EphA2 Inhibition Research Articles

A multi-model approach identifies ALW-II-41-27 as a promising therapy for osteoarthritis-associated inflammation and endochondral ossification

Low-grade inflammation and pathological endochondral ossification are key processes underlying the progression of osteoarthritis, the most prevalent joint disease worldwide. In this study, we employed a multi-faceted approach, integrating publicly available datasets, in silico analyses, in vitro experiments and in vivo models to identify new therapeutic candidates targeting these processes. Data mining of transcriptomic datasets identified EPHA2, a receptor tyrosine kinase associated with cancer, as being linked to both inflammation and endochondral ossification in osteoarthritis. A computational model of cellular signaling networks in chondrocytes predicted that in silico activation of EPHA2 in healthy chondrocytes increases inflammatory mediators and induces hypertrophic differentiation, a hallmark of endochondral ossification. We then evaluated the effect of EPHA2 inhibition using the tyrosine kinase inhibitor ALW-II-41-27 in cultured human chondrocytes from individuals with osteoarthritis, demonstrating significant reductions in both inflammation and hypertrophy. Additionally, systemic subcutaneous administration of ALW-II-41-27 in a mouse osteoarthritic model attenuated joint degeneration by reducing local inflammation and pathological endochondral ossification. Collectively, this study demonstrates a novel drug discovery pipeline that integrates computational, experimental, and animal models, paving the way for the development of disease-modifying treatments for osteoarthritis.

Erythropoietin-induced hepatocyte receptor A2 regulates effect of pyroptosis on gastrointestinal colorectal cancer occurrence and metastasis resistance.

Erythropoietin-induced hepatocyte receptor A2 (EphA2) is a receptor tyrosine kinase that plays a key role in the development and progression of a variety of tumors. This article reviews the expression of EphA2 in gastrointestinal (GI) colorectal cancer (CRC) and its regulation of pyroptosis. Pyroptosis is a form of programmed cell death that plays an important role in tumor suppression. Studies have shown that EphA2 regulates pyrodeath through various signaling pathways, affecting the occurrence, development and metastasis of GI CRC. The overexpression of EphA2 is closely related to the aggressiveness and metastasis of GI CRC, and the inhibition of EphA2 can induce pyrodeath and improve the sensitivity of cancer cells to treatment. In addition, EphA2 regulates intercellular communication and the microenvironment through interactions with other cytokines and receptors, further influencing cancer progression. The role of EphA2 in GI CRC and its underlying mechanisms provide us with new perspectives and potential therapeutic targets, which have important implications for future cancer treatment.

Catalpol ameliorates liver fibrosis via inhibiting aerobic glycolysis by EphA2/FAK/Src signaling pathway

BackgroundHepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis. ObjectiveThis study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms. MethodsTo explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl4). Additionally, LX-2 cells were stimulated with transforming growth factor-β (TGF-β) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT. ResultsResults showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl4. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-β-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl4-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro. ConclusionThe study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.

Computational insights into inhibiting EphA2: Integrating structure-based virtual screening, docking, and molecular dynamics simulations for small molecule discovery.

Elevated expression and dysfunction of ephrin type A receptor-2 (EphA2) have been implicated in the initiation and progression of cancer, metastasis, and unfavorable clinical outcomes. A promising strategy to counteract this dysregulation involves the development of small-molecule inhibitors that target EphA2. Our study focuses on this objective. To initiate Structure-Based Virtual Screening (SBVS), we leveraged an advanced online platform, the Mcule database, which houses an extensive collection of millions of chemical compounds. Using drug similarity filters, we efficiently identified ten thousand potential hits. By further refining the selection through toxicity profiling, we prudently narrowed down the candidates to a more manageable set of 100 molecules. Using the Mcule Single Click, DockThor, and SwissDock tools, we conducted multi-scoring docking assessments of thirty-seven compounds that satisfied the ADME standards. A comprehensive evaluation of Gibbs binding free energy terms, as derived from these docking tools, facilitated the identification of top-ranking docking hits. Remarkably, among the known inhibitors, dasatinib displayed the most robust binding to EphA2 with an average ΔG of -9.0 kcal/mol. Intriguingly, alternatives have emerged in recent years. Notably, small molecules such as Mcule-1579910267 (ΔG: -9.3 kcal/mol), Mcule-1893218381 (ΔG: -9.2 kcal/mol), Mcule-3981378344 (ΔG: -9.3 kcal/mol), and Mcule-8617639093 (ΔG: -9.1 kcal/mol) exhibited a notably strong binding affinity to EphA2, rivaling dasatinib. Subsequently, the four leading ligands along with dasatinib were selected for the MD simulations. Our rigorous analyses during the MD simulation phase encompassing RMSD, RMSF, SASA, ΔGsolv, and Rg underscored the favorable stability of Mcule-8617639093. This compelling evidence ultimately signifies the potential for selective EphA2 inhibition.

