Eph Receptor Tyrosine Kinases in Tumor Immunity Eileen Shiuan1,2 and Jin Chen1,3,4,5,6

Home , EPHB6, Ephrin, Ephrin A1, Ephrin A4, Ephrin B1, Ephrin B2

Published OnlineFirst November 3, 2016; DOI: 10.1158/0008-5472.CAN-16-1521 Cancer Review Research

Abstract

The family of Eph receptor tyrosine kinases and their ephrin area in which Eph receptors remain understudied. Here, we ligands regulate a diverse array of physiologic processes, such as provide the first comprehensive review of Eph receptors in the axonal guidance, bone remodeling, and immune cell develop- context of tumor immunity. With the recent rise of cancer ment and trafficking. Eph/ephrin interactions have also been immunotherapies as promising therapeutic interventions, fur- implicated in various pathologic processes, including inflam- ther elucidation of the roles of Eph receptors in the tumor mation, cancer, and tumor angiogenesis. Because Eph receptors immune microenvironment will be critical for understanding play prominent roles in both the immune system and cancer, and developing novel targets against tumor immune evasion. they likely impact the tumor immune microenvironment, an Cancer Res; 76(22); 1–6. 2016 AACR.

Introduction and anti-PD-1 mAbs (nivolumab and pembrolizumab) have proven successful in the clinic. However, these checkpoint inhi- Over the past several decades, the paradigm of cancer research bitors are collectively only approved for melanoma, renal cell and anticancer therapy has shifted from a focus on solely targeting carcinoma (RCC), non–small cell lung cancer, and Hodgkin lym- tumor cells to a broader approach of understanding and remodel- phoma, and head and neck squamous cell carcinoma. Typically, ing the tumor microenvironment. The tumor microenvironment only 15% to 40% of patients respond favorably to checkpoint contains a diverse population of host cells, among which include inhibitor monotherapy (2–4), although response rates up to 61% immune cells that are often hijacked by cancer cells to fulfill the have been reported for combination ipilimumab and nivolumab tumor's agenda for growth and metastasis. Recently, increasing therapy (4). Thus, it is clear that other critical factors in tumor attention has been directed toward investigating cancer cell eva- immunity have yet to be characterized and explored as targets. sion of immune destruction, and breakthroughs in this field have led to novel strategies for immunotherapy. The Eph Receptor Tyrosine Kinase Family Overview of Tumor Immunity The Eph receptor tyrosine kinase (RTK) family comprises the largest group of surface receptors and is categorized into EphA In 2013, Chen and Mellman proposed a model of the "cancer- or EphB subclasses based on sequence homology and prefer- immunity cycle," a multi-step process that includes the release of ential binding to their ephrin-A and ephrin-B ligands, respec- cancer cell antigens, antigen presentation, priming and activation tively (5). In humans, nine EphA (EphA1-8,10) and five EphB of T cells, trafficking of T cells to tumors, and recognition and (EphB1-4,6) receptors are expressed, along with five ephrin-A killing of cancer cells by cytotoxic T lymphocytes (CTL; ref. 1). This and three ephrin-B ligands. Unlike most RTKs, Eph receptors model of tumor immunity, although simplified and lacking other interact with ligands that are often membrane bound, allowing important players like natural killer cells, provides an organized both "forward signaling" in the receptor-bound cell and approach to identifying vulnerabilities capitalized by cancer cells. "reverse signaling" in the ephrin-bound cell (Fig. 1A). In The best-studied examples thus far include inhibition of T-cell addition to "forward signaling," Eph receptors can signal in activation through cytotoxic T lymphocyte antigen 4 (CTLA-4) and the absence of ligand binding and kinase activation through exhaustion of T-cell effector function through programmed death 1 cross-talk with other RTKs, such as HER2 (6, 7). Eph receptors (PD-1) receptor signaling. Therapeutic anti-CTLA-4 (ipilimumab) are involved in a wide range of physiologic and pathologic processes, including inflammation and cancer. Although their roles in immune cell development, migration, and activation 1Department of Cancer Biology, Vanderbilt University School of Med- icine, Nashville, Tennessee. 2Medical Scientist Training Program, Van- (8, 9), as well as their involvement in tumorigenesis, tumor derbilt University, Nashville, Tennessee. 3Division of Rheumatology angiogenesis, and cancer stem cells (10–12), have been and Immunology, Department of Medicine, Vanderbilt University, reviewed extensively, the junction between these two areas has Nashville, Tennessee. 4Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee. 5Department of yet to be rigorously examined. Here, we attempt to bridge this Cell & Developmental Biology, Vanderbilt University, Nashville, Ten- gap and provide the first comprehensive review of Eph RTKs in nessee. 6Veterans Affairs Medical Center,Tennessee Valley Healthcare the context of tumor immunity. System, Nashville, Tennessee. Corresponding Author: Jin Chen, T-3207E, Medical Center North, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232. Eph Receptor–Derived Tumor-Associated Phone: 615-343-3819; Fax: 615-343-8648; E-mail: [email protected] Antigens doi: 10.1158/0008-5472.CAN-16-1521 The first Eph receptor identified as a tumor-associated anti- 2016 American Association for Cancer Research. gen (TAA) was EphA3 (13), which is overexpressed in several

