Eph Receptor Family Research Articles

Biological Significance of EphB4 Expression in Cancer.

Eph receptors and their Eph receptor-interacting (ephrin) ligands comprise a vital cell communication system with several functions. In cancer cells, there was evidence of bilateral Eph receptor signaling with both tumor-suppressing and tumor-promoting actions. As a member of the Eph receptor family, EphB4 has been linked to tumor angiogenesis, growth, and metastasis, which makes it a viable and desirable target for drug development in therapeutic applications. Many investigations have been conducted over the last decade to elucidate the structure and function of EphB4 in association with its ligand ephrinB2 for its involvement in tumorigenesis. Although several EphB4-targeting drugs have been investigated, and some selective inhibitors have been evaluated in clinical trials. This article addresses the structure and function of the EphB4 receptor, analyses its possibility as an anticancer therapeutic target, and summarises knowledge of EphB4 kinase inhibitors. To summarise, EphB4 is a difficult but potential treatment option for cancers.

SASH1: A Novel Eph Receptor Partner and Insights into SAM-SAM Interactions

The Eph (erythropoietin-producing human hepatocellular) receptor family, the largest subclass of receptor tyrosine kinases (RTKs), plays essential roles in embryonic development and neurogenesis. The intracellular Sterile Alpha Motif (SAM) domain presents a critical structural feature that distinguishes Eph receptors from other RTKs and participates in recruiting and binding downstream molecules. This study identified SASH1 (SAM and SH3 domain containing 1) as a novel Eph receptor-binding partner through SAM-SAM domain interactions. Our comprehensive biochemical analyses revealed that SASH1 selectively interacts with Eph receptors via its SAM1 domain, displaying the highest affinity for EphA8. The high-resolution crystal structure of the EphA8-SASH1 complex provided insights into the specific intermolecular interactions between these proteins. Cellular assays confirmed that EphA8 and SASH1 co-localize and co-precipitate in mammalian cells, with cancer mutations (EphA8 R942H or G978D) impairing this interaction. We demonstrated that SAM-SAM interaction is critical for SASH1-mediated regulation of EphA8 kinase activity, shedding new light on the Eph signaling pathway and expanding our understanding of the molecular basis of the tumor suppressor gene SASH1.

Molecular and functional interaction of single pass ephrin receptor tyrosine kinase, EphA2 in lipid nanodisc using solution NMR.

Eph receptor, the largest subfamily of single pass trans membrane receptor tyrosine kinase family play crucial role as a regulator during embryonic development, cell maturation, and adulthood. EphA2, a member of the Eph family receptor tyrosine kinases (RTK) is a key regulator of tumorigenesis and cancer progression. EphA2 can be activated canonically by ephrin ligand which normally is a repulsive signal OR non-canonical (unliganded EphA2) promoting cell migration in several cancer types. Here we focus to dissect the effects of the sterile α motif (SAM) domain together with the phosphorylation on intracellular region (ICR) interactions as well as the transmembrane construct of EphA2 in solution to understand the signaling mechanism at molecular level. Our study of the ICR shows that the sterile α motif (SAM) domain acts as a regulator impeding kinase domains interaction and conversely increasing the kinase activity upon deletion of SAM domain. While the presence of the SAM domain hinders oligomerization of phosphorylated ICR, it appears that such protein-protein interactions are required for kinase activity regulation. Mutation studies of the kinase domain, SAM domain and the linker region between kinase domain and SAM domain give insight into its regulatory role for EphA2 activity. More importantly, we have recently optimized truncated construct with single Fibronectin-III, FN-III (Ecto)-Transmembrane (TM)-Juxta-Membrane, JM (ICR) in presence of DDM with well dispersed peaks in TROSY-HQSC indicating well folded protein. We will incorporate this truncated construct into MSP lipid nanodisc for NMR studies and biophysical characterization. In addition to the ICR regulation via SAM domain, we will have in-depth perspective on the configurational coupling of membrane-proximal transmembrane domains to each other.