Comprehensive analysis of EphA2 in pan-cancer: A prognostic biomarker associated with cancer immunity.

Several studies have reported a significant relationship between Ephrin receptor A2 (EphA2) and malignant progression in numerous cancers. However, there is a lack of comprehensive pan-cancer analysis on the prognostic value, mutation status, methylation landscape, and potential immunological function of EphA2. Using The Cancer Genome Atlas, Genotype Tissue Expression Database and GEO data, we analysed the differences in EphA2 expression between normal and tumour tissues and the effects of EphA2 on the prognosis of different tumours. Furthermore, using GSCALite, cBioPortal, TISDB, ULCLAN and TIMER 2.0 databases or platforms, we comprehensively analysed the potential oncogenic mechanisms or manifestations of EphA2 in 33 different tumour types, including tumour mutation status, DNA methylation status and immune cell infiltration. The correlation of EphA2 with immune checkpoints, tumour mutational burden, DNA microsatellite instability and DNA repair genes was also calculated. Finally, the effects of EphA2 inhibitors on the proliferation of human glioma and lung cancer cells were verified in cellular experiments. EphA2 is differentially expressed in different tumours, and patients with overexpression have poorer overall survival. In addition, gene mutations, gene copy number variation and DNA/RNA methylation of EphA2 have been identified in various tumours. Moreover, EphA2 is positively associated with immune infiltration involving macrophages and CD8+ T cells. Further, EphA2 mRNA expression is significantly associated with immune checkpoint in various cancers, especially programmed death-ligand 1. Finally, the EphA2 inhibitor ALW-II-41-27 shows potent anti-tumour activity. Our first pan-cancer study of EphA2 provides insight into the prognostic and immunological roles of EphA2 in different tumours, suggesting that EphA2 might be a potential biomarker for poor prognosis and immune infiltration in cancer.

Abstract 4538: Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4538.

Exploring the potential of EphA2 receptor signaling pathway: a comprehensive review in cancer treatment.

The protein encoded by the ephrin type-A receptor 2 (EphA2) gene is a member of the ephrin receptor subfamily of the receptor tyrosine kinase family (RTKs). Eph receptors play a significant role in various biological processes, particularly cancer progression, development, and pathogenesis. They have been observed to regulate cancer cell growth, migration, invasion, tumor development, invasiveness, angiogenesis, and metastasis. To target EphA2 activity, various molecular, genetic, biochemical, and pharmacological strategies have been extensively tested in laboratory cultures and animal models. Notably, drugs, such as dasatinib, initially designed to target the kinase family, have demonstrated an additional capability to target EphA2 activity. Additionally, a novel monoclonal antibody named EA5 has emerged as a promising option to counteract the effects of EphA2 overexpression and restore tamoxifen sensitivity in EphA2-transfected MCF-7 cells during in vitro experiments. This antibody mimicked the binding of Ephrin A to EphA2. These methods offer potential avenues for inhibiting EphA2 activity, which could significantly decelerate breast cancer progression and restore sensitivity to certain drugs. This review article comprehensively covers EphA2's involvement in multiple malignancies, including ovarian, colorectal, breast, lung, glioma, and melanoma. Furthermore, we discuss the structure of EphA2, the Eph-Ephrin signaling pathway, various EphA2 inhibitors, and the mechanisms of EphA2 degradation. This article provides an extensive overview of EphA2's vital role in different types of cancers and outlines potential therapeutic approaches to target EphA2, shedding light on the underlying molecular mechanisms that make it an attractive target for cancer treatment.

