Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

Targeted degradation of MERTK and other TAM receptor paralogs by heterobifunctional targeted protein degraders.

TAM receptors (TYRO3, AXL, and MERTK) comprise a family of homologous receptor tyrosine kinases (RTK) that are expressed across a range of liquid and solid tumors where they contribute to both oncogenic signaling to promote tumor proliferation and survival, as well as expressed on myeloid and immune cells where they function to suppress host anti-tumor immunity. In recent years, several strategies have been employed to inhibit TAM kinases, most notably small molecule tyrosine kinase inhibitors and inhibitory neutralizing monoclonal antibodies (mAbs) that block receptor dimerization. Targeted protein degraders (TPD) use the ubiquitin proteasome pathway to redirect E3 ubiquitin ligase activity and target specific proteins for degradation. Here we employ first-in-class TPDs specific for MERTK/TAMs that consist of a cereblon E3 ligase binder linked to a tyrosine kinase inhibitor targeting MERTK and/or AXL and TYRO3. A series of MERTK TPDs were designed and investigated for their capacity to selectively degrade MERTK chimeric receptors, reduce surface expression on primary efferocytic bone marrow-derived macrophages, and impact on functional reduction in efferocytosis (clearance of apoptotic cells). We demonstrate proof-of-concept and establish that TPDs can be tailored to either selectivity degrades MERTK or concurrently degrade multiple TAMs and modulate receptor expression in vitro and in vivo. This work demonstrates the utility of proteome editing, enabled by tool degraders developed here towards dissecting the therapeutically relevant pathway biology in preclinical models, and the ability for TPDs to degrade transmembrane proteins. These data also provide proof of concept that TPDs may serve as a viable therapeutic strategy for targeting MERTK and other TAMs and that this technology could be expanded to other therapeutically relevant transmembrane proteins.

Mertk: An emerging target in cancer biology and immuno-oncology.

Mertk, a type I Receptor Tyrosine Kinase (RTK) and member of the TAM (Tyro3, Axl, and Mertk) family of homologous tyrosine kinases, has important roles in signal transduction both homeostatically on normal cells as well as patho-physiologically on both tumor-associated macrophages and malignant cells by its overexpression in a wide array of cancers. The main ligands of Mertk are Vitamin K-modified endogenous proteins Gas6 and Protein S (ProS1), heterobifunctional modular proteins that bind Mertk via two carboxyl-terminal laminin-like globular (LG) domains, and an N-terminal Gla domain that binds anionic phospholipids, whereby externalized phosphatidylserine (PS) on stressed viable and caspase-activated apoptotic cells is most emblematic. Recent studies indicate that Vitamin K-dependent γ-carboxylation on the N-terminal Gla domain of Gas6 and Protein S is necessary for PS binding and Mertk activation, implying that Mertk is preferentially active in tissues where there is high externalized PS, such as the tumor microenvironment (TME) and acute virally infected tissues. Once stimulated, activated Mertk can provide a survival advantage for cancer cells as well as drive compensatory proliferation. On monocytes and tumor-associated macrophages, Mertk promotes efferocytosis and acts as an inhibitory receptor that impairs host anti-tumor immunity, functioning akin to a myeloid checkpoint inhibitor. In recent years, inhibition of Mertk has been implicated in a dual role to enhance the sensitivity of cancer cells to cytotoxic agents along with improving host anti-tumor immunity with anti-PD-1/PD-L1 immunotherapy. Here, we examine the rationale of Mertk-targeted immunotherapies, the current and potential therapeutic strategies, the clinical status of Mertk-specific therapies, and potential challenges and obstacles for Mertk-focused therapies.

Phosphatidylserine externalization by apoptotic cells is dispensable for specific recognition leading to innate apoptotic immune responses.

Surface determinants newly expressed by apoptotic cells that are involved in triggering potent immunosuppressive responses, referred to as "innate apoptotic immunity (IAI)" have not been characterized fully. It is widely assumed, often implicitly, that phosphatidylserine, a phospholipid normally cloistered in the inner leaflet of cells and externalized specifically during apoptosis, is involved in triggering IAI, just as it plays an essential role in the phagocytic recognition of apoptotic cells. It is notable, however, that the triggering of IAI in responder cells is not dependent on the engulfment of apoptotic cells by those responders. Contact between the responder and the apoptotic target, on the other hand, is necessary to elicit IAI. Previously, we demonstrated that exposure of protease-sensitive determinants on the apoptotic cell surface are essential for initiating IAI responses; exposed glycolytic enzyme molecules were implicated in particular. Here, we report our analysis of the involvement of externalized phosphatidylserine in triggering IAI. To analyze the role of phosphatidylserine, we employed a panel of target cells that either externalized phosphatidylserine constitutively, independently of apoptosis, or did not, as well as their WT parental cells that externalized the phospholipid in an apoptosis-dependent manner. We found that the externalization of phosphatidylserine, which can be fully uncoupled from apoptosis, is neither sufficient nor necessary to trigger the profound immunomodulatory effects of IAI. These results reinforce the view that apoptotic immunomodulation and phagocytosis are dissociable and further underscore the significance of protein determinants localized to the cell surface during apoptosis in triggering innate apoptotic immunity.

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.

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.

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.

Cell Death in the Tumor Microenvironment: Implications for Cancer Immunotherapy.

The physiological fate of cells that die by apoptosis is their prompt and efficient removal by efferocytosis. During these processes, apoptotic cells release intracellular constituents that include purine nucleotides, lysophosphatidylcholine (LPC), and Sphingosine-1-phosphate (S1P) that induce migration and chemo-attraction of phagocytes as well as mitogens and extracellular membrane-bound vesicles that contribute to apoptosis-induced compensatory proliferation and alteration of the extracellular matrix and the vascular network. Additionally, during efferocytosis, phagocytic cells produce a number of anti-inflammatory and resolving factors, and, together with apoptotic cells, efferocytic events have a homeostatic function that regulates tissue repair. These homeostatic functions are dysregulated in cancers, where, aforementioned events, if not properly controlled, can lead to cancer progression and immune escape. Here, we summarize evidence that apoptosis and efferocytosis are exploited in cancer, as well as discuss current translation and clinical efforts to harness signals from dying cells into therapeutic strategies.