{"gene":"ALKBH4","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2013,"finding":"ALKBH4 is a dioxygenase that demethylates monomethylated lysine-84 on actin (K84me1). This demethylation is required for non-muscle myosin II to interact with actin; non-muscle myosin II only binds unmethylated actin. ALKBH4 co-localizes with the actomyosin contractile ring and midbody via association with methylated actin, and its catalytic activity is essential for proper cytokinesis and cell migration. Reconstitution with wild-type but not catalytically inactive ALKBH4 rescues multinucleation and cleavage furrow disorganization in ALKBH4-deficient cells.","method":"In vitro demethylation assay, active-site mutagenesis, Co-IP (myosin II with actin), immunofluorescence localization, reconstitution rescue experiments, Alkbh4 knockout mice (early embryonic lethality)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including in vitro enzymatic assay, active-site mutagenesis, reconstitution rescue, Co-IP, and in vivo knockout, all in a single rigorous study","pmids":["23673617"],"is_preprint":false},{"year":2011,"finding":"Recombinant ALKBH4 is an active Fe(II)/2-oxoglutarate-dependent decarboxylase; it mediates decarboxylation of 2-oxoglutarate in the absence of a primary substrate. This activity requires Fe coordination at a histidine-carboxylate site. A unique N-terminal cysteine-rich motif conserved in ALKBH4 orthologues does not appear to coordinate Fe but may have other roles. An Fe(II)-binding mutant loses productive Fe binding, confirming the catalytic iron requirement.","method":"EPR spectroscopy, UV-visible spectroscopy, Fe(II)-binding mutant analysis, in vitro 2-oxoglutarate decarboxylation assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis and spectroscopic characterization, single lab but multiple orthogonal methods","pmids":["21166655"],"is_preprint":false},{"year":2012,"finding":"ALKBH4 interacts with multiple chromatin-associated and transcription-related proteins as identified by yeast two-hybrid screens. The regions of the interaction partners that mediate binding to ALKBH4 correspond to domains previously shown to interact with DNA or chromatin. Some of these partners show nuclear co-localization with ALKBH4.","method":"Yeast two-hybrid screen, nuclear co-localization (immunofluorescence)","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid only, no reciprocal Co-IP or functional validation of interactions in the abstract","pmids":["23145062"],"is_preprint":false},{"year":2014,"finding":"ALKBH4 is essential for spermatocyte development during prophase I of meiosis. Inducible Alkbh4 knockout mice show failure to establish the synaptonemal complex. ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia, and primary spermatocytes.","method":"Inducible Alkbh4 knockout mice, histological analysis of spermatogenesis, immunofluorescence localization","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO with defined meiotic phenotype and direct localization, single lab","pmids":["25153837"],"is_preprint":false},{"year":2017,"finding":"Zebrafish Alkbh4 regulates actomyosin contractile ring formation during embryonic epiboly; maternal depletion of Alkbh4 causes severe epiboly defects. Attractin (Atrn) was identified as a binding partner of Alkbh4 by yeast two-hybrid assay; Atrn preferentially interacts with the active (catalytically competent) form of Alkbh4 and cooperates with it to regulate actin demethylation and actomyosin formation during epiboly.","method":"CRISPR/Cas9 maternal mutants, morpholino knockdown, immunofluorescence, yeast two-hybrid assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — genetic loss-of-function with defined morphogenetic phenotype and yeast two-hybrid for binding partner, single lab","pmids":["28924386"],"is_preprint":false},{"year":2020,"finding":"ALKBH4 competitively binds WDR5, a key component of the histone H3K4 methyltransferase complex, thereby decreasing H3K4me3 modification at target gene promoters including MIR21. This interaction suppresses EMT and colorectal cancer metastasis.","method":"Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), shRNA knockdown, in vitro transwell invasion assay, in