Eph Receptor Tyrosine Kinases Research Articles

Abstract 5017: EphA2 is a clinically relevant target for breast cancer bone metastatic disease

Abstract While advances in molecular targeted therapies have increased survival for breast cancer patients, outcomes for patients with metastatic disease remain poor. EphA2 receptor tyrosine kinase (RTK) is highly expressed in breast tumor cells across multiple molecular subtypes and correlates with poor patient prognosis. As metastasis of breast cancer to bone is a major cause of morbidity and mortality in patients, we investigated the role of EphA2 in this clinically relevant phenomenon. Analysis of human patient gene expression datasets and human breast-to-bone metastasis samples revealed high level ephA2 expression in bone lesions relative to other sites of metastatic dissemination, and EphA2 positive staining on breast tumor cells in bone metastatic lesions, respectively. EphA2 loss of function in tumor cells reduced tumor-induced osteolysis in two independent intratibial tumor engraftment models (4T1 and MDA-MB-231) as well as in an intracardiac experimental metastasis model (MDA-MB-231) in vivo. We observed a significant reduction in osteoclasts in animals harboring EphA2 loss of function tumors relative to control tumors. Further in vitro analyses revealed that blocking EphA2 function resulted in defective precursor maturation into functional osteoclasts through an interleukin-6 (IL-6) dependent mechanism. Pharmacologic inhibition of EphA2 decreased breast tumor induced osteolytic disease severity in vivo. Together, these data provide pre-clinical validation of EphA2 as a clinically relevant molecular target for breast cancer bone metastatic, osteolytic disease, warranting further investigation of molecular mechanisms that link EphA2 to tumor-induced osteolysis and eventual clinical development. Citation Format: Dana M. Brantley-Sieders. EphA2 is a clinically relevant target for breast cancer bone metastatic disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5017.

Abstract 4139: Investigations of drug synergy reveal promising efficacy of EphA2 inhibition combined with CDK inhibition in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor prognosis relative to other breast cancers. Due to its lack of expression of commonly targeted cell surface markers and hormone receptors, there is a dearth of precision treatment options for this malignancy, with chemotherapy and radiation therapy remaining the major mainstays of care. Our group has previously reported that the receptor tyrosine kinase EphA2 is enriched in the basal-like/triple-negative breast cancer subtype and that this expression is associated with reduced recurrence-free survival. Furthermore, genetic knockdown or inhibition of EphA2 with the small molecule ALW-II-41-27 (ALW) reduces cancer cell growth in vitro and in vivo. In these studies, we sought to investigate whether the antitumor properties of ALW can be augmented by addition of other compounds to further reduce cancer cell growth and induce apoptosis. To this end, TNBC lines MDA-MB-231, BT-549, HCC1395, and HCC1187 were seeded in 96-well plates and exposed to ALW alone or in combination with chemotherapeutic agents doxorubicin and paclitaxel, CDK inhibitor SCH-727965, and MCL-1 inhibitor S63845 and cell viability assessed by MTT assay at 48 hours. While there was no significant reduction in viability for cells treated with ALW plus chemotherapy and no consistent improvement with addition of S63845 across cell lines, the combination of ALW with SCH-727965 performed significantly better than either drug alone (p < 0.05) in HCC1395 and BT-549 cells. To evaluate drug synergy, cells of all lines above were exposed to variable concentrations of SCH-727965, ALW, and the combination in a fixed ratio of their respective IC50 values to generate dose response curves, and these data analyzed via CompuSyn software. Combination indices (CI) were <1 for the majority of conditions across all cell lines, supporting synergistic interaction. Effects were most pronounced in HCC1395 cells with average CI 0.82. Furthermore, TUNEL assays show that addition of SCH-727965 to ALW significantly increases the induction of apoptosis by HCC1395 and HCC1187 cells. These preclinical studies demonstrate that combination of ALW and SCH-727965 potently reduces TNBC cell growth and promotes cell death, representing promising early data on the effects of EphA2 inhibition as part of a combination targeted therapeutic approach for triple-negative breast cancer. Citation Format: Kalin L. Wilson, Dana M. Brantley-Sieders. Investigations of drug synergy reveal promising efficacy of EphA2 inhibition combined with CDK inhibition in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4139.

