{"gene":"EFNA4","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1996,"finding":"EFNA4 (LERK-4/EPLG4) is anchored to the cell membrane via glycosyl-phosphatidylinositol (GPI) linkage, placing it in the GPI-anchored subgroup of Eph receptor ligands (LERKs), distinct from the type-1 transmembrane subgroup.","method":"cDNA cloning and sequence analysis identifying GPI-anchored membrane attachment","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — sequence-based classification with biochemical context, single lab but consistent with broad family characterization","pmids":["8660976"],"is_preprint":false},{"year":2021,"finding":"EFNA4 directly interacts with EPHA2 and promotes its phosphorylation at Ser897, leading to recruitment of PIK3R2 and activation of the GSK3β/β-catenin signaling pathway, which in turn promotes PIK3R2 expression forming a positive feedback loop that drives HCC cell proliferation and migration.","method":"Co-immunoprecipitation, overexpression and knockdown experiments in vitro and in vivo, phosphorylation assays","journal":"Molecular therapy. Nucleic acids","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction shown and pathway placement via gain/loss-of-function, single lab","pmids":["34484860"],"is_preprint":false},{"year":2022,"finding":"EFNA4 interacts with PYGO2 and positively regulates PYGO2 expression; EFNA4 knockdown suppresses Wnt/β-catenin signaling in HCC cells, and this effect is rescued by PYGO2 overexpression, placing EFNA4 upstream of PYGO2 in Wnt/β-catenin pathway regulation.","method":"Co-immunoprecipitation, gain- and loss-of-function experiments, Wnt signaling reporter assays, rescue experiments","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — protein interaction demonstrated and pathway placement by epistasis rescue, single lab","pmids":["36404439"],"is_preprint":false},{"year":2022,"finding":"EFNA4 overexpression contributes to lung tumor cell growth, migration, and adhesion, while EFNA4 knockdown or knockout suppresses cell growth and xenograft tumor growth in mice, demonstrating a functional oncogenic role in lung adenocarcinoma.","method":"EFNA4 overexpression and knockdown/knockout in cell lines, xenograft tumor models in mice","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular and in vivo phenotypes, single lab","pmids":["36077763"],"is_preprint":false},{"year":2024,"finding":"EFNA4 inhibits ferroptosis in HCC independently of Eph receptor binding by directly interacting with SLC7A11 through its domain (a.a. 161-201) binding to SLC7A11 domain (a.a. 222-501), recruiting the deubiquitinase USP9X, and thereby stabilizing SLC7A11 through deubiquitination, which suppresses reactive oxygen species accumulation and ferroptosis.","method":"CRISPR/Cas9 Eph receptor knockout environment, domain mapping by co-immunoprecipitation, ubiquitination assays, apoptosis/ROS/GPX4 assays, in vitro and in vivo experiments","journal":"Apoptosis : an international journal on programmed cell death","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-level interaction mapping and mechanistic pathway established in receptor-null background, single lab with multiple orthogonal methods","pmids":["39656358"],"is_preprint":false},{"year":2024,"finding":"EFNA4 activates β-catenin signaling and suppresses deoxycytidine kinase (dCK) expression in gemcitabine-resistant pancreatic cancer cells; hyperthermia reduces EFNA4 expression, which leads to decreased β-catenin activation and restored dCK activity, sensitizing cells to gemcitabine. β-catenin antagonist MSAB rescues the effect of EFNA4 overexpression on dCK suppression.","method":"GEM-resistant cell line engineering, MTT assay, dCK activity assay, lentiviral dCK silencing, EFNA4 overexpression with β-catenin antagonist rescue, xenograft tumor models","journal":"Heliyon","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway placement by pharmacological rescue with β-catenin antagonist, multiple assays, single lab","pmids":["38590861"],"is_preprint":false},{"year":2024,"finding":"EFNA4 knockdown in gastric cancer cells reduces PYGO2 protein expression and inactivates Wnt/β-catenin signaling; overexpression of PYGO2 reverses the effects of EFNA4 silencing on Wnt/β-catenin signaling, proliferation, apoptosis, migration, invasion, angiogenesis, and stemness, placing EFNA4 upstream of