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Shiuan and Chen

A Ephrin-A Ephrin-B

GPI anchor PDZ binding motif Eph-binding Ephrin-binding domain domain Eph-binding domain Reverse signaling Fibronectin III Juxtamembrane region repeats Forward signaling

Kinase domain Eph receptor

PDZ binding motif Figure 1. SAM domain A, structural elements of an Eph receptor and ephrin ligands. B, graphical summary of potential roles B of Eph receptors in the cancer- Tumor antigen immunity cycle. Eph receptors are recognition and induced sources of immunogenic TAAs. tumor cell apoptosis Interaction between ephrin and Eph receptor in the vasculature may regulate lymphocyte trafﬁcking to Cytotoxic T-cell tumor. In addition, EphB receptors may serve as costimulation molecules EphA1 to stimulate T-cell activation in the EphA4 TCR EphA2 tumor microenvironment. Eph epitope EphA3 T-cell Kill EphB4 EphB6 MHCI Naïve Integrins T-cell Tumor cell TCR VCAM-1 Immune cell Immune cell extravasation ICAM-1 activation and migration

Endothelial MHC II cell EphA2 Dendritic cell

þ malignancies, including melanoma. In this study, a CD4 T-cell Multiple EphA2-derived epitopes are recognized by human þ þ clone isolated from a melanoma patient with an exceptionally CD4 or CD8 T cells (15, 16). One particular epitope favorable clinical course was found to recognize an EphA3 (EphA2883-891) from the C-terminus of human EphA2 induces þ epitope and elicit selective immunoreactivity against melano- significant immunoreactivity in CD8 T cells via MHC I–restricted ma cell lines. presentation against RCC and glioma cell lines in vitro (15, 17, 18). Subsequently, EphA2 has become the best-studied source of Reactive T-cell clones were isolated from both healthy donors and TAAs in the Eph family (Fig. 1B). At baseline expression levels, patients with glioma or RCC; however, they were only identified EphA2 typically binds its main ligand ephrin-A1 and facilitates after ex vivo expansion and antigen stimulation. Whether these epithelial cell-to-cell adhesion. However, when overexpressed in EphA2883-891-specific T cells are generated spontaneously and many solid tumors, such as melanoma, glioma, RCC, and breast confer antitumor activity in humans remains unknown. Further and lung cancer, EphA2 signals in a ligand-independent fashion investigation will likely require a more direct and sensitive meth- through cross-talk with EGFR and HER2 and promotes tumor od of detecting specific T-cell clones while minimizing ex vivo growth and angiogenesis (7, 14). manipulation.