Evaluation of EphB4 as Target for Image-Guided Surgery of Breast Cancer.

Background: Targeted image-guided surgery is based on the detection of tumor cells after administration of a radio-active or fluorescent tracer. Hence, enhanced binding of a tracer to tumor tissue compared to healthy tissue is crucial. Various tumor antigens have been evaluated as possible targets for image-guided surgery of breast cancer, with mixed results. Methods: In this study we have evaluated tyrosine kinase receptor EphB4, a member from the Eph tyrosine kinase receptor family, as a possible target for image-guided surgery of breast cancers. Two independent tissue micro arrays, consisting of matched sets of tumor and normal breast tissue, were stained for EphB4 by immunohistochemistry. The intensity of staining and the percentage of stained cells were scored by two independent investigators. Results: Immunohistochemical staining for EphB4 shows that breast cancer cells display enhanced membranous expression compared to adjacent normal breast tissue. The enhanced tumor staining is not associated with clinical variables like age of the patient or stage or subtype of the tumor, including Her2-status. Conclusion: These data suggest that EphB4 is a promising candidate for targeted image-guided surgery of breast cancer, especially for Her2 negative cases.

Protein kinase C phosphorylates the EphA2 receptor on serine 892 in the regulatory linker connecting the kinase and SAM domains

The EphA2 receptor tyrosine kinase signals through two distinct mechanisms, one regulated by tyrosine phosphorylation and the other by serine/threonine phosphorylation. Serine 892 (S892) is one of the major serine/threonine phosphorylation sites in EphA2, but little is known about its regulation and function. S892 is located in the linker connecting the EphA2 kinase and SAM domains, and is part of a cluster of five phosphorylated residues that includes the well characterized S897. EphA2 can be phosphorylated on S897 by the RSK, AKT and PKA kinases to promote a non-canonical form of signaling that plays an important role in cancer malignancy. Here we show that the Protein Kinase C (PKC) family phosphorylates the EphA2 S892 motif in vitro and in cells. By using a newly developed phosphospecific antibody, we detected EphA2 S892 phosphorylation in a variety of cell lines. As expected for a PKC target site, the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) increases S892 phosphorylation whereas the broad-spectrum PKC inhibitor Go 6983 inhibits both basal and TPA-induced S892 phosphorylation. Besides phosphorylating S892, PKC can also increase EphA2 phosphorylation on S897 through the MEK kinase, which regulates the ERK-RSK signaling axis. We also found that S892 and S897 phosphorylation induced by PKC activation can be downregulated by ephrin ligand-induced EphA2 canonical signaling. Our data reveal that the PKC family contributes to the phosphorylation cluster in the EphA2 kinase-SAM linker, which regulates EphA2 non-canonical signaling and cancer malignancy.

Effect of EphA2 knockdown on melanoma metastasis depends on intrinsic ephrinA1 level.

Upregulation of receptor tyrosine kinase EphA2 has been found to be associated with a poor prognosis in many types of cancer and is considered an attractive therapeutic target. As yet, few efforts have been focused on its tumor suppressive activity triggered by its ligand, ephrinA1. Here, we aimed to determine the potential of ephrinA1 as an important player in melanoma metastasis. Data from the Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) were analyzed to explore the expression and prognostic implications of EphA2 and ephrinA1 in melanoma. Western blotting, shRNA, colony formation and immunofluorescence assays, as well as two in vivo xenograft models (subcutaneous and metastatic) were used to evaluate the role of EphA2 in melanoma progression. Akt inhibition and ephrinA1-Fc were used to confirm the influence of Akt activation and ephrinA1 levels on the EphA2 effects. Immunohistochemistry (IHC) was performed on xenograft and patient melanoma tissues. We found that high levels of ephrinA1, but not EphA2, were negatively correlated with melanoma metastasis. The expression levels of EphA2 and ephrinA1 were not correlated. After EphA2 downregulation, colony forming abilities and lung metastatic growth were reduced in melanoma cell lines with a low ephrinA1 expression, but were increased in melanoma cell lines with a high ephrinA1 expression. EphA2-mediated colony formation in EphA2-high/ephrinA1-low cells was found to be Akt-dependent and to be inhibited by the addition of ephrinA1-Fc. IHC staining of primary melanoma specimens revealed that EphA2-high/ephrinA1-low patients exhibited poorer outcomes than EphA2-high/ephrinA1-high patients. From our data we conclude that evaluation of ephrinA1 levels may be helpful for the application of EphA2-targeted therapies and for prognostic predictions in melanoma patients.