PYGO2/Wnt signaling in gastric cancer.","method":"siRNA knockdown, western blot, CCK-8/EDU proliferation, Transwell invasion, tube formation, sphere formation, rescue experiments with PYGO2 overexpression","journal":"Histology and histopathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistatic rescue with PYGO2, multiple orthogonal functional assays, single lab","pmids":["38953488"],"is_preprint":false},{"year":2020,"finding":"Efna4 knockout mice exhibit increased bone volume/trabecular volume (BV/TV) in the femur, increased bone formation rate, increased osteoblast labeling surfaces, and decreased osteoclast activity, demonstrating that EFNA4 plays a functional role in regulating bone modeling through the Ephrin-Eph receptor signaling axis between osteoblasts and osteoclasts.","method":"Efna4 knockout mouse, micro-CT, cryohistomorphometry, dynamic bone labeling, cellular histomorphometric analysis","journal":"Bone","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined skeletal phenotype and cellular analysis, single center screen","pmids":["33065355"],"is_preprint":false},{"year":2025,"finding":"Calcitriol supplementation in BTBR autism model mice corrects axon guidance abnormalities and hippocampal hypoplasia; these effects are mediated through modulation of the EfnA4-PI3K/AKT signaling pathway in hippocampal neural progenitor cells, placing EFNA4 in the PI3K/AKT pathway controlling axon guidance in neurodevelopment.","method":"BTBR mouse model, behavioral assessments, RNA sequencing, immunohistochemistry, biochemical assays, stripe guidance assays","journal":"CNS neuroscience & therapeutics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway modulation inferred from in vivo pharmacological treatment without direct EFNA4 manipulation, single lab","pmids":["40395150"],"is_preprint":false}],"current_model":"EFNA4 is a GPI-anchored membrane ligand that signals through Eph receptors (notably EPHA2, driving Ser897 phosphorylation and PIK3R2/GSK3β/β-catenin activation) but can also act receptor-independently by binding SLC7A11 and recruiting USP9X deubiquitinase to stabilize SLC7A11 and suppress ferroptosis; in multiple cancer contexts EFNA4 promotes proliferation, migration, and Wnt/β-catenin signaling via PYGO2, while in bone it limits osteoblast activity and promotes osteoclast function."},"narrative":{"mechanistic_narrative":"EFNA4 is a GPI-anchored member of the ephrin-A family of Eph receptor ligands [PMID:8660976] that functions as a context-dependent driver of cancer cell proliferation, migration, and survival across multiple tumor types [PMID:36077763]. As a canonical ligand it binds EPHA2 and promotes its phosphorylation at Ser897, recruiting PIK3R2 and activating GSK3β/β-catenin signaling in a positive feedback loop that drives hepatocellular carcinoma growth [PMID:34484860]. EFNA4 acts upstream of the Wnt/β-catenin co-activator PYGO2, which it physically binds and positively regulates to sustain Wnt signaling and oncogenic phenotypes in hepatocellular and gastric carcinoma [PMID:36404439, PMID:38953488]; in gemcitabine-resistant pancreatic cancer this β-catenin activation suppresses deoxycytidine kinase and confers drug resistance [PMID:38590861]. Independently of Eph receptor binding, EFNA4 directly interacts with SLC7A11 through a defined domain (a.a. 161-201) and recruits the deubiquitinase USP9X to stabilize SLC7A11, suppressing ROS accumulation and ferroptosis [PMID:39656358]. Beyond its oncogenic roles, genetic loss of Efna4 in mice increases bone volume and formation while decreasing osteoclast activity, implicating it in skeletal modeling via the ephrin-Eph axis [PMID:33065355].","teleology":[{"year":1996,"claim":"Established the molecular nature of EFNA4 as a membrane attachment, defining it as a GPI-anchored Eph receptor ligand rather than a transmembrane ligand.","evidence":"cDNA cloning and sequence analysis of LERK-4/EPLG4","pmids":["8660976"],"confidence":"Medium","gaps":["No receptor binding partner identified in this work","No functional or signaling readout established"]},{"year":2020,"claim":"Answered whether EFNA4 has a physiological in vivo role by showing it regulates bone modeling, balancing osteoblast and osteoclast activity through ephrin-Eph signaling.","evidence":"Efna4 knockout mouse with micro-CT, histomorphometry, and dynamic