling, in WT but not in Cbl-b knock-out NK cells. Together, these results suggest that TAM receptors may attenuate NK-cell function by phosphorylating Cbl-b, which in turn dampens NK-cell activation signaling by promoting the degradation of LAT1. Our data therefore support a mechanism by which RTKs attenuate, rather than stimulate, signaling pathways via the activation of ubiquitin ligases.

TAM Receptor Pathways at the Crossroads of Neuroinflammation and Neurodegeneration.

Increasing evidence suggests that pathogenic mechanisms underlying neurodegeneration are strongly linked with neuroinflammatory responses. Tyro3, Axl, and Mertk (TAM receptors) constitute a subgroup of the receptor tyrosine kinase family, cell surface receptors which transmit signals from the extracellular space to the cytoplasm and nucleus. TAM receptors and the corresponding ligands, Growth Arrest Specific 6 and Protein S, are expressed in different tissues, including the nervous system, playing complex roles in tissue repair, inflammation and cell survival, proliferation, and migration. In the nervous system, TAM receptor signalling modulates neurogenesis and neuronal migration, synaptic plasticity, microglial activation, phagocytosis, myelination, and peripheral nerve repair, resulting in potential interest in neuroinflammatory and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Multiple Sclerosis. In Alzheimer and Parkinson diseases, a role of TAM receptors in neuronal survival and pathological protein aggregate clearance has been suggested, while in Multiple Sclerosis TAM receptors are involved in myelination and demyelination processes. To better clarify roles and pathways involving TAM receptors may have important therapeutic implications, given the fine modulation of multiple molecular processes which could be reached. In this review, we summarise the roles of TAM receptors in the central nervous system, focusing on the regulation of immune responses and microglial activities and analysing in vitro and in vivo studies regarding TAM signalling involvement in neurodegeneration.

MERTK Acts as a Costimulatory Receptor on Human CD8+ T Cells.

The TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK) is known to be expressed on antigen-presenting cells and function as oncogenic drivers and as inhibitors of inflammatory responses. Both human and mouse CD8+ T cells are thought to be negative for TAM receptor expression. In this study, we show that T-cell receptor (TCR)-activated human primary CD8+ T cells expressed MERTK and the ligand PROS1 from day 2 postactivation. PROS1-mediated MERTK signaling served as a late costimulatory signal, increasing proliferation and secretion of effector and memory-associated cytokines. Knockdown and inhibition studies confirmed that this costimulatory effect was mediated through MERTK. Transcriptomic and metabolic analyses of PROS1-blocked CD8+ T cells demonstrated a role of the PROS1-MERTK axis in differentiation of memory CD8+ T cells. Finally, using tumor-infiltrating lymphocytes (TIL) from melanoma patients, we show that MERTK signaling on T cells improved TIL expansion and TIL-mediated autologous cancer cell killing. We conclude that MERTK serves as a late costimulatory signal for CD8+ T cells. Identification of this costimulatory function of MERTK on human CD8+ T cells suggests caution in the development of MERTK inhibitors for hematologic or solid cancer treatment.

Dead Cells Induce Innate Anergy Via Mertk after Acute Viral Infection

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, and this restriction limits pathogen control. Lipopolysaccharide (LPS) tolerance is the best studied mechanism of such unresponsiveness, however whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here we found that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days after infection. Innate anergy was associated with the induction of apoptotic cells which activated the TAM receptor Mertk and induced high levels of IL-10 and TGF-β. Lack of Mertk in Mertk–/– mice prevented the induction of IL-10 and TGF-β resulting in abrogation of innate anergy. Innate anergy was associated with enhanced VSV replication and poor survival after infection. Mechanistically Mertk-signaling upregulated SOCS1 and SOCS3. Dexamethasone treatment which upregulated Mertk enhanced innate anergy in wild-type but not in Mertk-deficient mice. In conclusion we found that Mertk is one major regulator of innate tolerance during infection with VSV.

Gas6/TAM Receptors in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a multiorgan autoimmune disease associated with impaired immune system regulation. The exact mechanisms of SLE development remain to be elucidated. TAM receptor tyrosine kinases (RTKs) are important for apoptotic cell clearance, immune homeostasis, and resolution of immune responses. TAM deficiency leads to lupus-like autoimmune diseases. Activation of TAM receptors leads to proteolytic cleavage of the receptors, generating soluble forms of TAM. Circulating TAM receptors have an immunoregulatory function and may also serve as biomarkers for disease prognosis. Here, we review the biological function and signaling of TAM RTKs in the development and pathogenesis of lupus and lupus nephritis. Targeting Gas6/TAM pathways may be of therapeutic benefit. A discussion of potential TAM activation and inhibition in the treatment of lupus and lupus nephritis is included.

Sensing of apoptotic cells through Axl causes lung basal cell proliferation in inflammatory diseases.

Epithelial cell proliferation, division, and differentiation are critical for barrier repair following inflammation, but the initial trigger for this process is unknown. Here we define that sensing of apoptotic cells by the TAM receptor tyrosine kinase Axl is a critical indicator for tracheal basal cell expansion, cell cycle reentry, and symmetrical cell division. Furthermore, once the pool of tracheal basal cells has expanded, silencing of Axl is required for their differentiation. Genetic depletion of Axl triggers asymmetrical cell division, leading to epithelial differentiation and ciliated cell regeneration. This discovery has implications for conditions associated with epithelial barrier dysfunction, basal cell hyperplasia, and continued turnover of dying cells in patients with chronic inflammatory pulmonary diseases.