Cataloguing the dead: breathing new life into pseudokinase research.

Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the ‘dark’ pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin‐producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co‐evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis‐independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species‐level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome.

Helicobacter Pylori Targets the EPHA2 Receptor Tyrosine Kinase in Gastric Cells Modulating Key Cellular Functions.

Helicobacter pylori, a stomach-colonizing Gram-negative bacterium, is the main etiological factor of various gastroduodenal diseases, including gastric adenocarcinoma. By establishing a life-long infection of the gastric mucosa, H. pylori continuously activates host-signaling pathways, in particular those associated with receptor tyrosine kinases. Using two different gastric epithelial cell lines, we show that H. pylori targets the receptor tyrosine kinase EPHA2. For long periods of time post-infection, H. pylori induces EPHA2 protein downregulation without affecting its mRNA levels, an effect preceded by receptor activation via phosphorylation. EPHA2 receptor downregulation occurs via the lysosomal degradation pathway and is independent of the H. pylori virulence factors CagA, VacA, and T4SS. Using small interfering RNA, we show that EPHA2 knockdown affects cell–cell and cell–matrix adhesion, invasion, and angiogenesis, which are critical cellular processes in early gastric lesions and carcinogenesis mediated by the bacteria. This work contributes to the unraveling of the underlying mechanisms of H. pylori–host interactions and associated diseases. Additionally, it raises awareness for potential interference between H. pylori infection and the efficacy of gastric cancer therapies targeting receptors tyrosine kinases, given that infection affects the steady-state levels and dynamics of some receptor tyrosine kinases (RTKs) and their signaling pathways.

Functional Oligomerization of the EphA2 Receptor Tyrosine Kinase

Eph receptor tyrosine kinases constitute the largest family of receptor tyrosine kinases (RTKs). Aberration of Eph/ephrin system leads to many pathologies, most notably in cancer. EphA2 is notorious for overexpression in a variety of human malignancies, in which the loss of ligand is often observed. Although the activation of EphA2 RTK is known to suppress the signaling of oncogenic kinases, such as Ras and Akt, the unliganded EphA2 receptor becomes a target for oncogenic kinases including Akt, ERK and PKA that phosphorylates EphA2 on serine 897 (S897). Remarkably the latter event turns EphA2 from a tumor suppressor into an oncogenic protein that drives tumor cell migration. We set out to understand the structural and cellular basis underlying the ligand-dependent tumor suppressive and ligand-independent pro-oncogenic functions of EphA2. We used a time-resolved fluorescence spectroscopy (PIE-FCCS) to study the assemblies of EphA2 in live cell membranes. The results show that EphA2 undergo multimerization prior to ligand binding, and polymerize upon ligand stimulation. We identified three sets of interfaces in the ecto-domain that drive the ligand-independent multimerization and ligand-induced polymerization of EphA2. These three sets of interfaces can be divided into two types of contact. The first one is symmetric contact driven by LBD-LBD and Sushi-Sushi interfaces. The second one is asymmetric contact via LBD interacting with FN2 domain on the neighboring molecule. Further studies reveal that the symmetric contact mediates the oligomerization and activation of kinases upon ligand stimulation, while the asymmetric contact keeps kinases separated facilitating the pro-migratory ligand-independent signaling via phosphorylation of S897. These results add a paradigm to the general framework of RTKs, and also suggest that the loss of ligands in many tumor tissues favors the asymmetric contact in the assemblies of overexpressed EphA2, promoting the ligand-independent oncogenic signaling.