bone labeling","pmids":["33065355"],"confidence":"Medium","gaps":["Specific Eph receptor partner in bone not identified","Cell-autonomous mechanism in osteoblasts vs osteoclasts not resolved"]},{"year":2021,"claim":"Defined the canonical signaling mechanism by which EFNA4 drives tumor growth, linking EPHA2 Ser897 phosphorylation to a PIK3R2/GSK3β/β-catenin feedback loop.","evidence":"Co-IP, gain/loss-of-function and phosphorylation assays in HCC in vitro and in vivo","pmids":["34484860"],"confidence":"Medium","gaps":["Single lab and tumor type","Direct enzymatic basis of Ser897 phosphorylation not dissected"]},{"year":2022,"claim":"Connected EFNA4 to the Wnt/β-catenin pathway through PYGO2 and demonstrated its broad oncogenic phenotype across liver and lung cancers.","evidence":"Co-IP, epistatic PYGO2 rescue, Wnt reporter assays in HCC; overexpression/KO with xenografts in lung adenocarcinoma","pmids":["36404439","36077763"],"confidence":"Medium","gaps":["Mechanism by which EFNA4 regulates PYGO2 protein levels unresolved","Whether PYGO2 regulation is receptor-dependent unclear"]},{"year":2024,"claim":"Revealed a receptor-independent function of EFNA4 in suppressing ferroptosis by stabilizing SLC7A11 via USP9X-mediated deubiquitination.","evidence":"Domain-mapping Co-IP, ubiquitination and ROS/GPX4 assays in an Eph receptor knockout background, in vitro and in vivo","pmids":["39656358"],"confidence":"Medium","gaps":["How a GPI-anchored ligand engages intracellular SLC7A11/USP9X is not mechanistically explained","Single lab"]},{"year":2024,"claim":"Extended the EFNA4/β-catenin axis to chemoresistance, showing it suppresses deoxycytidine kinase to confer gemcitabine resistance in pancreatic cancer.","evidence":"GEM-resistant cell lines, dCK activity assays, β-catenin antagonist rescue, xenografts","pmids":["38590861"],"confidence":"Medium","gaps":["Direct vs indirect regulation of dCK not distinguished","Single lab"]},{"year":2025,"claim":"Implicated EFNA4 in neurodevelopmental axon guidance through PI3K/AKT signaling, though only via indirect pharmacological modulation.","evidence":"BTBR autism model mice with calcitriol treatment, RNA-seq, IHC, stripe guidance assays","pmids":["40395150"],"confidence":"Low","gaps":["Pathway link inferred from drug treatment without direct EFNA4 manipulation","No demonstration of EFNA4 acting cell-autonomously in neural progenitors"]},{"year":null,"claim":"How EFNA4 switches between canonical Eph receptor-dependent signaling and receptor-independent intracellular partner stabilization, and what governs this choice across tissues, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of EFNA4 signaling states","Determinants of context-specific partner selection (EPHA2 vs SLC7A11 vs PYGO2) unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,6]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,5]}],"complexes":[],"partners":["EPHA2","PYGO2","SLC7A11","USP9X","PIK3R2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P52798","full_name":"Ephrin-A4","aliases":["EPH-related receptor tyrosine kinase ligand 4","LERK-4"],"length_aa":201,"mass_kda":22.4,"function":"Cell surface GPI-bound ligand for Eph receptors, a family of receptor tyrosine kinases which are crucial for migration, repulsion and adhesion during neuronal, vascular and epithelial development. Binds promiscuously Eph receptors residing on adjacent cells, leading to contact-dependent bidirectional signaling into neighboring cells. May play a role in the interaction between activated B-lymphocytes and dendritic cells in tonsils","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P52798/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EFNA4","classification":"Not Classified","n_dependent_lines":23,"n_total_lines":1208,"dependency_fraction":0.01903973509933775},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EFNA4","total_profiled":1310},"omim":[{"mim_id":"611123","title":"EPHRIN RECEPTOR EphA10; EPHA10","url":"https://www.omim.org/entry/611123"},{"mim_id":"610283","title":"CONE-ROD DYSTROPHY 10; CORD10","url":"https://www.omim.org/entry/610283"},{"mim_id":"610282","title":"RETINITIS PIGMENTOSA 35; RP35","url":"https://www.omim.org/entry/610282"},{"mim_id":"607292","title":"SEMAPHORIN 4A; SEMA4A","url":"https://www.omim.org/entry/607292"},{"mim_id":"602756","title":"EPHRIN A2; EFNA2","url":"https://www.omim.org/entry/602756"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skin 1","ntpm":29.2}],"url":"https://www.proteinatlas.org/search/EFNA4"},"hgnc":{"alias_symbol":["LERK4"],"prev_symbol":["EPLG4"]},"alphafold":{"accession":"P52798","domains":[{"cath_id":"2.60.40.420","chopping":"26-150","consensus_level":"high","plddt":95.7198,"start":26,"end":150}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P52798","model_url":"https://alphafold.ebi.ac.uk/files/AF-P52798-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P52798-F1-predicted_aligned_error_v6.png","plddt_mean":82.