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These early studies led to several preclinical investigations Eph Receptor–Mediated Immune Cell – adopting vaccinations with EphA2 peptide pulsed dendritic cells Trafficking (DC) in mouse tumor models of syngeneic glioma, sarcoma, melanoma, and colorectal carcinoma (19–21). In addition, Besides inducing immunologic responses, Eph receptors can ephrin-A1 has been used to activate DCs in a rat glioma model regulate immune cell trafficking. EphA2 is expressed on endothe- (22). The data from these preclinical models suggest that these DC lial cells and may mediate lymphocyte adhesion to vessel walls, vaccines induce immune responses and decrease tumor burden, the first step in lymphocyte extravasation. Expression of both although the findings invite further investigation. In these studies, EphA2 and its main ligand ephrin-A1 is upregulated in activated antigen-specific cytotoxic T-cell clones were isolated from vacci- endothelium in vitro, and stimulation of EphA2 signaling through þ nated animals, and in vivo depletion of CD8 T cells blocked the ephrin-A1 binding leads to increased expression of adhesion reduction in tumor size induced by vaccination. However, both proteins, such as E-selectin and VCAM-1, that bind to leukocyte primary tumor and metastasis models in these cases were non- integrins (27). Conversely, EphA2 binding to ephrin-A1 on T cells orthotopic and not ideal representations of human disease. results in reverse signaling that promotes T-cell adhesion to Furthermore, none of the studies analyzed tumor-infiltrating VCAM-1 and another vascular adhesion protein ICAM-1 immune populations or reported on the effects on survival. (Fig. 1B; ref. 28). However, these interactions have not been tested Interestingly, the mouse EphA2 peptides used to stimulate DCs in a coculture system or transendothelial migration model. in these studies are not homologous to EphA2883-891 in humans. Although cursory evidence suggests that EphA2 signaling may be This highlights the differences in MHC molecules across species modulating the NF-kB pathway to affect expression of adhesion and their different binding affinities for peptides, which becomes proteins (29), further mechanistic studies are needed to elucidate a significant barrier to translating these models into treatments for the signaling pathways downstream of both EphA2 and ephrin- patients. Thus, future research in this area will likely benefit from A1 that may lead to changes in lymphocyte adhesion to endo- using humanized mouse models. thelial cells. Furthermore, as these in vitro studies were conducted A few studies mentioned above (17–19) have led EphA2 to in the context of atherosclerosis, whether the adhesion induced by become a prominent target for immunotherapy in glioma and EphA2 and ephrin-A1 binding facilitates lymphocyte extravasa- provided rationale for clinical trials to test the safety of com- tion through activated tumor vessels remains to be investigated. in vitro bination peptide vaccines with EphA2883-891 plus other TAAs In addition to investigations, the interaction between in patients with gliomas (23–25). Reports on toxicity and ephrin-A1 and EphA2 has been studied in various lung injury and preliminary efficacy demonstrate that the peptide vaccinations inflammation mouse models (29, 30); however, their effects on are generally well tolerated and elicit clinical and immuno- lung vasculature remain equivocal and are likely model depen- logic T-cell responses. However, the data contain wide inter- dent. For example, Carpenter and colleagues report that EphA2 patient variability and