Abstract 4556: Targeting the TAM receptors on prostate cancer tumor-associated macrophages

Abstract Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages. Citation Format: Kayla V. Myers, Kenneth J. Pienta. Targeting the TAM receptors on prostate cancer tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4556.

Abstract 1555: Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases

Abstract The TAM receptors (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs) are important negative regulators of innate immunity. TAM receptor activation in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. TAM-deficient mice exhibit phenotypes consistent with systemic inflammation and autoimmunity. Importantly, individual ablation of TAM receptors can confer tumor immunity, increased pro-inflammatory cytokines and tumor lymphocyte infiltration, leading to the proposal that TAM receptors act as checkpoints of innate immunity. We hypothesize that pharmacological targeting of this family of receptors with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the antitumor effects observed in TAM-deficient mice. From a panel of human anti-MerTK, Axl or Tyro3 mAbs derived from phage-display libraries or human IgG expressing mice we identified unique mAbs that markedly enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Interestingly, the qualitative and quantitative pattern of cytokine response from dendritic cells was very similar using antibodies targeting the individual TAM receptors, and also similar to activation of dendritic cells using a CD40 agonist mAb, suggesting that TAM-targeting mAbs can promote immune activation. We identified surrogate mAbs targeting mouse TAM receptors that elicited similar responses in vivo, and demonstrated antitumor activity when dosed alone, or in combination with PD-1/L1 blockade in syngeneic tumor models. In addition, human MerTK transgenic, and TAM knockout mice have been generated and characterized in order to establish in vivo proof-of-concept with human TAM mAbs. Overall, pharmacological modulation of TAM receptors with mAbs enhances cytokine production in human and murine model systems consistent with the published role of TAMs as negative regulators of innate immunity. Mechanistic and proof-of-concept studies support further development of these mAbs as novel approaches to overcome these checkpoints of the innate immune response. Citation Format: Diego Alvarado, Laura Vitale, Mike Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Komal Patel, Anna Wasiuk, Jeff Weidlick, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, Russell A. Hammond, Li-Zhen He, Joel Goldstein, Lawrence J. Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1555.

Abstract CT167: A Phase Ib study to assess safety, tolerability, pharmacokinetics, and preliminary antitumor activity of sitravatinib in combination with tislelizumab in patients with advanced solid tumors

Abstract Background: Sitravatinib is an investigative, orally bioavailable, spectrum-selective receptor tyrosine kinase (RTK) inhibitor with potential antitumor activity that has been shown to potently inhibit split kinase receptors (eg, VEGFR2, KIT) and TAM receptors (eg, AXL, MER). Inhibition of RTKs by sitravatinib may also modulate effects on the tumor microenvironment to overcome resistance to checkpoint inhibitors, including enhanced M1/suppressed M2 macrophage cytokine response, abrogated negative regulation of antitumor NK cell activity, and depleted regulatory T- and myeloid-derived suppressor cells with enhanced antitumor cytotoxic T-cell activity. Tislelizumab is an investigational, humanized IgG4 monoclonal antibody that has been shown to have high affinity and binding specificity for programmed cell death receptor-1 (PD-1). Combining an agent that has both immune modulatory and antitumor properties with an immunotherapeutic PD-1 checkpoint inhibitor could enhance the antitumor efficacy observed with either agent alone. The primary objective of this trial is to examine the safety and tolerability of sitravatinib combined with tislelizumab in patients with advanced solid tumors. Methods: Adult patients with histologically or cytologically confirmed, locally advanced or metastatic, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), or ovarian cancer (OC) are enrolling in the open-label, multicenter, nonrandomized, phase 1b clinical trial. Entry criteria include Eastern Cooperative Oncology Group Performance Status ≤ 1 and adequate end-organ function. All patients will receive sitravatinib 120 mg PO QD in combination with tislelizumab 200 mg IV Q3W until progressive disease, unacceptable toxicity, death, withdrawal of consent, or study termination by the sponsor. Approximately 100 patients are expected to be enrolled in 5 cohorts (n=20 patients per cohort): Cohort A: anti-PD-(L)1 antibody-refractory/-resistant, metastatic, nonsquamous NSCLC; Cohort B: anti-PD-1(L)1 antibody-naive, metastatic, nonsquamous NSCLC; Cohort C: anti-PD-(L)1 antibody-refractory/-resistant, metastatic, clear cell RCC; Cohort D (China only): metastatic or advanced RCC without prior therapy; Cohort E: anti-PD-(L)1 antibody-naive, recurrent, platinum-resistant, epithelial OC. The primary endpoint is to assess safety and tolerability of the combination by monitoring adverse events (AEs) and serious AEs. Secondary endpoints include overall response rate, duration of response, disease control rate, progression-free survival assessed by RECIST version 1.1, and plasma concentrations and derived pharmacokinetic parameters of single- and repeated-dose sitravatinib. Seven patients have enrolled as of 3 January 2019 at the sites in Australia and China. Clinical trial registry number: NCT03666143 Citation Format: Jeffrey Goh, Ben Gao, Ben Markman, Hui Gan, Mark Voskoboynik, Yi-Long Wu, Jun Guo, Qing Zhou, Jun Zhao, Cheng Chen, Jingjun Qiu, Yingying Xu, Liu Yang, Michael Millward. A Phase Ib study to assess safety, tolerability, pharmacokinetics, and preliminary antitumor activity of sitravatinib in combination with tislelizumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT167.