Targeting EphA2: a promising strategy to overcome chemoresistance and drug resistance in cancer.

Erythropoietin-producing hepatocellular A2 (EphA2) is a vital member of the Eph tyrosine kinase receptor family and has been associated with developmental processes. However, it is often overexpressed in tumors and correlates with cancer progression and worse prognosis due to the activation of its noncanonical signaling pathway. Throughout cancer treatment, the emergence of drug-resistant tumor cells is relatively common. Since the early 2000s, researchers have focused on understanding the role of EphA2 in promoting drug resistance in different types of cancer, as well as finding efficient and secure EphA2 inhibitors. In this review, the current knowledge regarding induced resistance by EphA2 in cancer treatment is summarized, and the types of cancer that lead to the most cancer-related deaths are highlighted. Some EphA2 inhibitors were also investigated. Regardless of whether the cancer treatment has reached a drug-resistance stage in EphA2-overexpressing tumors, once EphA2 is involved in cancer progression and aggressiveness, targeting EphA2 is a promising therapeutic strategy, especially in combination with other target-drugs for synergistic effect. For that reason, monoclonal antibodies against EphA2 and inhibitors of this receptor should be investigated for efficacy and drug toxicity.

EphA2- and HDAC-Targeted Combination Therapy in Endometrial Cancer.

Endometrial cancer is the most frequent malignant tumor of the female reproductive tract but lacks effective therapy. EphA2, a receptor tyrosine kinase, is overexpressed by various cancers including endometrial cancer and is associated with poor clinical outcomes. In preclinical models, EphA2-targeted drugs had modest efficacy. To discover potential synergistic partners for EphA2-targeted drugs, we performed a high-throughput drug screen and identified panobinostat, a histone deacetylase inhibitor, as a candidate. We hypothesized that combination therapy with an EphA2 inhibitor and panobinostat leads to synergistic cell death. Indeed, we found that the combination enhanced DNA damage, increased apoptosis, and decreased clonogenic survival in Ishikawa and Hec1A endometrial cancer cells and significantly reduced tumor burden in mouse models of endometrial carcinoma. Upon RNA sequencing, the combination was associated with downregulation of cell survival pathways, including senescence, cyclins, and cell cycle regulators. The Axl-PI3K-Akt-mTOR pathway was also decreased by combination therapy. Together, our results highlight EphA2 and histone deacetylase as promising therapeutic targets for endometrial cancer.

TARGETING HYPERTROPHIC CHONDROCYTES IN OSTEOARTHRITIS: THE ROLE OF INFLAMMATION AND THE USE OF A MULTI-MODEL APPROACH IN SEARCH OF PHARMACOLOGICAL TREATMENTS

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780).In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis.Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants.In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis.

ProNGF promotes brain metastasis through TrkA/EphA2 induced Src activation in triple negative breast cancer cells

BackgroundTriple-Negative Breast Cancer is particularly aggressive, and its metastasis to the brain has a significant psychological impact on patients' quality of life, in addition to reducing survival. The development of brain metastases is particularly harmful in triple-negative breast cancer (TNBC). To date, the mechanisms that induce brain metastasis in TNBC are poorly understood.MethodsUsing a human blood–brain barrier (BBB) in vitro model, an in vitro 3D organotypic extracellular matrix, an ex vivo mouse brain slices co-culture and in an in vivo xenograft experiment, key step of brain metastasis were recapitulated to study TNBC behaviors.ResultsIn this study, we demonstrated for the first time the involvement of the precursor of Nerve Growth Factor (proNGF) in the development of brain metastasis. More importantly, our results showed that proNGF acts through TrkA independent of its phosphorylation to induce brain metastasis in TNBC. In addition, we found that proNGF induces BBB transmigration through the TrkA/EphA2 signaling complex. More importantly, our results showed that combinatorial inhibition of TrkA and EphA2 decreased TBNC brain metastasis in a preclinical model.ConclusionsThese disruptive findings provide new insights into the mechanisms underlying brain metastasis with proNGF as a driver of brain metastasis of TNBC and identify TrkA/EphA2 complex as a potential therapeutic target.