Molecular Dynamics Simulation Investigation of the Binding and Interaction of the EphA6-Odin Protein Complex.

Protein-protein interaction plays an important role in the development of almost all cells. Elucidating the dynamic binding and affinity of a protein-protein complex is essential for understanding the biological functions of proteins. EphA6 and Odin proteins are members of the Eph (erythropoietin-producing hepatocyte) receptor family and the Anks (ankyrin repeat and sterile α motif domain-containing) family, respectively. Odin significantly functions in regulating endocytosis, degradation, and stability of EphA receptors. In this work, the key residues of the interaction interface were determined through a hydrogen bond, root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and dynamic correlation analysis of the conventional molecular dynamics (MD) simulations. The calculated standard binding free energy, -7.92 kcal/mol, between EphA6 and Odin is quite consistent with the experimental measurement value, -8.73 kcal/mol. By the combination of several MD simulation techniques, our investigation of the binding process reveals the detailed representative characteristics of the entire binding pathway at the molecular level. Based on the obtained potential of the mean force (PMF) curve, the analysis of the simulation trajectories shows that the residue Arg1013 in the receptor EphA6 is responsible for capturing Asp739 and Asp740 in the ligand Odin during the initial stage of binding. In the later stage of binding, the hydrogen bonds and salt bridges between a series of residues Lys973, Leu1007, Gly1009, His1010, and Arg1012 in the receptor and residues Leu735, Asn736, Asp739, Asp740, and Asp753 in the ligand mainly contribute to the stability of the protein complex. In addition, the specific change process of the receptor-ligand-binding mode is also clarified during the binding process. Our present simulation will promote a deep understanding of the protein-protein interaction, and the identified key interresidue interaction will be theoretical guidance for the design of protein drugs.

Utilizing Exosomal-EPHs/Ephrins as Biomarkers and as a Potential Platform for Targeted Delivery of Therapeutic Exosomes.

Exosomes are cell-secreted nanoparticles containing various molecules including small vesicles, microRNAs (miRNAs), messenger RNAs or bioactive proteins which are thought to be of paramount importance for intercellular communication. The unique effects of exosomes in terms of cell penetration capacity, decreased immunogenicity and inherent stability, along with their key role in mediating information exchange among tumor cells and their surrounding tumor microenvironment (TME), render them a promising platform for drug targeted delivery. Compared to synthetic drugs, exosomes boast a plethora of advantages, including higher biocompatibility, lower toxicity and increased ability of tissue infiltration. Nevertheless, the use of artificial exosomes can be limited in practice, partly due to their poor targeting ability and partly due to their limited efficacy. Therefore, efforts have been made to engineer stem cell-derived exosomes in order to increase selectiveness and effectivity, which can then become loaded with various active substances depending on the therapeutic approach followed. Erythropoietin-producing human hepatocellular receptors (EPHs), along with their ligands, the EPH family receptor interacting proteins (ephrins), have been extensively investigated for their key roles in both physiology and cancer pathogenesis. EPHs/ephrins exhibit both tumorigenic and tumor suppressing properties, with their targeting representing a promising, novel therapeutic approach in cancer patients’ management. In our review, the use of ephrin-loaded exosomes as a potential therapeutic targeted delivery system in cancer will be discussed.