Transmembrane and Juxtamembrane Interactions of EphA2 with Lipid Membranes in the Active and Inactive States

Aberrant signaling of the receptor tyrosine kinase EphA2 is linked to tumor aggressiveness in many types of cancers. Upon binding of an extracellular ephrin ligand EphA2 undergoes oligomerization and phosphorylation inducing an intracellular signaling cascade. Little else is known of the mechanisms by which the signal is conferred across the membrane to drive phosphorylation or how in the absence of ligand the receptor remains inactive. Evidence suggests that prior to activation EphA2 dimers exhibit a distinct 15° interhelical crossing angle between the transmembrane domains (TMD) and that upon activation a rotation of the TMDs occurs and it widens to 45°. It is hypothesized that positively charged residues on the intracellular juxtamembrane (JM) segment interact with negatively charged Phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on the inner leaflet of the cell membrane inhibiting activation in the absence of bound ligand. Here we use a method of studying a peptide comprised of the TMD and first five JM residues of EphA2 in the putative active and inactive conformations by varying membrane thickness using 14:1 PC and 22:1 PC. We established that the TMD orientation is more tilted in thin bilayers and a less tilted in thick bilayers. We used this system to investigate the interaction of JM residues with membranes containing PIP2 in thin and thick bilayers. Fluorescence experiments indicate that JM residues experience different environments in thin vs. thick bilayers in the presence of PIP2. FRET results suggest that the JM is buried deeper into the membrane in the presence of PIP2 in 22:1 PC but not 14:1 PC. The extent of burial has been further examined by quenching experiments using brominated lipids. These results provide new mechanistic insights into the inhibition and activation of EphA2 signaling.

EphA4/Tie2 crosstalk regulates leptomeningeal collateral remodeling following ischemic stroke.

Leptomeningeal anastomoses or pial collateral vessels play a critical role in cerebral blood flow (CBF) restoration following ischemic stroke. The magnitude of this adaptive response is postulated to be controlled by the endothelium, although the underlying molecular mechanisms remain under investigation. Here we demonstrated that endothelial genetic deletion, using EphA4fl/fl/Tie2-Cre and EphA4fl/fl/VeCahderin-CreERT2 mice and vessel painting strategies, implicated EphA4 receptor tyrosine kinase as a major suppressor of pial collateral remodeling, CBF, and functional recovery following permanent middle cerebral artery occlusion. Pial collateral remodeling is limited by the crosstalk between EphA4-Tie2 signaling in vascular endothelial cells, which is mediated through p-Akt regulation. Furthermore, peptide inhibition of EphA4 resulted in acceleration of the pial arteriogenic response. Our findings demonstrate that EphA4 is a negative regulator of Tie2 receptor signaling, which limits pial collateral arteriogenesis following cerebrovascular occlusion. Therapeutic targeting of EphA4 and/or Tie2 represents an attractive new strategy for improving collateral function, neural tissue health, and functional recovery following ischemic stroke.

Conditional knockout of ephrinB1 in osteogenic progenitors delays the process of endochondral ossification during fracture repair.