vivo metastatic tumor model","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and ChIP with functional KD/OE, single lab, two orthogonal methods","pmids":["32478065"],"is_preprint":false},{"year":2022,"finding":"ALKBH4 acts as a 6mA (N6-methyladenine) DNA demethylase. Arsenic promotes ALKBH4 protein stability through reduced autophagy, leading to increased ALKBH4 levels and decreased genomic 6mA. ALKBH4 deletion impairs arsenic-induced keratinocyte malignant transformation and tumorigenicity both in vitro and in mice.","method":"Western blot, ALKBH4 deletion (genetic ablation), in vitro transformation assay, xenograft mouse model, autophagy inhibition experiments","journal":"Water","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with functional tumor phenotype and mechanistic link to autophagy-regulated protein stability, single lab","pmids":["37207134"],"is_preprint":false},{"year":2022,"finding":"ALKBH4 depletion in HEK293T cells leads to increased DNMT1 protein expression, which in turn elevates cytosine methylation in the promoter regions of GSTP1 and HSPB1, reducing their protein levels. Thus ALKBH4 modulates DNA cytosine methylation indirectly through regulation of DNMT1 protein levels.","method":"Quantitative proteomics (ALKBH4 knockout cells), Western blot, bisulfite sequencing of promoter regions","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative proteomics plus biochemical validation in KO cells, single lab, two orthogonal methods","pmids":["34951099"],"is_preprint":false},{"year":2023,"finding":"ALKBH4 interacts with small RNA (tRNA) and regulates the formation and metabolism of (R)-5-carboxyhydroxymethyl uridine methyl ester (a modified uridine). This reaction with small RNA enhances protein translation efficiency in an in vitro assay system.","method":"RNA binding assay, in vitro translation efficiency assay, identification of tRNA-associated uridine modification","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro assay with RNA modification identification, single lab, novel activity claimed but limited orthogonal validation described in abstract","pmids":["37507018"],"is_preprint":false},{"year":2024,"finding":"ALKBH4 inhibits GSDME (gasdermin E) activation at the transcriptional level by suppressing H3K4me3 histone modification at the GSDME promoter region, thereby reducing 5-FU-induced pyroptosis in gastric cancer cells.","method":"ChIP assay (H3K4me3 at GSDME promoter), ALKBH4 knockdown/overexpression, cell proliferation and pyroptosis assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with functional KD/OE assays, single lab, two methods","pmids":["38902235"],"is_preprint":false},{"year":2026,"finding":"ALKBH4 maintains GPX4 protein levels in a demethylase activity-dependent manner in breast cancer cells, thereby conferring resistance to ferroptosis. GPX4 overexpression reverses ferroptotic cell death and growth defects caused by ALKBH4 knockdown.","method":"ALKBH4 knockdown, GPX4 overexpression rescue, ferroptosis markers (MDA, Fe2+, GSH, ROS), catalytically inactive ALKBH4 mutant, in vivo xenograft","journal":"In vitro cellular & developmental biology. Animal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — active-site mutagenesis combined with rescue experiments and in vivo model, single lab","pmids":["42207440"],"is_preprint":false},{"year":2026,"finding":"ALKBH4 promotes biogenesis of 5'-tsRNAGlu in NSCLC cells. Overexpression of ALKBH4 inhibits cell proliferation, induces cell cycle arrest, and reduces global translational efficiency; knockdown of 5'-tsRNAGlu attenuates these ALKBH4-mediated tumor-suppressive effects, placing 5'-tsRNAGlu downstream of ALKBH4 in this pathway.","method":"CCK-8 and colony formation assays, flow cytometry, polysome profiling, Northern blot, RNA pulldown, RNA immunoprecipitation, dual-luciferase reporter, ALKBH4 overexpression/knockdown","journal":"Frontiers in bioscience (Landmark edition)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (tsRNA KD rescues ALKBH4 OE phenotype) plus multiple orthogonal assays, single lab","pmids":["42052838"],"is_preprint":false}],"current_model":"ALKBH4 is an Fe(II)/2-oxoglutarate-dependent dioxygenase that demethylates monomethylated lysine-84 on actin (K84me1), promoting non-muscle myosin II binding to actin and thereby regulating actomyosin contractility, cytokinesis, cell migration, and embryonic morphogenesis; additionally, ALKBH4 acts as a 6mA DNA demethylase and tRNA uridine modification enzyme, modulates H3K4me3 levels at gene promoters by competing with WDR5, regulates DNMT1-dependent cytosine methylation, and maintains GPX4 protein levels to confer ferroptosis resistance, with its demethylase catalytic activity required for most of these functions."