Identification of potent pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology.

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.

Transmembrane helix interactions regulate oligomerization of the receptor tyrosine kinase EphA2.

The transmembrane helices of receptor tyrosine kinases (RTKs) have been proposed to switch between two different dimeric conformations, one associated with the inactive RTK and the other with the active RTK. Furthermore, recent work has demonstrated that some full-length RTKs are associated into oligomers that are larger than dimers, raising questions about the roles of the TM helices in the assembly and function of these oligomers. Here we probe the roles of the TM helices in the assembly of EphA2 RTK oligomers in the plasma membrane. We employ mutagenesis to evaluate the relevance of a published NMR dimeric structure of the isolated EphA2 TM helix in the context of the full-length EphA2 in the plasma membrane. We use two fluorescence methods, Förster Resonance Energy Transfer and Fluorescence Intensity Fluctuations spectrometry, which yield complementary information about the EphA2 oligomerization process. These studies reveal that the TM helix mutations affect the stability, structure, and size of EphA2 oligomers. However, the effects are multifaceted and point to a more complex role of the TM helix than the one expected from the "TM dimer switch" model.

EphA2 promotes the transcription of KLF4 to facilitate stemness in oral squamous cell carcinoma.

Ephrin receptor A2 (EphA2), a member of the Ephrin receptor family, is closely related to the progression of oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play essential roles in OSCC development and occurrence. The underlying mechanisms between EphA2 and CSCs, however, are not yet fully understood. Here, we found that EphA2 was overexpressed in OSCC tissues and was associated with poor prognosis. Knockdown of EphA2 dampened the CSC phenotype and the tumour-initiating frequency of OSCC cells. Crucially, the effects of EphA2 on the CSC phenotype relied on KLF4, a key transcription factor for CSCs. Mechanistically, EphA2 activated the ERK signalling pathway, promoting the nuclear translocation of YAP. Subsequently, YAP was bound to TEAD3, leading to the transcription of KLF4. Overall, our findings revealed that EphA2 can enhance the stemness of OSCC cells, and this study identified the EphA2/KLF4 axis as a potential target for treating OSCC.

Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway.

The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK) via the MEK-ERK pathway in ligand- and tyrosine kinase-independent manners. Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.

LDHA-mediated M2-type macrophage polarization via tumor-derived exosomal EPHA2 promotes renal cell carcinoma progression.

Lactate dehydrogenase A (LDHA) is known to promote the growth and invasion of various types of tumors, affects tumor resistance, and is associated with tumor immune escape. But how LDHA reshapes the tumor microenvironment and promotes the progression of renal cell carcinoma (RCC) remains unclear. In this study, we found that LDHA was highly expressed in clear cell RCC (ccRCC), and this high expression was associated with macrophage infiltration, while macrophages were highly infiltrated in ccRCC, affecting patient prognosis via M2-type polarization. Our in vivo and in vitro experiments demonstrated that LDHA and M2-type macrophages could enhance the proliferation, invasion, and migration abilities of ccRCC cells. Mechanistically, high expression of LDHA in ccRCC cells upregulated the expression of EPHA2 in exosomes derived from renal cancer. Exosomal EPHA2 promoted M2-type polarization of macrophages by promoting activation of the PI3K/AKT/mTOR pathway in macrophages, thereby promoting the progression of ccRCC. All these findings suggest that EPHA2 may prove to be a potential therapeutic target for advanced RCC.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Targeting the EphA2 pathway: could it be the way for bone sarcomas?

Bone sarcomas are malignant tumors of mesenchymal origin. Complete surgical resection is the cornerstone of multidisciplinary treatment. However, advanced, unresectable forms remain incurable. A crucial step towards addressing this challenge involves comprehending the molecular mechanisms underpinning tumor progression and metastasis, laying the groundwork for innovative precision medicine-based interventions. We previously showed that tyrosine kinase receptor Ephrin Type-A Receptor 2 (EphA2) is overexpressed in bone sarcomas. EphA2 is a key oncofetal protein implicated in metastasis, self-renewal, and chemoresistance. Molecular, genetic, biochemical, and pharmacological approaches have been developed to target EphA2 and its signaling pathway aiming to interfere with its tumor-promoting effects or as a carrier for drug delivery. This review synthesizes the main functions of EphA2 and their relevance in bone sarcomas, providing strategies devised to leverage this receptor for diagnostic and therapeutic purposes, with a focus on its applicability in the three most common bone sarcoma histotypes: osteosarcoma, chondrosarcoma, and Ewing sarcoma.

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.

Sesamol-mediated targeting of EPHA2 sensitises cervical cancer for cisplatin treatment by regulating mitochondrial dynamics, autophagy, and mitophagy.