The Eph receptor tyrosine kinase ligand, ephrinB1 (EfnB1) is important for correct skeletal and cartilage development, however, the role of EfnB1 in fracture repair is unknown. This study investigated the role of EfnB1 during fracture repair where EfnB1 expression increased significantly at 1 and 2weeks post fracture in C57Bl/6 wildtype mice, coinciding with the haematoma, soft callus formation/remodelling stages, respectively. To investigate the specific role of EfnB1 within the osteogenic lineage during fracture repair, male mice with a conditional deletion of EfnB1 in the osteogenic lineage (EfnB1OBfl/O), driven by the Osterix (Osx) promoter, and their male Osx:Cre counterparts were subject to a femoral fracture with internal fixation. Two weeks post fracture micro computed tomography (μCT) analysis revealed that EfnB1OBfl/O mice displayed a significant decrease in bone volume relative to tissue volume within the fracture callus. This was attributed to an alteration in the distribution of osteoclasts within the fracture site, a significant elevation in cartilaginous tissue and reduction in the osteoprogenitor population and calcein labelled bone within the fracture site of EfnB1OBfl/O mice. Supportive in vitro studies demonstrated that under osteogenic conditions, cultured EfnB1OBfl/O stromal cells derived from the 2week fracture site exhibited a reduced capacity to produce mineral and decreased expression of the osteogenic gene, Osterix, when compared to Osx:Cre controls. These findings suggest that the loss of EfnB1 delays the fracture repair process. The present study confirmed that EFNB1 activation in human BMSC, following stimulation with soluble-EphB2 resulted in de-phosphorylation of TAZ, demonstrating similarities in EfnB1 signalling between human and mouse stromal populations. Overall, the present study provides evidence that loss of EfnB1 in the osteo/chondrogenic lineages delays the soft callus formation/remodelling stages of the fracture repair process.

Abstract GMM-041: OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH

Abstract Despite increased understanding of chemotherapy resistance, the effects of the chemotherapy itself on the oncogenic cancer cell properties and adaptive resistance mechanisms of the therapy-escaping metastatic cancer cells remain largely undefined. In this study, we show that the platinum-based chemotherapeutic drugs cisplatin and carboplatin induce a switch from the canonical EphA2 receptor tyrosine kinase activity to the stemness and invasion-associated, ligand independent signaling via EphA2 S897 phosphorylation. In patient-derived high grade serous ovarian cancer (HGSOC) cells and established ovarian cancer cell lines, the chemotherapy treatment led to a significant increase in the ratio between S897 and Y588 phosphorylation of EphA2 in conjunction with GPRC5A protein upregulation and beta1-integrin activation. Notably, high protein levels of GPRC5A were significantly associated to poor survival in patients with metastatic HGSOC, revealing a previously unknown association of this EphA2 interacting receptor with the clinical HGSOC outcome. Moreover, pharmacological inhibition of RSK serine-threonine kinases blocked both the EphA2 S897 phosphorylation and the GPRC5A induction coincident with markedly increased cisplatin and carboplatin sensitivity of the HGSOC cells ex vivo. These results identify a novel mechanism of chemotherapy-induced adaptive resistance via the RSK-EphA2-GPRC5A oncogenic signaling axis in HGSOC. Citation Format: Lidia Moyano-Galceran, Siri Pauliina Turunen, Elina Pietilä, Twana Alkasalias, Sara Corvigno, Elisabet Hjerpe, Ulrika Joneborg, Daria Bulanova, Mina Eriksson, Hanna Dahlstrand, Joseph Carlson, Kaisa Lehti. OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-041.

EphA4 Negatively Regulates Myelination by Inhibiting Schwann Cell Differentiation in the Peripheral Nervous System.

Myelin plays a crucial role in axon function recovery following nerve damage, and the interaction between Schwann cells (SCs) and regenerating axons profoundly affects myelin formation. Eph receptor A4 (EphA4), a member of the Eph tyrosine kinase receptor family, regulates cell-cell interactions via its ligand ephrins. However, our current knowledge on how EphA4 regulates the formation of myelin sheaths remains limited. In order to explore the roles of EphA4 in myelination in the peripheral nervous system, we used a combination of (1) a co-culture model of dorsal root ganglion (DRG) explants and SCs, (2) a SC differentiation model induced by db-cAMP, and (3) a regeneration model of crushed sciatic nerves in rats. Our results demonstrated that EphA4 inhibited myelination by inhibiting SC differentiation and facilitating SC proliferation in vitro. The in vivo experiments revealed that EphA4 expression in SCs is upregulated following nerve crush injury and then downregulated during remyelination. Moreover, silencing of EphA4 by siRNA or overexpression of EphA4 by genetic manipulation can accelerate or slow down nerve remyelination in crushed sciatic nerves. Taken together, our results suggest that EphA4 may negatively regulate myelination by abrogating SC differentiation.