Inhibition of EphA2 protects against atherosclerosis by synergizing with statins to mitigate macrophage inflammation

Statins are highly prevalent in patients with coronary artery disease. Statins exert their anti-inflammatory effects on the vascular wall and circulating levels of pro-inflammatory cytokines. However, increasing attention revealed the exacerbation of macrophage inflammation induced by statins, and a clear mechanistic explanation of whether the detrimental effects of statins on macrophage inflammatory phenotypes outweigh the beneficial effects is has not yet been established. Here, RNA-sequencing and RT-qPCR analyses demonstrated that statins significantly upregulated EphA2, Nlrp3, IL-1β and TNF-α expression in macrophages. Mechanistically, we found that atorvastatin reduced KLF4 binding to the EphA2 promoter using KLF4-chromatin immunoprecipitation, suppressed HDAC11-mediated deacetylation and subsequently led to enhanced EphA2 transcription. The 4D-label-free proteomics analysis further confirmed the upregulated EphA2 and inflammatory signals. Furthermore, the proinflammatory effect of atorvastatin was neutralized by an addition of recombinant Fc-ephrinA1, a selective Eph receptor tyrosine kinase inhibitor (ALW-II-41–27) or EphA2-silencing adenovirus (siEphA2). In vivo, EphA2 was identified a proatherogenic factor and apoE-/- mice placed on a high-fat diet following gastric gavage with atorvastatin exhibited a consistent elevation in EphA2 expression. We further observed that the transfection with siEphA2 in atorvastatin-treated mice significantly attenuated atherosclerotic plaque formation and abrogated statin-orchestrated macrophages proinflammatory genes expression as compared to that in atorvastatin alone. Increased plaque stability index was also observed following the addition of siEphA2, as evidenced by increased collagen and smooth muscle content and diminished lipid accumulation and macrophage infiltration. The data suggest that blockage of EphA2 provides an additional therapeutic benefit for further improving the anti-atherogenic effects of statins.

Targeting EPHA2 with Kinase Inhibitors in Colorectal Cancer.

The ephrin type-A 2 receptor tyrosine kinase (EPHA2) is involved in the development and progression of various cancer types, including colorectal cancer (CRC). There is also evidence that EPHA2 plays a key role in the development of resistance to the endothelial growth factor receptor (EGFR) monoclonal antibody Cetuximab used clinically in CRC. Despite the promising pharmacological potential of EPHA2, only a handful of specific inhibitors are currently available. In this concept paper, general strategies for EPHA2 inhibition with molecules of low molecular weight (small molecules) are described. Furthermore, available examples of inhibiting EPHA2 in CRC using small molecules are summarized, highlighting the potential of this approach.

Curcumin inhibits vascular mimicry to prevent thyroid cancer progression through regulation of EPH receptor A2/phosphoinositide 3-kinase