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EFNA4","jax_strain_url":"https://www.jax.org/strain/search?query=EFNA4"},"sequence":{"accession":"P52798","fasta_url":"https://rest.uniprot.org/uniprotkb/P52798.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P52798/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P52798"}},"corpus_meta":[{"pmid":"26015513","id":"PMC_26015513","title":"Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.","date":"2015","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/26015513","citation_count":65,"is_preprint":false},{"pmid":"30680712","id":"PMC_30680712","title":"First-in-human, phase I study of PF-06647263, an anti-EFNA4 calicheamicin antibody-drug conjugate, in patients with advanced solid tumors.","date":"2019","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30680712","citation_count":35,"is_preprint":false},{"pmid":"34484860","id":"PMC_34484860","title":"EFNA4 promotes cell proliferation and tumor metastasis in hepatocellular carcinoma through a PIK3R2/GSK3β/β-catenin positive feedback loop.","date":"2021","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/34484860","citation_count":24,"is_preprint":false},{"pmid":"8660976","id":"PMC_8660976","title":"The genes encoding the eph-related receptor tyrosine kinase ligands LERK-1 (EPLG1, Epl1), LERK-3 (EPLG3, Epl3), and LERK-4 (EPLG4, Epl4) are clustered on human chromosome 1 and mouse chromosome 3.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8660976","citation_count":13,"is_preprint":false},{"pmid":"36404439","id":"PMC_36404439","title":"Interference of EFNA4 suppresses cell proliferation, invasion and angiogenesis in hepatocellular carcinoma by downregulating PYGO2.","date":"2022","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/36404439","citation_count":10,"is_preprint":false},{"pmid":"36077763","id":"PMC_36077763","title":"Oncogenic EFNA4 Amplification Promotes Lung Adenocarcinoma Lymph Node Metastasis.","date":"2022","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/36077763","citation_count":10,"is_preprint":false},{"pmid":"39656358","id":"PMC_39656358","title":"EFNA4-enhanced deubiquitination of SLC7A11 inhibits ferroptosis in hepatocellular carcinoma.","date":"2024","source":"Apoptosis : an international journal on programmed cell death","url":"https://pubmed.ncbi.nlm.nih.gov/39656358","citation_count":4,"is_preprint":false},{"pmid":"38590861","id":"PMC_38590861","title":"Hyperthermia improves gemcitabine sensitivity of pancreatic cancer cells by suppressing the EFNA4/β-catenin axis and activating dCK.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/38590861","citation_count":4,"is_preprint":false},{"pmid":"40395150","id":"PMC_40395150","title":"Calcitriol Modulates Hippocampal Axon Guidance Through Enhanced EfnA4-Mediated PI3K/AKT Signaling in an Autism Mouse Model.","date":"2025","source":"CNS neuroscience & therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/40395150","citation_count":4,"is_preprint":false},{"pmid":"36140816","id":"PMC_36140816","title":"TBX3 and EFNA4 Variant in a Family with Ulnar-Mammary Syndrome and Sagittal Craniosynostosis.","date":"2022","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/36140816","citation_count":4,"is_preprint":false},{"pmid":"38953488","id":"PMC_38953488","title":"EFNA4 deletion suppresses the migration, invasion, stemness, and angiogenesis of gastric cancer cells through the inactivation of Pygo2/Wnt signaling.","date":"2024","source":"Histology and histopathology","url":"https://pubmed.ncbi.nlm.nih.gov/38953488","citation_count":3,"is_preprint":false},{"pmid":"33065355","id":"PMC_33065355","title":"Skeletal screening IMPC/KOMP using μCT and computer automated cryohistology: Application