Exome Sequencing Defines the Molecular Pathogenesis of Vein of Galen Malformation

Abstract INTRODUCTION Vein of Galen malformations (VOGMs) are morbid arteriovenous malformations, with poorly described genetis. 1 Despite improvement in endovascular treatment, VOGM mortality remains high. 2 VOGM has been reported as a rare finding in Capillary Malformation-Arteriovenous Malformation Syndrome (RASA1; OMIM #605384) and Hereditary Hemorrhagic Telangiectasia (ENG, ACVRL1; OMIM #187300, #600376). 3-4 Our previous work has identified EPH receptor tyrosine kinase as also playing a role in VOGM pathogenesis. 5 Here, we report a larger cohort of probands and identify a new gene in the same pathway as EPHB4 as playing a role in VOGM. METHODS Germline DNA was isolated from 84 unrelated probands harboring radiographically confirmed VOGMs Both parents were available for 69/84 probands. Exome capture and paired-end WES was performed on DNA samples from participating individuals (n = 237). Data was bioinformatically analyzed to identify rare de-novo and transmitted mutations. Binomial analysis tested for exome-wide significance of mutational burden. RESULTS Only 3/75 patients harbored mutations in previously reported VOGM-associated genes (2.3%; RASA1 n = 2, 1.1%; ACVRL1 n = 1). Significant enrichment of rare damaging mutations was found for a member of the EPH receptor tyrosine kinase family (EPHB4, n = 5; 6.0%; P = 3.31 × 10–7, 36.62-fold enrichment). Entirely novel mutations in the integrin family were also identified (n = 2; 1.1%, P = 6.03 × 10–5, 179.6-fold enrichment). Both of these mutations are located in the c-terminal domain and disrupt a binding motif. Furthermore, this integrin protein mutation is involved in the same pathway as EPHB4, although whether or not they directly interact remains unknown. CONCLUSION This work represents an expansion upon the largest phenotyped, exome-sequenced VOGM cohort in the world. Having discovered a new gene in the same pathway as EPHB4 strongly implicates said pathway in VOMG development. We are currently pioneering tissue sampling from endovascular instruments used during treatment to explore potential somatic mutations. Our findings continue to uncover genetic determinants of VOGM pathogenesis, providing novel insight into vascular developmental biology.

The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cellular collapse and spinal motor axon guidance

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 proximity-dependent biotin identification (BioID) interactome, and confirmed this association using co-immunoprecipitation. HD-PTP loss attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo. HD-PTP depletion abrogates ephrin-B2-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP loss also accelerates ligand-induced EphB2 degradation, contrasting the effects of HD-PTP loss on the relay of signals from other cell surface receptors. Our results link Eph function to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.

Tight Junction Protein Junctional Adhesion Molecule-A Regulates the Expression of Receptor Tyrosine Kinase EPHA2 In Triple-Negative Breast Cancer Cells

Breast tumors lacking expression of the human epidermal growth factor receptor-2 (HER2), progesterone receptor (PR) and estrogen receptor (ER?) are defined as triple negative breast cancers (TNBC). A lack of targeted therapies has impaired TNBC patient prognosis. It has previously been shown that high expression of Junctional Adhesion Molecule-A (JAM-A) correlates with aggressive breast cancer patient phenotypes, and that JAM-A regulates the expression of HER2 in breast cancer cells. Accordingly, we hypothesized that JAM-A might regulate the expression of other receptor tyrosine kinases. We show for the first time that JAM-A may regulate the expression of the EPHA2 receptor in TNBC cells and propose that this pathway merits deeper investigation for its therapeutic value in TNBC settings.