Antibodies Targeting KSHV gH/gL Reveal Distinct Neutralization Mechanisms

Kaposi’s sarcoma herpesvirus (KSHV) is associated with a significant disease burden, in particular in Sub-Sahara Africa. A KSHV vaccine would be highly desirable, but the mechanisms underlying neutralizing antibody responses against KSHV remain largely unexplored. The complex made of glycoproteins H and L (gH/gL) activates gB for the fusion of viral and cellular membranes in all herpesviruses. KSHV gH/gL also interacts with cellular Eph family receptors. To identify optimal antigens for vaccination and to elucidate neutralization mechanisms, we primed mice with recombinantly expressed, soluble gH/gL (gHecto/gL) that was either wildtype (WT), lacking defined glycosylation sites or bearing modified glycosylation, followed by boosts with WT gHecto/gL. We also immunized with a gL-gHecto fusion protein or a gHecto-ferritin/gL nanoparticle. Immune sera neutralized KSHV and inhibited EphA2 receptor binding. None of the regimens was superior to immunization with WT gHecto/gL with regard to neutralizing activity and EphA2 blocking activity, the gL-gHecto fusion protein was equally effective, and the ferritin construct was inferior. gH/gL-targeting sera inhibited gB-mediated membrane fusion and inhibited infection also independently from receptor binding and gL, as demonstrated by neutralization of a novel KSHV mutant that does not or only marginally incorporate gL into the gH/gL complex and infects through an Eph-independent route.

The Roles of EphB2 in Cancer.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors and their Eph receptor-interacting (ephrin) ligands together constitute a vital cell communication system with diverse roles. Experimental evidence revealed Eph receptor bidirectional signaling with both tumor-promoting and tumor-suppressing activities in different cancer types and surrounding environment. Eph receptor B2 (EphB2), an important member of the Eph receptor family, has been proved to be aberrantly expressed in many cancer types, such as colorectal cancer, gastric cancer and hepatocellular carcinoma, resulting in tumor occurrence and progression. However, there are no reviews focusing on the dual roles of EphB2 in cancer. Thus, in this paper we systematically summarize and discuss the roles of EphB2 in cancer. Firstly, we review the main biological features and the related signaling regulatory mechanisms of EphB2, and then we summarize the roles of EphB2 in cancer through current studies. Finally, we put forward our viewpoint on the future prospects of cancer research focusing on EphB2, especially with regard to the effects of EphB2 on tumor immunity.

The role of EphA7 in different tumors.

Ephrin receptor A7 (EphA7) is a member of the Eph receptor family. It is widely involved in signal transduction between cells, regulates cell proliferation and differentiation, and participates in developing neural tubes and brain. In addition, EphA7 also has a dual role of tumor promoter and tumor suppressor. It can participate in cell proliferation, migration and apoptosis through various mechanisms, and affect tumor differentiation, staging and prognosis. EphA7 may be a potential diagnostic marker and tumor treatment target. This article reviews the effects of EphA7 on a variety of tumor biological processes and pathological characteristics, as well as specific effects and regulatory mechanisms.

Investigating the effects of compound paralogous EPHB receptor mutations on mouse facial development.

Variation in facial shape may arise from the combinatorial or overlapping actions of paralogous genes. Given its many members, and overlapping expression and functions, the EPH receptor family is a compelling candidate source of craniofacial morphological variation. We performed a detailed morphometric analysis of an allelic series of E14.5 Ephb1-3 receptor mutants to determine the effect of each paralogous receptor gene on craniofacial morphology. We found that Ephb1, Ephb2, and Ephb3 genotypes significantly influenced facial shape, but Ephb1 effects were weaker than Ephb2 and Ephb3 effects. Ephb2-/- and Ephb3-/- mutations affected similar aspects of facial morphology, but Ephb3-/- mutants had additional facial shape effects. Craniofacial differences across the allelic series were largely consistent with predicted additive genetic effects. However, we identified a potentially important nonadditive effect where Ephb1 mutants displayed different morphologies depending on the combination of other Ephb paralogs present, where Ephb1+/- , Ephb1-/- , and Ephb1-/- ; Ephb3-/- mutants exhibited a consistent deviation from their predicted facial shapes. This study provides a detailed assessment of the effects of Ephb receptor gene paralogs on E14.5 mouse facial morphology and demonstrates how the loss of specific receptors contributes to facial dysmorphology.

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs.

EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell–cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.

Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling.

Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor's cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8-related diseases.

The Clinical Impact of the EPH/Ephrin System in Cancer: Unwinding the Thread.