},"narrative":{"mechanistic_narrative":"ALKBH4 is an Fe(II)/2-oxoglutarate-dependent dioxygenase that governs actomyosin contractility through site-specific protein demethylation [PMID:23673617, PMID:21166655]. It is a catalytically active enzyme whose iron requirement depends on coordination at a histidine-carboxylate site [PMID:21166655], and its defining substrate is monomethylated lysine-84 on actin (K84me1): by removing this mark, ALKBH4 enables non-muscle myosin II to engage actin, since myosin II binds only unmethylated actin [PMID:23673617]. Through this activity ALKBH4 localizes to the actomyosin contractile ring and midbody and is required for proper cytokinesis and cell migration, with loss causing multinucleation and cleavage furrow disorganization that is rescued only by catalytically active enzyme [PMID:23673617]. This function extends to development and reproduction: ALKBH4 controls contractile ring formation during zebrafish epiboly in cooperation with its partner Attractin [PMID:28924386] and is essential for synaptonemal complex formation during meiotic prophase I in spermatocytes [PMID:25153837]. Beyond actin, ALKBH4 has been linked to chromatin and RNA regulation, where it competes with WDR5 to lower H3K4me3 at target promoters [PMID:32478065, PMID:38902235], modulates DNMT1-dependent cytosine methylation [PMID:34951099], demethylates 6mA DNA [PMID:37207134], and influences tRNA-derived small RNA biogenesis and translational efficiency [PMID:37507018, PMID:42052838], and it sustains GPX4 levels to confer ferroptosis resistance in a demethylase-dependent manner [PMID:42207440]. The unifying biochemical principle across these activities is its 2-oxoglutarate/Fe(II) dioxygenase catalysis [PMID:23673617, PMID:21166655], though the substrate basis for its nuclear and RNA-associated roles is less defined than its actin demethylase function.","teleology":[{"year":2011,"claim":"Established that ALKBH4 is a genuine catalytically active enzyme before any substrate was known, defining its cofactor requirements.","evidence":"EPR/UV-visible spectroscopy, Fe(II)-binding mutagenesis, and in vitro 2-oxoglutarate decarboxylation assay on recombinant protein","pmids":["21166655"],"confidence":"High","gaps":["No physiological substrate identified at this stage","Function of the conserved N-terminal cysteine-rich motif unresolved"]},{"year":2013,"claim":"Identified actin K84me1 as the physiological substrate and connected ALKBH4 demethylase activity to myosin II engagement and cytokinesis, defining its core cellular function.","evidence":"In vitro demethylation assay, active-site mutagenesis, Co-IP, immunofluorescence, reconstitution rescue, and Alkbh4 knockout mice","pmids":["23673617"],"confidence":"High","gaps":["Structural basis of substrate recognition not resolved","Embryonic lethality of full knockout limits in vivo dissection"]},{"year":2012,"claim":"Raised the possibility of a nuclear/chromatin-associated role by identifying chromatin and transcription-related interactors.","evidence":"Yeast two-hybrid screen with nuclear co-localization by immunofluorescence","pmids":["23145062"],"confidence":"Low","gaps":["Yeast two-hybrid only, no reciprocal Co-IP or functional validation","Interactions not linked to a demethylation activity"]},{"year":2014,"claim":"Extended ALKBH4 function to meiosis, showing it is required for synaptonemal complex establishment in spermatocytes.","evidence":"Inducible Alkbh4 knockout mice, histology of spermatogenesis, immunofluorescence localization to nucleolar structures","pmids":["25153837"],"confidence":"Medium","gaps":["Molecular substrate underlying the meiotic phenotype not