EphA7 Functions as Receptor on BJAB Cells for Cell-to-Cell Transmission of the Kaposi's Sarcoma-Associated Herpesvirus and for Cell-Free Infection by the Related Rhesus Monkey Rhadinovirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma and is associated with two B cell malignancies, primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease. On several adherent cell types, EphA2 functions as a cellular receptor for the gH/gL glycoprotein complex of KSHV. KSHV gH/gL also has previously been found to interact weakly with other members of the Eph family of receptor tyrosine kinases (Ephs), and other A-type Ephs have been shown to be able to compensate for the absence of EphA2 using overexpression systems. However, whether these interactions are of functional consequence at endogenous protein levels has remained unclear so far. Here, we demonstrate for the first time that endogenously expressed EphA7 in BJAB B cells is critical for the cell-to-cell transmission of KSHV from producer iSLK cells to BJAB target cells. The BJAB lymphoblastoid cell line often serves as a model for B cell infection and expresses only low levels of all Eph family receptors other than EphA7. Endogenous EphA7 could be precipitated from the cellular lysate of BJAB cells using recombinant gH/gL, and knockout of EphA7 significantly reduced transmission of KSHV into BJAB target cells. Knockout of EphA5, the second most expressed A-type Eph in BJAB cells, had a similar, although less pronounced, effect on KSHV infection. Receptor function of EphA7 was conserved for cell-free infection by the related rhesus monkey rhadinovirus (RRV), which is relatively even more dependent on EphA7 for infection of BJAB cells.IMPORTANCE Infection of B cells is relevant for two KSHV-associated malignancies, the plasmablastic variant of multicentric Castleman's disease and PEL. Therefore, elucidating the process of B cell infection is important for the understanding of KSHV pathogenesis. While the high-affinity receptor for the gH/gL glycoprotein complex, EphA2, has been shown to function as an entry receptor for various types of adherent cells, the gH/gL complex can also interact with other Eph receptor tyrosine kinases with lower avidity. We analyzed the Eph interactions required for infection of BJAB cells, a model for B cell infection by KSHV. We identified EphA7 as the principal Eph receptor for infection of BJAB cells by KSHV and the related rhesus monkey rhadinovirus. While two analyzed PEL cell lines exhibited high EphA2 and low EphA7 expression, a third PEL cell line, BCBL-1, showed high EphA7 and low EphA2 expression, indicating a possible relevance for KSHV pathology.

Abstract 5035: Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers

Abstract Introduction: Identifying targets in the tumor microenvironment (TME) that act as barriers to an effective anti-tumor immune response has become an area of intense investigation. In the current study, we established EphB4-ephrin-B2 signaling as a key pathway that regulates both innate and adaptive arms of the immune system. Eph receptor tyrosine kinases and their membrane-bound ephrin ligands have been implicated in human malignancies and in immune cell development, migration, and activation in inflammatory models. However, direct evidence that supports the role of Eph-ephrin interaction in cancer-related immune response is lacking. We hypothesized that EphB4-ephrin-B2 interaction regulates TME by sustaining immunosuppressive cells-Tregs and TAMs thus negatively impacting the functional ability of CD8 T cells. Materials and methods: We used orthotopic models of head and neck squamous cell carcinoma to determine the role of EphB4-ephrin-B2 interaction in tumor immune microenvironment. Mice were treated with control agent or an EphB4-ephrin-B2 blocker in the absence or presence of radiation (RT). Tumor immune cell infiltrates were analyzed using mass cytometry and flow cytometry applications. ELISA or multiplex cytokine array were utilized to determine circulating cytokine/chemokine levels in plasma. Results: We observed that inhibition of EphB4-ephrin-B2 signaling in vivo significantly reduced tumor growth and decreased intratumoral Tregs, TAMs, and increased the activation of Teffector cells, without affecting CD4 T cell numbers. This was correlated with decreased Treg proliferation and activation when EphB4-ephrin-B2 signaling is inhibited. Since RT remains the mainstay in treatment of head and neck squamous cell cancer (HNSCC) patients, we combined EphB4-ephrin-B2 inhibitor with RT in our tumor model and observed further increase in CD8 and CD4 T cell infiltrates and activation status, and a significant decline in circulating TGF-β1 levels and an increase in CXCL10 levels compared to the control group. A significant reduction of TAMs, favoring a polarization towards an anti-tumoral M1 phenotype, was also observed in EphB4-ephrin-B2 inhibitor+RT group. We also compared the efficacy of combining EphB4-ephrin-B2 inhibitor with RT to anti-PDL1+RT in an in vivo model known to develop resistance to anti-PDL1+RT therapy. Our data demonstrated that combining EphB4-ephrin-B2 inhibitor with RT was equally effective to that of anti-PDL1+RT in terms of anti-tumor growth response. Conclusions: Our study provides the first insight into a novel role for EphB4-ephrin-B2 interaction in modulating tumor immune microenvironment in HNSCC. Our findings present a potential alternative in the form of EphB4-ephrin-B2 targeted therapeutics that can be tested in clinical trials in combination with RT for HNSCC patients. Citation Format: Shilpa Bhatia, Ayman Oweida, Shelby Lennon, Laurel Darragh, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Natalie Serkova, Antonio Jimeno, Eric Clambey, Elena Pasquale, Sana Karam. Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5035.