Erythropoietin-producing human hepatocellular receptors (EPHs) compose the largest known subfamily of receptor tyrosine kinases (RTKs). They bind and interact with the EPH family receptor interacting proteins (ephrins). EPHs/ephrins are implicated in a variety of physiological processes, as well as in cancer pathogenesis. With neoplastic disease remaining a leading cause of death world-wide, the development of novel biomarkers aiding in the field of diagnosis, prognosis, and disease monitoring is of utmost importance. A multitude of studies have proven the association between the expression of members of the EPH/ephrin system and various clinicopathological parameters, including disease stage, tumor histologic grade, and patients’ overall survival. Besides their utilization in timely disease detection and assessment of outcome, EPHs/ephrins could also represent possible novel therapeutic targets. The aim of the current review of the literature was to present the existing data regarding the association between EPH/ephrin system expression and the clinical characteristics of malignant tumors.

EphA4 is highly expressed in the atria of heart and its deletion leads to atrial hypertrophy and electrocardiographic abnormalities in rats

AimsEphA4 is a member of the Eph receptor family, and expressed mainly in central nervous system (CNS), which is involved in CNS development and multiple diseases. Due to the variability in EphA4 expression, we wondered if EphA4 is expressed in other tissues, and what role does EphA4 play? Materials and methodsWe generated an EphA4 knockout (KO) rat line with red fluorescent marker protein encoded by the mCherry cassette inserted downstream of the EphA4 promoter as a reporter. Using this system, we observed high expression of EphA4 in the heart atria and in the brain. Key findingsEphaA4 KO rats (EphA4−/−) developed obvious atrial hypertrophy with an increased atria-to-heart weight ratio and atrial cardiomyocyte cross-sectional area at six months of age. EphA4−/− rats had reduced atrial end diastolic volume (EDV), atrial ejection fraction (EF) and left ventricular EF. They also exhibited increased amplitude of QRS complexes and QT intervals, with invisible p waves. RNA sequencing revealed that EphA4 KO altered the transcription of multiple genes involved in regulation of transcription and translation, ion binding, metabolism and cell adhesion. Deletion of EphA4 reduced IGF1 mRNA and protein expression, which is involved in cardiac remodeling. SignificanceOur data demonstrated that EphA4 was highly expressed in the atria and its deletion caused atrial dysfunction. Our findings also suggested that the EphA4 KO rat could be a potential model for studies on atrial remodeling.

The Role of the Eph Receptor Family in Tumorigenesis.

Simple SummaryThe Eph receptor family is implicated in both tumour promotion and suppression, depending on the tissue-specific context of available receptor interactions with ligands, adaptor proteins and triggered downstream signalling pathways. This complex interplay has not only consequences for tumorigenesis but also offers a basis from which new cancer-targeting strategies can be developed. This review comprehensively summarises the current knowledge of Eph receptor implications in oncogenesis in a tissue- and receptor-specific manner, with the aim to develop a better understanding of Eph signalling pathways for potential targeting in novel cancer therapies.The Eph receptor tyrosine kinase family, activated by binding to their cognate ephrin ligands, are important components of signalling pathways involved in animal development. More recently, they have received significant interest due to their involvement in oncogenesis. In most cases, their expression is altered, affecting the likes of cell proliferation and migration. Depending on the context, Eph receptors have the potential to act as both tumour promoters and suppressors in a number of cancers, such as breast cancer, colorectal cancer, lung cancer, prostate cancer, brain cancer and Kaposi’s sarcoma (KS), the latter being intrinsically linked to EphA2 as this is the receptor used for endothelial cell entry by the Kaposi’s sarcoma-associated herpesvirus (KSHV). In addition, EphA2 deregulation is associated with KS, indicating that it has a dual role in this case. Associations between EphA2 sequence variation and KSHV infection/KS progression have been detected, but further work is required to formally establish the links between EphA2 signalling and KS oncogenesis. This review consolidates the available literature of the role of the Eph receptor family, and particularly EphA2, in tumorigenesis, with the aim to develop a better understanding of Eph signalling pathways for potential targeting in novel cancer therapies.