identified","Relationship to actin demethylase activity unclear"]},{"year":2017,"claim":"Demonstrated conservation of the actomyosin-regulatory role in vivo and identified Attractin as a functional partner that prefers the active enzyme.","evidence":"Zebrafish CRISPR maternal mutants, morpholino knockdown, immunofluorescence, yeast two-hybrid","pmids":["28924386"],"confidence":"Medium","gaps":["Mechanism by which Atrn modulates demethylation not defined","Direct biochemical interaction not validated reciprocally"]},{"year":2020,"claim":"Introduced a chromatin-regulatory mechanism in which ALKBH4 competes with WDR5 to lower H3K4me3 at promoters, linking it to cancer metastasis.","evidence":"Reciprocal Co-IP, ChIP, shRNA knockdown, invasion assays, and in vivo metastasis model in colorectal cancer","pmids":["32478065"],"confidence":"Medium","gaps":["Whether demethylase catalytic activity is required for WDR5 competition unclear","Single lab, single cancer context"]},{"year":2022,"claim":"Expanded substrate scope to nucleic acids, proposing ALKBH4 as a 6mA DNA demethylase whose stability is regulated by arsenic-modulated autophagy.","evidence":"Western blot, genetic ablation, in vitro transformation, xenograft, and autophagy inhibition","pmids":["37207134"],"confidence":"Medium","gaps":["Direct enzymatic demethylation of 6mA not shown biochemically in the abstract","Relationship to actin substrate undefined"]},{"year":2022,"claim":"Showed an indirect route to DNA cytosine methylation control via regulation of DNMT1 protein levels and downstream promoter methylation.","evidence":"Quantitative proteomics of knockout cells, Western blot, bisulfite sequencing in HEK293T","pmids":["34951099"],"confidence":"Medium","gaps":["Mechanism by which ALKBH4 controls DNMT1 levels not defined","Indirect effect, no direct substrate"]},{"year":2023,"claim":"Linked ALKBH4 to tRNA uridine modification and translational efficiency, broadening its activity to RNA metabolism.","evidence":"RNA binding assay, identification of a modified uridine, in vitro translation efficiency assay","pmids":["37507018"],"confidence":"Medium","gaps":["Limited orthogonal validation of the RNA modification activity","In vivo relevance not established"]},{"year":2024,"claim":"Connected the H3K4me3-lowering activity to suppression of GSDME transcription and pyroptosis resistance in gastric cancer.","evidence":"ChIP at GSDME promoter, knockdown/overexpression, proliferation and pyroptosis assays","pmids":["38902235"],"confidence":"Medium","gaps":["Direct catalytic mechanism on H3K4me3 not shown","Single cancer model"]},{"year":2026,"claim":"Established a demethylase-dependent role in sustaining GPX4 and conferring ferroptosis resistance in breast cancer.","evidence":"Knockdown, GPX4 overexpression rescue, ferroptosis markers, catalytically inactive mutant, xenograft","pmids":["42207440"],"confidence":"Medium","gaps":["Molecular substrate connecting ALKBH4 to GPX4 levels unknown","Single lab"]},{"year":2026,"claim":"Placed 5'-tsRNAGlu biogenesis downstream of ALKBH4 in a tumor-suppressive, translation-modulating axis in NSCLC.","evidence":"Proliferation/colony assays, flow cytometry, polysome profiling, Northern blot, RNA pulldown/IP, luciferase reporter, epistasis with tsRNA knockdown","pmids":["42052838"],"confidence":"Medium","gaps":["Direct enzymatic step in tsRNA biogenesis not defined","Reconciliation with tumor-promoting roles in other cancers unresolved"]},{"year":null,"claim":"It remains unresolved how a single 2-oxoglutarate/Fe(II) dioxygenase mechanism accommodates substrates as diverse as actin lysine, 6mA DNA, and tRNA uridine, and which substrate underlies each cellular and disease phenotype.