do not control for key confounding knockout mice have reduced vascular permeability, immune cell factors, such as prior chemotherapy and/or radiation regimens, infiltration, and chemokine production in a bleomycin-induced that can have a notable impact on immune function. Later lung injury model (29), whereas Okazaki and colleagues con- phase trials will hopefully provide greater insight on the clude that the same knockout mice have increased immune cell efficacy of combination peptide vaccines in patients with infiltration and inflammatory cytokine production in Mycoplas- glioma. Currently, a vaccine trial testing a group of TAAs, ma and ovalbumin-induced inflammatory lung models com- in vivo including EphA2883-891, in combination with dasatinib, an pared with their wild-type counterparts (30). Further inhibitor of Src and EphA2, is ongoing in patients with studies are needed to clarify the effect of EphA2 in the lung melanoma (26). Despite these advances, EphA2883-891 and vasculature. other TAAs used in peptide vaccines can only be presented Two other binding partners of ephrin-A1 are EphA1 and EphA4, which are expressed in T cells and mediate T-cell chemotaxis in and potentially induce responses in patients with the MHC I þ vitro (Fig. 1B; refs. 31–33). Ephrin-A1 stimulation of CD4 and haplotype HLA-A2. This greatly limits the patient population þ that may respond to such therapies. CD8 T cells promotes chemotaxis in response to stromal cell- Although the discussion thus far has focused on CTL function in derived factor 1a (SDF-1a)/CXCL12 and macrophage inflamma- relation to TAA immunogenicity, a few studies have also exam- tory protein 3b (MIP-3b)/CCL19 in vitro. EphA1 is expressed on þ þ þ ined the roles of other immune cell subsets, such as CD4 T cells. CD4 and CD8 T cells and facilitates migration of both T-cell For example, Tatsumi and colleagues identified MHC II–restricted subsets via recruitment of FAK-like kinase Pyk2, Src kinase Lck, þ Rho-GEF Vav1, and PI3K (31, 32). On the other hand, EphA4 is EphA2 epitopes and examined the immunoreactivity of CD4 þ þ T-cell subsets from RCC patients against these peptides (15). They not expressed on CD8 T cells but facilitates CD4 T-cell migra- þ found that Th1 polarization of CD4 T cells is greater in patients tion through activation of Vav1 and Src kinases Lck and Fyn, as with stage I disease, while Th2 polarization and regulatory T cell well as potential dimerization with EphA1 (32, 33). Surprisingly, (Treg) differentiation, both generally considered to be immuno- administration of a pan-Src inhibitor appears to increase T-cell suppressive and tumor-promoting responses, are increased in chemotaxis in response to CCL19 and CXCL12 (31, 32). These patients with more advanced disease. In in vivo models, Yama- chemokines both regulate lymphocyte homing, although þ guchi and colleagues provided preliminary evidence that CD4 CXCL12 in particular is overexpressed in several types of cancer T cells can partially mediate antitumor responses induced by and can promote tumor growth, metastasis, and angiogenesis EphA2 peptide–pulsed DCs (20, 21). Further elucidation of the through signaling of its receptor CXCR4 on cancer cells (34). Thus, effects of EphA2 peptide and DC vaccines on T-cell subsets, as well evaluation of EphA receptor roles in T-cell recruitment to tumors, as novel investigations on other immune cell populations, will especially those that are driven by CXCL12/CXCR4, may provide broaden our understanding of the mechanisms and advance additional insight in the immune microenvironment of these development of these anticancer therapies. tumors.