Biochemical and NMR characterization of the interactions of Vav2-SH2 domain with lipids and the EphA2 juxtamembrane region on membrane.

Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases that is involved in regulating a wide range of biological processes. It interacts with several tyrosine-phosphorylated cell surface receptors, including the Eph family receptors, through its SH2 domain. The interaction of Vav2 with EphA2 is crucial for EphA2-mediated tumor angiogenesis. Here we show that Vav2-SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically. The specific lipid-binding site in Vav2-SH2 domain was identified by NMR chemical shift perturbation experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3, both of which bind to Vav2-SH2 with millimolar binding affinities. In addition, the interaction between Vav2-SH2 and the phosphorylated juxtamembrane region (JM) of EphA2 (Y594 phosphorylated) was investigated using NMR techniques. Furthermore, by using a nickel-lipid containing peptide-based nanodiscs system, we studied the binding of Vav2-SH2 to the phosphorylated JM region of EphA2 on lipid membrane and uncovered a role of membrane environment in modulating this protein-protein recognition.

Epstein-Barr Virus gH/gL and Kaposi's Sarcoma-Associated Herpesvirus gH/gL Bind to Different Sites on EphA2 To Trigger Fusion.

Both Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses and are important in a variety of malignancies. Eph family receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV and EBV. Previous studies identified five conserved residues (ELEFN50-54) in the N-terminal domain of KSHV gH that are critical for Eph binding and KSHV infection. However, the specific domains of EBV gH/gL important for EphA2 binding are not well described. We found that the KSHV gH (ELEFN50-54) motif is important for higher KSHV fusion and that EBV gH/gL does not utilize a similar motif for fusion activity. We previously identified that an EBV gL N-glycosylation mutant (gL-N69L/S71V) was hyperfusogenic in epithelial cells but not in B cells. To determine whether this glycosylation site may be the binding region for EphA2, we compared the EphA2 binding activity of EBV gH/gL and the EBV gH/gL-N69L/S71V mutant. We found that EBV gH/gL-N69L/S71V had higher binding affinity for EphA2, indicating that the EBV gL N-glycosylation site might be responsible for inhibiting the binding of gH/gL to EphA2. Loss of N-glycosylation at this site may remove steric hindrance that reduces EBV gH/gL binding to EphA2. In addition, the mutations located in the large groove of EBV gH/gL (R152A and G49C) also have decreased binding with EphA2. Taken together, our data indicate that the binding site of EphA2 on EBV gH/gL is at least in part proximal to the EBV gL glycosylation site, which in part accounts for differences in EphA2 binding affinity by KSHV.IMPORTANCE Virus entry into target cells is the first step for virus infection. Understanding the overall entry mechanism, including the binding mechanism of specific virus glycoproteins with cellular receptors, can be useful for the design of small molecule inhibitors and vaccine development. Recently, EphA2 was identified as an important entry receptor for both KSHV and EBV. In the present study, we investigated the required binding sites within EphA2 and EBV gH/gL that mediate the interaction of these two proteins allowing entry into epithelial cells and found that it differed in compared to the interaction of KSHV gH/gL with EphA2. Our discoveries may uncover new potential interventional strategies that block EBV and KSHV infection of target epithelial cells.

Enhanced YB1/EphA2 axis signaling promotes acquired resistance to sunitinib and metastatic potential in renal cell carcinoma