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural or substrate-specificity model across protein, DNA, and RNA activities","Context-dependent tumor-suppressive versus tumor-promoting roles not reconciled","Catalytic requirement not directly tested for several nuclear/RNA functions"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016491","term_label":"oxidoreductase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[6]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[8,11]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,4]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,5]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[5,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[8,11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[9,10]}],"complexes":[],"partners":["ACTB","MYH9","ATRN","WDR5","DNMT1","GPX4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NXW9","full_name":"Alpha-ketoglutarate-dependent dioxygenase alkB homolog 4","aliases":["Alkylated DNA repair protein alkB homolog 4","DNA N6-methyl adenine demethylase ALKBH4","Lysine-specific demethylase ALKBH4"],"length_aa":302,"mass_kda":33.8,"function":"Dioxygenase that mediates demethylation of actin monomethylated at 'Lys-84' (K84me1), thereby acting as a regulator of actomyosin-processes (PubMed:23673617). Demethylation of actin K84me1 is required for maintaining actomyosin dynamics supporting normal cleavage furrow ingression during cytokinesis and cell migration (PubMed:23673617). In addition to proteins, also demethylates DNA: specifically demethylates DNA methylated on the 6th position of adenine (N(6)-methyladenosine) DNA, thereby regulating Polycomb silencing (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Nucleus, nucleolus; Midbody","url":"https://www.uniprot.org/uniprotkb/Q9NXW9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ALKBH4","classification":"Not Classified","n_dependent_lines":15,"n_total_lines":1208,"dependency_fraction":0.012417218543046357},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ALKBH4","total_profiled":1310},"omim":[{"mim_id":"619626","title":"METHYLTRANSFERASE 4, N6-ADENOSINE; METTL4","url":"https://www.omim.org/entry/619626"},{"mim_id":"613302","title":"AlkB HOMOLOG 4, LYSINE DEMETHYLASE; ALKBH4","url":"https://www.omim.org/entry/613302"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ALKBH4"},"hgnc":{"alias_symbol":["FLJ20013"],"prev_symbol":[]},"alphafold":{"accession":"Q9NXW9","domains":[{"cath_id":"-","chopping":"42-71_191-208_220-236","consensus_level":"medium","plddt":90.434,"start":42,"end":236},{"cath_id":"2.60.120.590","chopping":"73-189_238-302","consensus_level":"medium","plddt":97.0356,"start":73,"end":302}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXW9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXW9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NXW9-F1-predicted_aligned_error_v6.png","plddt_mean":89.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ALKBH4","jax_strain_url":"https://www.jax.org/strain/search?query=ALKBH4"},"sequence":{"accession":"Q9NXW9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NXW9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NXW9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NXW9"}},"corpus_meta":[{"pmid":"23673617","id":"PMC_23673617","title":"ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.","date":"2013","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/23673617","citation_count":70,"is_preprint":false},{"pmid":"23145062","id":"PMC_23145062","title":"Human ALKBH4 interacts with proteins associated with transcription.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23145062","citation_count":27,"is_preprint":false},{"pmid":"21166655","id":"PMC_21166655","title":"Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4.","date":"2011","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/21166655","citation_count":20,"is_preprint":false},{"pmid":"38902235","id":"PMC_38902235","title":"ALKBH4 impedes 5-FU Sensitivity through suppressing GSDME induced pyroptosis in gastric cancer.","date":"2024","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/38902235","citation_count":19,"is_preprint":false},{"pmid":"25153837","id":"PMC_25153837","title":"ALKBH4 depletion in mice leads to spermatogenic defects.