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Although the association between EphA receptors and T-cell ephrin-B1 and B2 may modulate costimulatory function. Kawano trafficking is well established, some evidence suggests that EphB and colleagues show that high concentrations of ephrin-B1 and receptors regulate monocyte migration. Interactions between B2, although not ephrin-B3, can inhibit instead of stimulate T-cell ephrin-B2 on endothelial cells and EphB4 expressed on mono- activation through EphB4 signaling (46). Nguyen and colleagues cytes inhibit their chemotaxis and transendothelial migration report that ephrin-B2 and EphB2 expressed on mesenchymal (35, 36). Although these studies are framed in the context of stromal/stem cells (MSC) can suppress T-cell activation via inter- atherosclerosis, ephrin-B2 also plays a crucial role in angiogenesis action with EphB4 and ephrin-B1 on T cells, respectively (47). The through regulation of VEGFR signaling (37, 38) and may affect collective interactions of EphBs and ephrin-Bs between T cells and monocyte trafficking through the tumor vasculature. In addition MSCs increase expression of TGFb1, indoleamine 2,3-dioxygen- to T-cell and monocyte recruitment, there is sparse literature on ase, and inducible nitric oxide synthase, all three of which are Eph/ephrin involvement in trafficking of other immune cells, immunosuppressive factors that can promote tumor immune mainly B cells (39) and DCs (40). evasion (48). Although these investigations examining Ephs/ephrins in Besides EphB/ephrin-B involvement in T-cell costimulation immune cell chemotaxis and migration have not utilized cancer and suppression, very little is known about the roles of other models, much of the understanding gained from this work can Eph receptors in activating immune cells, except scant evidence be easily applied and tested in the context of tumor immunity. suggesting that EphA2 expressed on DCs may help activate B cells EphA/ephrin-A1 interactions between lymphocytes and endo- (49). Given the overwhelming evidence of EphB/ephrin-B thelial cells may play a role in lymphocyte infiltration of engagement in T-cell costimulation, the activation of T cells tumors, whereas EphB/ephrin-B2 may have similar functions against TAAs and their differentiation into tumor-suppressing in monocytes and tumor-associated macrophages. Additional CTLs may be affected by EphB/ephrin-B interactions. Further research in Eph/ephrin interactions in the immune system will investigation in this area using cancer models may provide insight help redress inconsistencies in our current understanding and into how we may capitalize on Eph/ephrin relationships to perhaps reveal novel targets in the tumor microenvironment or accentuate the antitumor immune response. treatment strategies. Concluding Remarks Eph Receptors in Immune Cell Activation Eph receptors are sources of immunogenic TAAs and have The balance between costimulatory and inhibitory signals from critical functions in the immune system that likely impact the DCs to na€ve T cells can direct the fate of T cells toward differ- tumor immune microenvironment (Fig. 1B). Thus, modulating entiation into a tumor fighting or immunosuppressive subclass, Eph receptor activities, in principle, could be leveraged to improve like Tregs, or apoptosis, leading to immune tolerance of tumor tumor immune therapies. However, much is still unknown about cells. Although little is known about EphB/ephrin-B interactions how Eph receptors regulate tumor immunity. First, many of the in facilitating T-cell trafficking, their role in T-cell costimulation studies investigating Eph receptors in immune responses have yet has been well characterized (Fig. 1B; refs. 41–45). to be translated in cancer models. Second, as the EphA2 receptor is In murine T cells, in vitro stimulation of EphB receptors by often not mutated in human cancer, it is unclear how the immune ephrin-B1, B2, or B3 leads to EphB colocalization with the system distinguishes EphA2 peptide–MHC complexes on tumor T-cell receptor (TCR) complex and T-cell activation, indicated cells from those on normal tissue. Third, Eph receptor kinase by MAPK pathway signaling, T-cell proliferation, IFNg secre- inhibitors have been developed (50, 51), but their impact on the tion, and cytotoxic activity (41–43). Likewise, stimulation of immune system is unknown. Finally, although EphA2 is EphB6 expressed on human T cells with ephrin-B2 yields expressed on DCs (40), its role in tumor immunity remains to similar results (44). EphB6 activation leads to increased be investigated. In summary, although there is abundant literature expression of T-cell activation markers CD25 and CD69 and on Eph receptors and ephrins in cancer biology, as well as secretion of several proinflammatory cytokines, including immunology, this family of RTKs is highly understudied in the IFNg and TNFa, although not IL2, the main cytokine upregu- context of tumor immunity. This gap in our current knowledge lated in T cells by canonical APC costimulation. The traditional identifies a distinct opportunity for new discoveries that may costimulatory B7 molecules on DCs induce IL2 transcription advance our understanding of the tumor microenvironment and in T cells and their survival, whereas other costimulatory pave the way for novel immunotherapeutic targets. receptors, such as those in the TNF receptor family, promote proliferation and differentiation without affecting IL2 levels. Thus, EphB6 appears to function similarly to this last group of Disclosure of Potential Conflicts of Interest fl costimulatory receptors and, because it promotes IFNg secre- No potential con icts of interest were disclosed. tion and cytotoxic activity, may promote CTL differentiation. Further evidence of this is demonstrated in EphB6 knockout Grant Support mice that have impaired T-cell activation and function and This work was supported by the Department of Veterans Affairs through a VA reduced TCR downstream signaling proteins, such as activated Merit Award and a Research Career Scientist Award (J. Chen), NIH grants R01 ZAP-70, LAT, PLC-g, and Erk1/2 (45). CA95004 and CA177681 (J. Chen) and T32 GM0734 (NIH/National Institute Despite the evidence presented in these studies, EphB/ephrin-B of General Medical Sciences; E. Shiuan), and a Breast Cancer Program pilot involvement in T-cell activation is likely more complex. For project from the Vanderbilt-Ingram Cancer Center. example, other EphB receptors expressed on T cells, such as EphB4, may share redundant functions with EphB6 in stimulating Received June 3, 2016; revised July 15, 2016; accepted July 20, 2016; T-cell proliferation (45). Moreover, the concentration of available published OnlineFirst November 3, 2016.

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OF6 Cancer Res; 76(22) November 15, 2016 Cancer Research

Eph Receptor Tyrosine Kinases in Tumor Immunity

Eileen Shiuan and Jin Chen

Cancer Res Published OnlineFirst November 3, 2016.

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