Phosphatidylserine-Targeting Monoclonal Antibodies Exhibit Distinct Biochemical and Cellular Effects on Anti-CD3/CD28-Stimulated T Cell IFN-γ and TNF-α Production.

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.

Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

BACKGROUND: Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. METHODS: In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. RESULTS: Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. CONCLUSION: These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

Allosteric MAPKAPK2 inhibitors improve plaque stability in advanced atherosclerosis.

Atherosclerotic vascular disease resulting from unstable plaques is the leading cause of morbidity and mortality in subjects with type 2 diabetes (T2D), and thus a major therapeutic goal is to discover T2D drugs that can also promote atherosclerotic plaque stability. Genetic or pharmacologic inhibition of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2 or MK2) in obese mice improves glucose homeostasis and enhances insulin sensitivity. We developed two novel orally active small-molecule inhibitors of MK2, TBX-1 and TBX-2, and tested their effects on metabolism and atherosclerosis in high-fat Western diet (WD)-fed Ldlr-/- mice. Ldlr-/- mice were first fed the WD to allow atherosclerotic lesions to become established, and the mice were then treated with TBX-1 or TBX-2. Both compounds improved glucose metabolism and lowered plasma cholesterol and triglyceride, without an effect on body weight. Most importantly, the compounds decreased lesion area, lessened plaque necrosis, and increased fibrous cap thickness in the aortic root lesions of the mice. Thus, in a preclinical model of high-fat feeding and established atherosclerosis, MK2 inhibitors improved metabolism and also enhanced atherosclerotic plaque stability, suggesting potential for further clinical development to address the epidemic of T2D associated with atherosclerotic vascular disease.

Efferocytosis induces macrophage proliferation to help resolve tissue injury.

Apoptotic cell clearance by macrophages (efferocytosis) promotes resolution signaling pathways, which can be triggered by molecules derived from the phagolysosomal degradation of apoptotic cells. We show here that nucleotides derived from the hydrolysis of apoptotic cell DNA by phagolysosomal DNase2a activate a DNA-PKcs-mTORC2/Rictor pathway that increases Myc to promote non-inflammatory macrophage proliferation. Efferocytosis-induced proliferation expands the pool of resolving macrophages in vitro and in mice, including zymosan-induced peritonitis, dexamethasone-induced thymocyte apoptosis, and atherosclerosis regression. In the dexamethasone-thymus model, hematopoietic Rictor deletion blocked efferocytosing macrophage proliferation, apoptotic cell clearance, and tissue resolution. In atherosclerosis regression, silencing macrophage Rictor or DNase2a blocked efferocyte proliferation, apoptotic cell clearance, and plaque stabilization. In view of previous work showing that other types of apoptotic cell cargo can promote resolution in individual efferocytosing macrophages, the findings here suggest that signaling-triggered apoptotic cell-derived nucleotides can amplify this benefit by increasing the number of these macrophages.

Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis.

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet-fed Ldlr-/- mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps - all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Axl and Mertk Receptors Cooperate to Promote Breast Cancer Progression by Combined Oncogenic Signaling and Evasion of Host Antitumor Immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

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.

Cyclophilin A Inhibitor Debio-025 Targets Crk, Reduces Metastasis, and Induces Tumor Immunogenicity in Breast Cancer.

The Crk adaptor protein, a critical modifier of multiple signaling pathways, is overexpressed in many cancers where it contributes to tumor progression and metastasis. Recently, we have shown that Crk interacts with the peptidyl prolyl cis-trans isomerase, Cyclophilin A (CypA; PP1A) via a G219P220Y221 (GPY) motif in the carboxyl-terminal linker region of Crk, thereby delaying pY221 phosphorylation and preventing downregulation of Crk signaling. Here, we investigate the physiologic significance of the CypA/Crk interaction and query whether CypA inhibition affects Crk signaling in vitro and in vivo. We show that CypA, when induced under conditions of hypoxia, regulates Crk pY221 phosphorylation and signaling in cancer cell lines. Using nuclear magnetic resonance spectroscopy, we show that CypA binds to the Crk GPY motif via the catalytic PPII domain of CypA, and small-molecule nonimmunosuppressive inhibitors of CypA (Debio-025) disrupt the CypA-CrkII interaction and restores phosphorylation of Crk Y221. In cultured cell lines, Debio-025 suppresses cell migration, and when administered in vivo in an orthotopic model of triple-negative breast cancer, Debio-025 showed antitumor efficacy either alone or in combination with anti-PD-1 mAb, reducing both tumor volume and metastatic lung dispersion. Furthermore, when analyzed by NanoString immune profiling, treatment of Debio-025 with anti-PD-1 mAb increased both T-cell signaling and innate immune signaling in tumor microenvironment. IMPLICATIONS: These data suggest that pharmacologic inhibition of CypA may provide a promising and unanticipated consequence in cancer biology, in part by targeting the CypA/CrkII axis that regulates cell migration, tumor metastasis, and host antitumor immune evasion.