Abstract 1174: Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer

Abstract Background: Given the success of both targeted and immunotherapies against cancer, there is increasing utility for identifying targeted agents that also promote anti-tumor immunity. EphA2 is a receptor tyrosine kinase that contributes to tumor growth and metastasis in various cancer types and has been identified as a viable target for non-small cell lung cancer (NSCLC). Here, we examine how tumor-specific EphA2 affects the infiltration of different immune populations, the activation or inhibition of those populations, and the cytokine and chemokine milieu in mouse models of NSCLC. Methods: We generated two murine NSCLC cell lines, one derived from Lewis Lung Carcinoma and the other derived from a primary lung tumor harboring a KRAS mutation, as well as p53 and LKB1 loss of function alterations created using CRISPR-Cas9 gene editing. Effects of EphA2 overexpression in these cells were evaluated in both in vitro assays and in vivo models via subcutaneous tumor implantation and tail vein-injected orthotopic tumor formation in immunocompetent, syngeneic mice. Tumor immune infiltrate was assessed by flow cytometry, and cytokine and chemokine RNA expression levels were evaluated using Nanostring’s PanCancer Immune Profiling Panel. Results: Although EphA2 overexpression in these cell lines did not display significant proliferative advantage in vitro in MTT and colony formation assays, it did confer a growth advantage in vivo. Analysis of lung tumor immune infiltrate revealed decreased NK and T cells in the EphA2-overexpressing tumors, as well as increased myeloid populations, such as macrophages and monocytes. Furthermore, T cell activation markers, such as CD25, CD44, and CD69, in both CD4 and CD8 T cells were decreased in the EphA2-overexpressing tumors, while the percentage of regulatory T cells was increased. These changes in T cell activity were accompanied by increased presence of monocyte-derived CD11b+ Ly6C+ Ly6G- cells and Gr1- F4/80+ MHCII+ tumor-associated macrophages (TAMs). Higher infiltration of these myeloid populations coincided with increased monocyte-attracting chemokine and receptor expression, including CCL2, CCL7, CCL8, CCL12, and CCR2 and increased expression of immunosuppressive proteins, including TGFβ and arginase 1. Conclusion: Our studies suggest tumor-specific EphA2 inhibits infiltration and activation of key lymphocytic populations, especially T cells, while recruiting monocytes and likely promoting their transformation into myeloid-derived suppressor cells (MDSCs) and TAMs. Further functional studies are needed to validate the role of these myeloid cells. Elucidation of EphA2’s role in regulating immune recruitment and function will advance our understanding of tumor immune evasion and the potentially beneficial consequences of targeting EphA2 in cancer on the tumor microenvironment. Citation Format: Eileen Shiuan, Wenqiang Song, Shan Wang, Mark Boothby, Jin Chen. Tumor-specific EphA2 receptor tyrosine kinase inhibits anti-tumor immunity by recruiting suppressive myeloid populations in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1174.

361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.