This study explored the impact of curcumin on vascular mimicry in thyroid cancer (TC) cells and underlying mechanism. TC cells (TPC-1) were exposed to curcumin at 15 and 30 μmol/L (low-dose and high-dose group) concentrations, respectively, with control group exposed to culture medium. After treatment, cell migration, invasion, proliferation and apoptosis were detected along with analysis of EphA2 and PI3K expressions. The amount of migrated and invaded cells, EphA2 and PI3K protein expressions, Matrix metalloproteinase 2(MMP2), MMP9, CyclinD1, EphA2 and PI3K of TPC-1 cells in curcumin groups were decreased (P < 0.05). Apoptosis, as well as Cleaved-caspase-3, Bax and p21 expressions increased (P < 0.05). Curcumin dose-dependently suppressed angiogenesis of TPC-1 cells (P < 0.05) and lumen formation. In addition, TPC-1 cells showed cell rearrangement and scattered tubules after conditioned medium of neurotrophic factor (NTF-CM) treatment, while the CM of cancer-associated fibroblasts (CAF-CM) (50%) and CAF-CM (100%) both significantly promoted vasculogenic mimicry in TPC-1 cells, with elevated amount of tubules and junctions (P < 0.05). Moreover, treatment with ALW-II-41-27, which is an EphA2 inhibitor, alleviated the CAF-CM’s effect on vasculogenic mimicry (P < 0.05), and EphA2 knockdown decreased MMP2, MMP9, EphA2 and PI3K protein expressions when decelerating tube formation in NTF-CM and CAF-CM groups (P < 0.05). Collectively, Curcumin suppresses malignant behaviors and hinders vascular mimicry of TC cells by inhibiting the EPHA2/PI3K pathway, thereby preventing malignant progression of TC.

EphA2 is a functional entry receptor for HCMV infection of glioblastoma cells.

Human cytomegalovirus (HCMV) infection is associated with human glioblastoma, the most common and aggressive primary brain tumor, but the underlying infection mechanism has not been fully demonstrated. Here, we show that EphA2 was upregulated in glioblastoma and correlated with the poor prognosis of the patients. EphA2 silencing inhibits, whereas overexpression promotes HCMV infection, establishing EphA2 as a crucial cell factor for HCMV infection of glioblastoma cells. Mechanistically, EphA2 binds to HCMV gH/gL complex to mediate membrane fusion. Importantly, the HCMV infection was inhibited by the treatment of inhibitor or antibody targeting EphA2 in glioblastoma cells. Furthermore, HCMV infection was also impaired in optimal glioblastoma organoids by EphA2 inhibitor. Taken together, we propose EphA2 as a crucial cell factor for HCMV infection in glioblastoma cells and a potential target for intervention.

Abstract 321: Altered Epha2 Signaling In Non-alcoholic Fatty Liver Disease Progression

Background: Nonalcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease worldwide, results from diet-induced hepatic steatosis that can result in hepatocyte ballooning, inflammation and fibrosis driving nonalcoholic steatohepatitis (NASH). The Eph receptors, the largest family of receptor tyrosine kinases in the mammalian genome, affect inflammation and fibrosis in other model systems, and we have shown that EphA2 deletion reduces atherosclerosis despite increasing plasma cholesterol levels. While a GWAS study has linked EphA2 to NAFLD, the potential role of EphA2 in NAFLD has never been investigated. Methods: EphA2 expression and signaling was assessed in mice fed a high fat diet (HFD) for 8 and 24 weeks, in human liver samples from NASH patients, and in HuH7 cells treated with palmitic acid (FA), low density lipoprotein (LDL), and/or fructose. Global EphA2 KO mice were fed a HFD for 12 weeks followed by histological and molecular analyses. Results: Mice with early-stage non-alcoholic fatty liver disease (NAFLD) showed elevated EphA2 expression along with markers of EphA2 ligand-dependent signaling (EphA2 tyrosine phosphorylation). Consistently, HuH7 cells treated with FA, LDL, and/or fructose displayed a similar enhancement of EphA2 expression upon treatment. However, a mouse model of late-stage NASH exhibited reduced expression of the EphA2 ligand ephrinA1 and elevated markers of ligand-independent signaling (Ser897 phosphorylation). Similarly, human liver samples from NASH patients show reduced ephrinA1 expression and elevated markers of EphA2 ligand independent signaling. Consistent with a role for EphA2 in NAFLD/NASH, global EphA2 KO mice show significantly lower indices of NAFLD following high fat diet feeding, including reduced liver-to-body weight ratios, hepatic steatosis, and inflammation. Conclusion: Taken together, our data demonstrate that EphA2 expression and signaling are altered during NAFLD/NASH pathogenesis and suggest that EphA2 inhibition may reduce NAFLD/NASH disease progression.