Macrophage MerTK Promotes Liver Fibrosis in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is emerging as a leading cause of chronic liver disease. However, therapeutic options are limited by incomplete understanding of the mechanisms of NASH fibrosis, which is mediated by activation of hepatic stellate cells (HSCs). In humans, human genetic studies have shown that hypomorphic variations in MERTK, encoding the macrophage c-mer tyrosine kinase (MerTK) receptor, provide protection against liver fibrosis, but the mechanisms remain unknown. We now show that holo- or myeloid-specific Mertk targeting in NASH mice decreases liver fibrosis, congruent with the human genetic data. Furthermore, ADAM metallopeptidase domain 17 (ADAM17)-mediated MerTK cleavage in liver macrophages decreases during steatosis to NASH transition, and mice with a cleavage-resistant MerTK mutant have increased NASH fibrosis. Macrophage MerTK promotes an ERK-TGFß1 pathway that activates HSCs and induces liver fibrosis. These data provide insights into the role of liver macrophages in NASH fibrosis and provide a plausible mechanism underlying MERTK as a genetic risk factor for NASH fibrosis.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

The role of non-resolving inflammation in atherosclerosis.

Non-resolving inflammation drives the development of clinically dangerous atherosclerotic lesions by promoting sustained plaque inflammation, large necrotic cores, thin fibrous caps, and thrombosis. Resolution of inflammation is not merely a passive return to homeostasis, but rather an active process mediated by specific molecules, including fatty acid-derived specialized pro-resolving mediators (SPMs). In advanced atherosclerosis, there is an imbalance between levels of SPMs and proinflammatory lipid mediators, which results in sustained leukocyte influx into lesions, inflammatory macrophage polarization, and impaired efferocytosis. In animal models of advanced atherosclerosis, restoration of SPMs limits plaque progression by suppressing inflammation, enhancing efferocytosis, and promoting an increase in collagen cap thickness. This Review discusses the roles of non-resolving inflammation in atherosclerosis and highlights the unique therapeutic potential of SPMs in blocking the progression of clinically dangerous plaques.

MerTK signaling in macrophages promotes the synthesis of inflammation resolution mediators by suppressing CaMKII activity.

Inflammation resolution counterbalances excessive inflammation and restores tissue homeostasis after injury. Failure of resolution contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated by endogenous specialized proresolving mediators (SPMs), which are derived from long-chain fatty acids by lipoxygenase (LOX) enzymes. 5-LOX plays a critical role in the biosynthesis of two classes of SPMs: lipoxins and resolvins. Cytoplasmic localization of the nonphosphorylated form of 5-LOX is essential for SPM biosynthesis, whereas nuclear localization of phosphorylated 5-LOX promotes proinflammatory leukotriene production. We previously showed that MerTK, an efferocytosis receptor on macrophages, promotes SPM biosynthesis by increasing the abundance of nonphosphorylated, cytoplasmic 5-LOX. We now show that activation of MerTK in human macrophages led to ERK-mediated expression of the gene encoding sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2), which decreased the cytosolic Ca2+ concentration and suppressed the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). This, in turn, reduced the activities of the mitogen-activated protein kinase (MAPK) p38 and the kinase MK2, resulting in the increased abundance of the nonphosphorylated, cytoplasmic form of 5-LOX and enhanced SPM biosynthesis. In a zymosan-induced peritonitis model, an inflammatory setting in which macrophage MerTK activation promotes resolution, inhibition of ERK activation delayed resolution, which was characterized by an increased number of neutrophils and decreased amounts of SPMs in tissue exudates. These findings contribute to our understanding of how MerTK signaling induces 5-LOX-derived SPM biosynthesis and suggest a therapeutic strategy to boost inflammation resolution in settings where defective resolution promotes disease progression.

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.

Phosphatidylserine Sensing by TAM Receptors Regulates AKT-Dependent Chemoresistance and PD-L1 Expression.

Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In addition, we leveraged this system to engineer epithelial cells that express wild-type TAM receptors and show that although each receptor can promote PS-mediated efferocytosis, AKT-mediated chemoresistance, as well as upregulate the immune checkpoint molecule PD-L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM receptor-mediated efferocytosis could be partially blocked by PS-targeting antibody 11.31 and Annexin V, demonstrating the existence of a PS/PS receptor (i.e., TAM receptor)/PD-L1 axis that operates in epithelial cells to foster immune escape. These data provide a rationale that PS-targeting, anti-TAM receptor, and anti-PD-L1-based therapeutics will have merit as combinatorial checkpoint inhibitors.Implications: Many tumor cells are known to upregulate the immune checkpoint inhibitor PD-L1. This study demonstrates a role for PS and TAM receptors in the regulation of PD-L1 on cancer cells. Mol Cancer Res; 15(6); 753-64. ©2017 AACR.

Contribution of Defective PS Recognition and Efferocytosis to Chronic Inflammation and Autoimmunity.

The rapid and efficient clearance of apoptotic cells results in the elimination of auto-antigens and provides a strong anti-inflammatory and immunosuppressive signal to prevent autoimmunity. While professional and non-professional phagocytes utilize a wide array of surface receptors to recognize apoptotic cells, the recognition of phosphatidylserine (PS) on apoptotic cells by PS receptors on phagocytes is the emblematic signal for efferocytosis in metazoans. PS-dependent efferocytosis is associated with the production of anti-inflammatory factors such as IL-10 and TGF-ß that function, in part, to maintain tolerance to auto-antigens. In contrast, when apoptotic cells fail to be recognized and processed for degradation, auto-antigens persist, such as self-nucleic acids, which can trigger immune activation leading to autoantibody production and autoimmunity. Despite the fact that genetic mouse models clearly demonstrate that loss of PS receptors can lead to age-dependent auto-immune diseases reminiscent of systemic lupus erythematosus (SLE), the link between PS and defective clearance in chronic inflammation and human autoimmunity is not well delineated. In this perspective, we review emerging questions developing in the field that may be of relevance to SLE and human autoimmunity.