to the Efna4 KO mouse line.","date":"2020","source":"Bone","url":"https://pubmed.ncbi.nlm.nih.gov/33065355","citation_count":3,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.07.25333199","title":"Plasma proteomic signatures of social support and their association with cardiovascular disease and mortality","date":"2025-08-11","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.07.25333199","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9395,"output_tokens":2275,"usd":0.031155,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9426,"output_tokens":2672,"usd":0.056965,"stage2_stop_reason":"end_turn"},"total_usd":0.08812,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"EFNA4 (LERK-4/EPLG4) is anchored to the cell membrane via glycosyl-phosphatidylinositol (GPI) linkage, placing it in the GPI-anchored subgroup of Eph receptor ligands (LERKs), distinct from the type-1 transmembrane subgroup.\",\n      \"method\": \"cDNA cloning and sequence analysis identifying GPI-anchored membrane attachment\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — sequence-based classification with biochemical context, single lab but consistent with broad family characterization\",\n      \"pmids\": [\"8660976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EFNA4 directly interacts with EPHA2 and promotes its phosphorylation at Ser897, leading to recruitment of PIK3R2 and activation of the GSK3β/β-catenin signaling pathway, which in turn promotes PIK3R2 expression forming a positive feedback loop that drives HCC cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and knockdown experiments in vitro and in vivo, phosphorylation assays\",\n      \"journal\": \"Molecular therapy. Nucleic acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction shown and pathway placement via gain/loss-of-function, single lab\",\n      \"pmids\": [\"34484860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EFNA4 interacts with PYGO2 and positively regulates PYGO2 expression; EFNA4 knockdown suppresses Wnt/β-catenin signaling in HCC cells, and this effect is rescued by PYGO2 overexpression, placing EFNA4 upstream of PYGO2 in Wnt/β-catenin pathway regulation.\",\n      \"method\": \"Co-immunoprecipitation, gain- and loss-of-function experiments, Wnt signaling reporter assays, rescue experiments\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — protein interaction demonstrated and pathway placement by epistasis rescue, single lab\",\n      \"pmids\": [\"36404439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EFNA4 overexpression contributes to lung tumor cell growth, migration, and adhesion, while EFNA4 knockdown or knockout suppresses cell growth and xenograft tumor growth in mice, demonstrating a functional oncogenic role in lung adenocarcinoma.\",\n      \"method\": \"EFNA4 overexpression and knockdown/knockout in cell lines, xenograft tumor models in mice\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular and in vivo phenotypes, single lab\",\n      \"pmids\": [\"36077763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EFNA4 inhibits ferroptosis in HCC independently of Eph receptor binding by directly interacting with SLC7A11 through its domain (a.a. 161-201) binding to SLC7A11 domain (a.a. 222-501), recruiting the deubiquitinase USP9X, and thereby stabilizing SLC7A11 through deubiquitination, which suppresses reactive oxygen species accumulation and ferroptosis.\",\n      \"method\": \"CRISPR/Cas9 Eph receptor knockout environment, domain mapping by co-immunoprecipitation, ubiquitination assays, apoptosis/ROS/GPX4 assays, in vitro and in vivo experiments\",\n      \"journal\": \"Apoptosis : an international journal on programmed cell death\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-level interaction mapping and mechanistic pathway established in receptor-null background, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39656358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EFNA4 activates β-catenin signaling and suppresses deoxycytidine kinase (dCK) expression in gemcitabine-resistant pancreatic cancer cells; hyperthermia reduces EFNA4 expression, which leads to decreased β-catenin activation and restored dCK activity, sensitizing cells to gemcitabine. β-catenin antagonist MSAB rescues the effect of EFNA4 overexpression on dCK suppression.