Abstract 1324: EphA2 Expression in Breast Cancer Mediates Osteoclast Expansion and Promotes Osteolytic Bone Metastasis

Abstract Breast cancer is the most diagnosed cancer in the U.S., with a 5-year survival rate of less than 30% for patients with distant metastases. Bone metastases are present in over 70% of metastatic breast cancer patients, and current treatments treat the symptoms of osteolytic bone metastasis (OBM), but do not improve survival rates. OBM is characterized by an increase in osteoclast proliferation and bone-resorbing activity. Moreover, receptor tyrosine kinase, Ephrin-type-A 2 receptor (EphA2), is highly expressed in bone metastases, and previous evidence suggests its involvement in OBM and anti-tumor immunity. EphA2 participates in both forward and reverse signaling during receptor/ligand engagement and mediates various biological processes such as tissue organization and homeostasis and inflammation, as well as oncogenic processes like epithelial-mesenchymal transition. However, the mechanism by which EphA2 mediates the expansion of osteoclasts is not well understood. Analysis of transcriptomic data of patients with bone metastases in The Metastatic Breast Cancer Project (Provisional, December 2021) resulted in over 3500 genes that significantly correlated with EphA2 expression (Spearman's Correlation ≥ 0.35). Using a publicly available Gene Ontology reference list for myeloid differentiation, we identified 155 genes that have a significant positive correlation with EphA2 expression. To assess the effect of EphA2 expression on genes associated with osteoclasts, we compared the same samples to reference lists for osteoclast differentiation and proliferation. We identified 17 genes of interest that significantly correlate with EphA2 expression, further underscoring the possible involvement of EphA2 in OBM. We then compared the patients without bone metastases to the same osteoclast reference lists and found no significant correlations. We hypothesize that increased expression of EphA2 in breast cancer cells at the site of bone metastasis promotes osteoclast expansion, leading to OBM. We aim to investigate EphA2 reverse signaling in myeloid progenitor cells, osteoclasts, and bone metastasis-associated macrophages. We will utilize breast cancer mouse models to elucidate the effects of EphA2 inhibition on immune cell populations, bone metastasis, and tumor progression. We aim to reduce OBM and tumor burden by reducing the expansion of osteoclasts and shifting the immune cells to an anti-tumorigenic phenotype, which will offer new treatment strategies for osteolytic breast cancer. Citation Format: Dominique Parker, Verra Ngwa, Logan Northcutt, Natalie Bennett, Erik Beadle, Jade Miller, JIn Chen, Julie Rhoades. EphA2 Expression in Breast Cancer Mediates Osteoclast Expansion and Promotes Osteolytic Bone Metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1324.

The EphA1 and EphA2 Signaling Modulates the Epithelial Permeability in Human Sinonasal Epithelial Cells and the Rhinovirus Infection Induces Epithelial Barrier Dysfunction via EphA2 Receptor Signaling.

Deficiencies in epithelial barrier integrity are involved in the pathogenesis of chronic rhinosinusitis (CRS). This study aimed to investigate the role of ephrinA1/ephA2 signaling on sinonasal epithelial permeability and rhinovirus-induced epithelial permeability. This role in the process of epithelial permeability was evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to rhinovirus infection. EphrinA1 treatment increased epithelial permeability, which was associated with decreased expression of ZO-1, ZO-2, and occludin. These effects of ephrinA1 were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. Furthermore, rhinovirus infection upregulated the expression levels of ephrinA1 and ephA2, increasing epithelial permeability, which was suppressed in ephA2-deficient cells. These results suggest a novel role of ephrinA1/ephA2 signaling in epithelial barrier integrity in the sinonasal epithelium, suggesting their participation in rhinovirus-induced epithelial dysfunction.