VHL mutations are the most common tumorigenic lesions in clear cell renal cell carcinoma (ccRCC) and result in continued activation of the HIF/VEGF pathway and uncontrolled cancer progression. Receptor tyrosine kinase (RTK) inhibitors such as sunitinib have been demonstrated to target tumorigenic signaling pathways, delay tumor progression, and improve patient prognosis in metastatic renal cell carcinoma (mRCC). Although several mechanisms of sunitinib resistance have been reported, the solutions to overcome this resistance remain unclear. In our study, we found that increased expression of Y-box binding protein 1 (YB1, a multidrug resistance associated protein) and EphA2 (a member of the erythropoietin-producing hepatocellular (Eph) receptor family, belonging to the RTK family) mediated sunitinib resistance and mRCC exhibited a large phenotypic dependence on YB1 and EphA2. In addition, our findings confirm that YB1 promotes the invasion, metastasis and sunitinib resistance of ccRCC by regulating the EphA2 signaling pathway. Furthermore, pharmacological inhibition of EphA2 through the small molecule inhibitor ALW-II-41-27 reduced the proliferation of sunitinib-resistant tumor cells, suppressed tumor growth in vivo, and restored the sensitivity of sunitinib-resistant tumor cells to sunitinib in vitro and in vivo. Mechanistically, YB1 increases the protein levels of EphA2 by maintaining the protein stability of EphA2 through inhibition of the proteasomal degradation pathway. Collectively, our findings provide the theoretical rationale that ccRCC metastasis and RTK-directed therapeutic resistance could be prospectively and purposefully targeted.

Abstract A42: EphB2 a potential therapeutic target for pediatric medulloblastoma

Abstract Aim: Medulloblastoma (MB) is the most frequent malignant brain tumor to occur in children and remains the leading cause of cancer-related mortality in childhood. The current standard of care for patients with MB includes surgical resection and radiation therapy followed by high-dose chemotherapy. Overall survival rates have reached 70%, but the outcome for young children, especially infants, is worse. Those who do survive suffer from long-term therapy-induced side effects. Thus, more effective and less toxic therapies are needed. Eph family receptors predominantly function during embryonic development and are typically either not expressed or expressed at very low levels in normal healthy tissues. However, it is now established that many Eph receptors are re-expressed and functional in human cancers, making them attractive, relatively tumor-specific targets. Thus, in MB anti-Eph therapy has the potential to target tumor cells and not normal brain, reducing off-target toxicity and potentially increasing response rates. We have generated compelling preliminary data showing that EphB2 is functional and overexpressed in pediatric MB. We now propose to extend these studies and validate antibody-based strategies to target this receptor. Methods: 1. EphB receptor expression was characterized using a number of approaches: RNA (RNASeq and QPCR), protein (flow cytometry and Western blot) and receptor localization by immunofluorescence in MB ATCC lines, primary lines, and patient tissue. 2. To better understand the function of the EphB2, gene expression was downregulated using shRNA in primary MB cell line models. Following EphB downregulation, we assessed in vitro growth and cell death induction and tumor-forming capacity using orthotopic MB intracranial xenografts. Moreover, following EphB activation, using soluble ligand we assessed in vitro growth via direct cell count and MTT assay, cell migration via scratch assay, and cell death induction via Annexin V staining. Following EphB inhibition, using peptide strategies we assessed in vitro growth, cell migration, and cell death induction. Results: Our study shows that EphB2 is both functional and highly expressed in MB. Furthermore, EphB2 appears to be expressed in a subtype-specific manner. We further confirmed our expression studies using the MB Advanced Genomics International Consortium (MAGIC) dataset. The EphB2 high-affinity ligands ephrinB1/B2-Fc were used to induce kinase activation. Following receptor activation, we observed reduced tumor cell proliferation and invasion in vitro. EphB2 receptor blocking studies, using the SNEW blocking peptide, effectively inhibited kinase phosphorylation and subsequently FAK phosphorylation. Most importantly, the small peptide rescues MB cell growth upon ephrin B ligand stimulation, indicating the specific mitogenic capacity of this receptor. Conclusion: In summary, this study furthers our understanding of EphB2 expression and function in MB and paves the way for future receptor-targeting studies in MB. Citation Format: Yuchen Li, Carolin Offenhauser, Rochelle D’Souza, Seckin Akgul, Bryan W. Day. EphB2 a potential therapeutic target for pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A42.

A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses.

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ versus CD20+ versus CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro, in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag181-189CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.