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25153837","citation_count":19,"is_preprint":false},{"pmid":"32478065","id":"PMC_32478065","title":"ALKBH4 Functions as a Suppressor of Colorectal Cancer Metastasis via Competitively Binding to WDR5.","date":"2020","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/32478065","citation_count":16,"is_preprint":false},{"pmid":"28924386","id":"PMC_28924386","title":"Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly.","date":"2017","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/28924386","citation_count":12,"is_preprint":false},{"pmid":"34951099","id":"PMC_34951099","title":"Quantitative proteomics revealed new functions of ALKBH4.","date":"2022","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/34951099","citation_count":7,"is_preprint":false},{"pmid":"37207134","id":"PMC_37207134","title":"ALKBH4 Stabilization Is Required for Arsenic-Induced 6mA DNA Methylation Inhibition, Keratinocyte Malignant Transformation, and Tumorigenicity.","date":"2022","source":"Water","url":"https://pubmed.ncbi.nlm.nih.gov/37207134","citation_count":6,"is_preprint":false},{"pmid":"37507018","id":"PMC_37507018","title":"ALKBH4 is a novel enzyme that promotes translation through modified uridine regulation.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37507018","citation_count":5,"is_preprint":false},{"pmid":"35966331","id":"PMC_35966331","title":"Clinical significance of ALKBH4 expression in non-small cell lung cancer.","date":"2022","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35966331","citation_count":4,"is_preprint":false},{"pmid":"39400679","id":"PMC_39400679","title":"ALKBH4 functions as a hypoxia-responsive tumor suppressor and inhibits metastasis and tumorigenesis.","date":"2024","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/39400679","citation_count":4,"is_preprint":false},{"pmid":"42052838","id":"PMC_42052838","title":"Demethylase ALKBH4 Inhibits Cell Proliferation via Induction of 5'-tsRNAGlu in Non-Small Cell Lung Cancer.","date":"2026","source":"Frontiers in bioscience (Landmark edition)","url":"https://pubmed.ncbi.nlm.nih.gov/42052838","citation_count":0,"is_preprint":false},{"pmid":"42207440","id":"PMC_42207440","title":"ALKBH4 confers ferroptosis resistance and drives tumorigenesis via dysregulation of GPX4 in breast cancer cells.","date":"2026","source":"In vitro cellular & developmental biology. Animal","url":"https://pubmed.ncbi.nlm.nih.gov/42207440","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.06.25327057","title":"Identification of (ultra-)rare functional promoter mutations in cancer using sequence-based deep learning models","date":"2025-05-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.06.25327057","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9622,"output_tokens":3187,"usd":0.038336,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10592,"output_tokens":3862,"usd":0.074755,"stage2_stop_reason":"end_turn"},"total_usd":0.113091,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"ALKBH4 is a dioxygenase that demethylates monomethylated lysine-84 on actin (K84me1). This demethylation is required for non-muscle myosin II to interact with actin; non-muscle myosin II only binds unmethylated actin. ALKBH4 co-localizes with the actomyosin contractile ring and midbody via association with methylated actin, and its catalytic activity is essential for proper cytokinesis and cell migration. Reconstitution with wild-type but not catalytically inactive ALKBH4 rescues multinucleation and cleavage furrow disorganization in ALKBH4-deficient cells.\",\n      \"method\": \"In vitro demethylation assay, active-site mutagenesis, Co-IP (myosin II with actin), immunofluorescence localization, reconstitution rescue experiments, Alkbh4 knockout mice (early embryonic lethality)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including in vitro enzymatic assay, active-site mutagenesis, reconstitution rescue, Co-IP, and in vivo knockout, all in a single rigorous study\",\n      \"pmids\": [\"23673617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Recombinant ALKBH4 is an active Fe(II)/2-oxoglutarate-dependent decarboxylase; it mediates decarboxylation of 2-oxoglutarate in the absence of a primary substrate. This activity requires Fe coordination at a histidine-carboxylate site. A unique N-terminal cysteine-rich motif conserved in ALKBH4 orthologues does not appear to coordinate Fe but may have other roles. An Fe(II)-binding mutant loses productive Fe binding, confirming the catalytic iron requirement.