EPHA2 is a Potential Target for the Treatment of NF2-/-Vestibular Schwannoma

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer predisposition syndrome characterized by the development of bilateral vestibular (VS) and spinal schwannomas secondary to loss of heterozygosity of NF2 in Schwann cells or their precursors. While these tumors are largely benign, they can cause considerable morbidity due to compromised auditory, vestibular, facial, and vertebral nerve function. This may result in deafness, vertigo, facial muscle weakness, chronic neuropathic pain, and even death.There are currently no pharmacotherapies for VS, and surgical resection remains the standard of care, which is associated with significant morbidity. Thus, there is an urgent need to develop pharmaceutical approaches to halt or reverse the progression of tumor growth in NF2 patients who develop VS. Our lab previously identified the receptor tyrosine kinase inhibitors brigatinib and dasatinib as potentially efficacious agents for the treatment of VS and demonstrated that both agents targeted the Ephrin A2 receptor (EPHA2). EPHA2is a transmembrane receptor tyrosine kinase thatis involved in cell contact-mediated motility, adhesion,and migration. Additionally, EPHA2 modulates axon guidance, and synaptogenesis in developing brain. Here we demonstrate that EPHA2expression is increased in NF2-/-Schwanncells and NF2-/-cancers. We identify ponatinib, a receptor tyrosine kinase inhibitor targeting ABL1 that is FDA-approved for CML, as an additional agent that targets EPHA2. We demonstrate that ponatinib treatment impairs the viability of both human and murine NF2-/-Schwanncells in vitro and decreases EPHA2 protein expression. Accordingly, pharmacologic,and siRNA-mediated inhibition of EPHA2 also impaired the growth of human NF2-/-Schwann cells in vitro. Lastly, we demonstrate that both ponatinib and EPHA2 inhibition induce morphological changes in NF2-/-Schwann cells. Our findings suggest that ponatinib or the direct targeting of EPHA2 may be efficacious for the treatment of NF2-associated vestibular schwannoma. Future in vivo efficacystudies are warranted.

A mechanism study of DUSP1 in inhibiting malignant progression of endometrial carcinoma by regulating ERK/AP-1 axis and dephosphorylation of EPHA2.

Background: Endometrial carcinoma is one of the most common female malignancies worldwide. Based on our preliminary investigation, DUSP1 was identified as a potential biomarker for endometrial carcinoma prognosis, but its function and mechanism remained unclear. Methods: In this study, genes highly correlated with DUSP1 in endometrial cancer were found through correlation analysis, and the promoter sequence of DUSP1 was analyzed by PROMO program. Next-generation phosphorylation mass spectrometry was used to explore new downstream target proteins and pathways of DUSP1 in endometrial carcinoma. The mRNA and protein expression levels were detected by real-time quantitative PCR, immunohistochemistry and Western blotting. The cell survival and proliferation were analyzed by CCK8 assay, cell apoptosis was analyzed by Annexin-V-APC and PI dual staining assay, and the cell invasion was analyzed by Transwell method. Results: (1) There was a high correlation between the expression of DUSP1 and the genes involved in AP-1 complex and its co-expression network. (2) Promoter sequence analysis predicted that the members of AP-1 complex might be the upstream transcriptional regulators of DUSP1. (3) Transfection experiments proved DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating ERK pathway. (4) The results of phosphorylation mass spectrometry showed that overexpression of DUSP1 mainly dephosphorylated EPHA2 in endometrial carcinoma, and co-immunoprecipitation verified the protein interaction between DUSP1 and EPHA2. (5) Overexpression or knockdown of EPHA2 significantly changed the phosphorylation level of EPHA2. (6) The expression of EPHA2 protein was high in patients with more aggressive endometrial cancer. (7) Using EPHA2 inhibitor could significantly slow down the growth rate of tumor cells. Conclusion: (1) There exists a mutual regulation relationship between DUSP1 and AP-1 co-expression network in endometrial carcinoma. (2) It is reported for the first time that DUSP1 phosphatase acts on the ser899 site of EphA2 in endometrial carcinoma. (3) DUSP1 can inhibit tumor growth and invasion, and promote apoptosis by regulating MAPK pathway through directly dephosphorylating ERK, or by dephosphorylating EPHA2.