\",\n      \"method\": \"GEM-resistant cell line engineering, MTT assay, dCK activity assay, lentiviral dCK silencing, EFNA4 overexpression with β-catenin antagonist rescue, xenograft tumor models\",\n      \"journal\": \"Heliyon\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway placement by pharmacological rescue with β-catenin antagonist, multiple assays, single lab\",\n      \"pmids\": [\"38590861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EFNA4 knockdown in gastric cancer cells reduces PYGO2 protein expression and inactivates Wnt/β-catenin signaling; overexpression of PYGO2 reverses the effects of EFNA4 silencing on Wnt/β-catenin signaling, proliferation, apoptosis, migration, invasion, angiogenesis, and stemness, placing EFNA4 upstream of PYGO2/Wnt signaling in gastric cancer.\",\n      \"method\": \"siRNA knockdown, western blot, CCK-8/EDU proliferation, Transwell invasion, tube formation, sphere formation, rescue experiments with PYGO2 overexpression\",\n      \"journal\": \"Histology and histopathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistatic rescue with PYGO2, multiple orthogonal functional assays, single lab\",\n      \"pmids\": [\"38953488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Efna4 knockout mice exhibit increased bone volume/trabecular volume (BV/TV) in the femur, increased bone formation rate, increased osteoblast labeling surfaces, and decreased osteoclast activity, demonstrating that EFNA4 plays a functional role in regulating bone modeling through the Ephrin-Eph receptor signaling axis between osteoblasts and osteoclasts.\",\n      \"method\": \"Efna4 knockout mouse, micro-CT, cryohistomorphometry, dynamic bone labeling, cellular histomorphometric analysis\",\n      \"journal\": \"Bone\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined skeletal phenotype and cellular analysis, single center screen\",\n      \"pmids\": [\"33065355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Calcitriol supplementation in BTBR autism model mice corrects axon guidance abnormalities and hippocampal hypoplasia; these effects are mediated through modulation of the EfnA4-PI3K/AKT signaling pathway in hippocampal neural progenitor cells, placing EFNA4 in the PI3K/AKT pathway controlling axon guidance in neurodevelopment.\",\n      \"method\": \"BTBR mouse model, behavioral assessments, RNA sequencing, immunohistochemistry, biochemical assays, stripe guidance assays\",\n      \"journal\": \"CNS neuroscience & therapeutics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway modulation inferred from in vivo pharmacological treatment without direct EFNA4 manipulation, single lab\",\n      \"pmids\": [\"40395150\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EFNA4 is a GPI-anchored membrane ligand that signals through Eph receptors (notably EPHA2, driving Ser897 phosphorylation and PIK3R2/GSK3β/β-catenin activation) but can also act receptor-independently by binding SLC7A11 and recruiting USP9X deubiquitinase to stabilize SLC7A11 and suppress ferroptosis; in multiple cancer contexts EFNA4 promotes proliferation, migration, and Wnt/β-catenin signaling via PYGO2, while in bone it limits osteoblast activity and promotes osteoclast function.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EFNA4 is a GPI-anchored member of the ephrin-A family of Eph receptor ligands [#0] that functions as a context-dependent driver of cancer cell proliferation, migration, and survival across multiple tumor types [#3]. As a canonical ligand it binds EPHA2 and promotes its phosphorylation at Ser897, recruiting PIK3R2 and activating GSK3\\u03b2/\\u03b2-catenin signaling in a positive feedback loop that drives hepatocellular carcinoma growth [#1]. EFNA4 acts upstream of the Wnt/\\u03b2-catenin co-activator PYGO2, which it physically binds and positively regulates to sustain Wnt signaling and oncogenic phenotypes in hepatocellular and gastric carcinoma [#2, #6]; in gemcitabine-resistant pancreatic cancer this \\u03b2-catenin activation suppresses deoxycytidine kinase and confers drug resistance [#5]. Independently of Eph receptor binding, EFNA4 directly interacts with SLC7A11 through a defined domain (a.a. 161-201) and recruits the deubiquitinase USP9X to stabilize SLC7A11, suppressing ROS accumulation and ferroptosis [#4]. Beyond its oncogenic roles, genetic loss of Efna4 in mice increases bone volume and formation while decreasing osteoclast activity, implicating it in skeletal modeling via the ephrin-Eph axis [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established the molecular nature of EFNA4 as a membrane attachment, defining it as a GPI-anchored Eph receptor ligand rather than a transmembrane ligand.