BACKGROUND: Cervical cancer ranks as the fourth most prevalent cancer among women globally, presenting a significant therapeutic challenge due to its resistance to cisplatin. Ephrin type-A receptor 2 (EPHA2) is prominently overexpressed in cervical cancer and plays a vital role in cisplatin resistance, although the underlying mechanisms remain incompletely elucidated. Mitochondrial dynamics, autophagy, and mitophagy are critical in mediating cisplatin resistance. Sesamol, a phytochemical compound, has exhibited promising anticancer properties. This study aims to investigate the regulatory role of EPHA2 in these pathways underlying cisplatin resistance and to investigate the potential of sesamol in overcoming this resistance and inhibiting cervical cancer progression. METHODS AND RESULT: In this study, we knocked down EPHA2 in the SiHa cell line and evaluated the resulting changes in molecular markers associated with mitochondrial dynamics, mitophagy, and autophagy. Our results indicated that EPHA2 knockdown (EPHA2-KD) led to enhanced mitochondrial fusion and reduced mitochondrial fission, mitophagy, and autophagy. Furthermore, we investigated the effect of EPHA2-KD and sesamol treatment on sensitising cervical cancer to cisplatin treatment. Our data revealed that EPHA2-KD and sesamol treatment significantly increases cellular sensitivity to cisplatin-induced cytotoxicity. Additionally, we demonstrated that sesamol effectively targets EPHA2, as evidenced by decreased EPHA2 expression levels following sesamol treatment. CONCLUSION: In summary, targeting EPHA2 through knockdown or sesamol treatment enhances cisplatin sensitivity in cervical cancer by modulating mitochondrial dynamics, autophagy and mitophagy, suggesting promising therapeutic strategies to overcome chemoresistance.

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.

TYRO3 and EPHA2 Expression Are Dysregulated in Breast Cancer.

Receptor tyrosine kinases (RTKs) are involved in cell growth, motility, and differentiation. Deregulation of RTKs signaling is associated with tumor development and therapy resistance. Potential RTKs like TAM (TYRO3, AXL, MERTK), RON, EPH, and MET have been evaluated in many cancers like lung, prostate, and colorectal, but little is known in breast tumors. In this study, 51 luminal breast cancer tissue and 8 triple negative breast cancer (TNBC) subtypes were evaluated by qPCR for the expression of TAM, RON, EPHA2, and MET genes. Statistical analysis was performed to determine the correlation to clinical data. TYRO3 is related to tumor subtype and stage, patient's age, smoking habits, and obesity. MET expression is correlated to EPHA2 and TAM gene expression. EPHA2 expression is also related to aging and smoking habits. The expression levels of the TAM and EPHA2 genes seem to play an important role in breast cancer, being also influenced by the patient's lifestyle.

Exploring a Potential Optimization Route for Peptide Ligands of the Sam Domain from the Lipid Phosphatase Ship2.

The Sam (Sterile alpha motif) domain of the lipid phosphatase Ship2 (Ship2-Sam) is engaged by the Sam domain of the receptor tyrosine kinase EphA2 (EphA2-Sam) and, this interaction is principally linked to procancer effects. Peptides able to hinder the formation of the EphA2-Sam/Ship2-Sam complex could possess therapeutic potential. Herein, by employing the FoldX software suite, we set up an in silico approach to improve the peptide targeting of the so-called Mid Loop interface of Ship2-Sam, representing the EphA2-Sam binding site. Starting from a formerly identified peptide antagonist of the EphA2-Sam/Ship2-Sam association, first, the most stabilizing mutations that could be inserted in each peptide position were predicted. Then, they were combined, producing a list of potentially enhanced Ship2-Sam ligands. A few of the in silico generated peptides were experimentally evaluated. Interaction assays with Ship2-Sam were performed using NMR and BLI (BioLayer Interferometry). In vitro assays were conducted as well to check for cytotoxic effects against both cancerous and healthy cells, and also to assess the capacity to regulate EphA2 degradation. This study undoubtedly enlarges our knowledge on how to properly target EphA2-Sam/Ship2-Sam associations with peptide-based tools and provides a promising strategy that can be used to target any protein-protein interaction.

The efficacy of receptor tyrosine kinase EphA2 autophosphorylation increases with EphA2 oligomer size.

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms high-order oligomers upon ligand binding. Here, we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used fluorescence intensity fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Our comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, an autophosphorylation response contributing to EphA2 activation, correlates with EphA2 mean oligomer size. However, we found that other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. Taken together, this work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids.

Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.

Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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.

Competition for shared downstream signaling molecules establishes indirect negative feedback between EGFR and EphA2.

Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remain largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single-molecule imaging, to measure the competition between ephrin-A1:EphA2 and epidermal growth factor (EGF):EGF receptor (EGFR) ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.

PIP2 promotes conformation-specific dimerization of the EphA2 membrane region.

The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces antioncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using styrene maleic acid lipid particles to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TMs are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition.

Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1-ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.

Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer.

BACKGROUND: The key process of mesenchymal to amoeboid transition (MAT), which enables prostate cancer (PCa) transendothelial migration and subsequent development of metastases in red bone marrow stroma, is driven by phosphorylation of EphA2S897 by pAkt, which is induced by the omega-6 polyunsaturated fatty acid arachidonic acid. Here we investigate the influence of EphA2 signalling in PCa progression and long-term survival. METHODS: The mechanisms underpinning metastatic biopotential of altered EphA2 signalling in relation to PTEN status were assessed in vitro using canonical (EphA2D739N) and non-canonical (EphA2S897G) PC3-M mutants, interrogation of publicly available PTEN-stratified databases and clinical validation using a PCa TMA (n = 177) with long-term follow-up data. Spatial heterogeneity of EphA2 was assessed using a radical prostatectomy cohort (n = 67). RESULTS: Non-canonical EphA2 signalling via pEphA2S897 is required for PCa transendothelial invasion of bone marrow endothelium. High expression of EphA2 or pEphA2S897 in a PTENlow background is associated with poor overall survival. Expression of EphA2, pEphA2S897 and the associated MAT marker pMLC2 are spatially regulated with the highest levels found within lesion areas within 500 µm of the prostate margin. CONCLUSION: EphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.