\",\n      \"evidence\": \"cDNA cloning and sequence analysis of LERK-4/EPLG4\",\n      \"pmids\": [\"8660976\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No receptor binding partner identified in this work\", \"No functional or signaling readout established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Answered whether EFNA4 has a physiological in vivo role by showing it regulates bone modeling, balancing osteoblast and osteoclast activity through ephrin-Eph signaling.\",\n      \"evidence\": \"Efna4 knockout mouse with micro-CT, histomorphometry, and dynamic bone labeling\",\n      \"pmids\": [\"33065355\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Specific Eph receptor partner in bone not identified\", \"Cell-autonomous mechanism in osteoblasts vs osteoclasts not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the canonical signaling mechanism by which EFNA4 drives tumor growth, linking EPHA2 Ser897 phosphorylation to a PIK3R2/GSK3\\u03b2/\\u03b2-catenin feedback loop.\",\n      \"evidence\": \"Co-IP, gain/loss-of-function and phosphorylation assays in HCC in vitro and in vivo\",\n      \"pmids\": [\"34484860\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Single lab and tumor type\", \"Direct enzymatic basis of Ser897 phosphorylation not dissected\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected EFNA4 to the Wnt/\\u03b2-catenin pathway through PYGO2 and demonstrated its broad oncogenic phenotype across liver and lung cancers.\",\n      \"evidence\": \"Co-IP, epistatic PYGO2 rescue, Wnt reporter assays in HCC; overexpression/KO with xenografts in lung adenocarcinoma\",\n      \"pmids\": [\"36404439\", \"36077763\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which EFNA4 regulates PYGO2 protein levels unresolved\", \"Whether PYGO2 regulation is receptor-dependent unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a receptor-independent function of EFNA4 in suppressing ferroptosis by stabilizing SLC7A11 via USP9X-mediated deubiquitination.\",\n      \"evidence\": \"Domain-mapping Co-IP, ubiquitination and ROS/GPX4 assays in an Eph receptor knockout background, in vitro and in vivo\",\n      \"pmids\": [\"39656358\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How a GPI-anchored ligand engages intracellular SLC7A11/USP9X is not mechanistically explained\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the EFNA4/\\u03b2-catenin axis to chemoresistance, showing it suppresses deoxycytidine kinase to confer gemcitabine resistance in pancreatic cancer.\",\n      \"evidence\": \"GEM-resistant cell lines, dCK activity assays, \\u03b2-catenin antagonist rescue, xenografts\",\n      \"pmids\": [\"38590861\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct vs indirect regulation of dCK not distinguished\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated EFNA4 in neurodevelopmental axon guidance through PI3K/AKT signaling, though only via indirect pharmacological modulation.\",\n      \"evidence\": \"BTBR autism model mice with calcitriol treatment, RNA-seq, IHC, stripe guidance assays\",\n      \"pmids\": [\"40395150\"],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Pathway link inferred from drug treatment without direct EFNA4 manipulation\", \"No demonstration of EFNA4 acting cell-autonomously in neural progenitors\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EFNA4 switches between canonical Eph receptor-dependent signaling and receptor-independent intracellular partner stabilization, and what governs this choice across tissues, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structural model of EFNA4 signaling states\", \"Determinants of context-specific partner selection (EPHA2 vs SLC7A11 vs PYGO2) unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 6]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EPHA2\", \"PYGO2\", \"SLC7A11\", \"USP9X\", \"PIK3R2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":5,"faith_pct":80.0}}