\",\n      \"method\": \"EPR spectroscopy, UV-visible spectroscopy, Fe(II)-binding mutant analysis, in vitro 2-oxoglutarate decarboxylation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with mutagenesis and spectroscopic characterization, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21166655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ALKBH4 interacts with multiple chromatin-associated and transcription-related proteins as identified by yeast two-hybrid screens. The regions of the interaction partners that mediate binding to ALKBH4 correspond to domains previously shown to interact with DNA or chromatin. Some of these partners show nuclear co-localization with ALKBH4.\",\n      \"method\": \"Yeast two-hybrid screen, nuclear co-localization (immunofluorescence)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid only, no reciprocal Co-IP or functional validation of interactions in the abstract\",\n      \"pmids\": [\"23145062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ALKBH4 is essential for spermatocyte development during prophase I of meiosis. Inducible Alkbh4 knockout mice show failure to establish the synaptonemal complex. ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia, and primary spermatocytes.\",\n      \"method\": \"Inducible Alkbh4 knockout mice, histological analysis of spermatogenesis, immunofluorescence localization\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO with defined meiotic phenotype and direct localization, single lab\",\n      \"pmids\": [\"25153837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Zebrafish Alkbh4 regulates actomyosin contractile ring formation during embryonic epiboly; maternal depletion of Alkbh4 causes severe epiboly defects. Attractin (Atrn) was identified as a binding partner of Alkbh4 by yeast two-hybrid assay; Atrn preferentially interacts with the active (catalytically competent) form of Alkbh4 and cooperates with it to regulate actin demethylation and actomyosin formation during epiboly.\",\n      \"method\": \"CRISPR/Cas9 maternal mutants, morpholino knockdown, immunofluorescence, yeast two-hybrid assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — genetic loss-of-function with defined morphogenetic phenotype and yeast two-hybrid for binding partner, single lab\",\n      \"pmids\": [\"28924386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ALKBH4 competitively binds WDR5, a key component of the histone H3K4 methyltransferase complex, thereby decreasing H3K4me3 modification at target gene promoters including MIR21. This interaction suppresses EMT and colorectal cancer metastasis.\",\n      \"method\": \"Co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), shRNA knockdown, in vitro transwell invasion assay, in vivo metastatic tumor model\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and ChIP with functional KD/OE, single lab, two orthogonal methods\",\n      \"pmids\": [\"32478065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH4 acts as a 6mA (N6-methyladenine) DNA demethylase. Arsenic promotes ALKBH4 protein stability through reduced autophagy, leading to increased ALKBH4 levels and decreased genomic 6mA. ALKBH4 deletion impairs arsenic-induced keratinocyte malignant transformation and tumorigenicity both in vitro and in mice.\",\n      \"method\": \"Western blot, ALKBH4 deletion (genetic ablation), in vitro transformation assay, xenograft mouse model, autophagy inhibition experiments\",\n      \"journal\": \"Water\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with functional tumor phenotype and mechanistic link to autophagy-regulated protein stability, single lab\",\n      \"pmids\": [\"37207134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH4 depletion in HEK293T cells leads to increased DNMT1 protein expression, which in turn elevates cytosine methylation in the promoter regions of GSTP1 and HSPB1, reducing their protein levels. Thus ALKBH4 modulates DNA cytosine methylation indirectly through regulation of DNMT1 protein levels.\",\n      \"method\": \"Quantitative proteomics (ALKBH4 knockout cells), Western blot, bisulfite sequencing of promoter regions\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative proteomics plus biochemical validation in KO cells, single lab, two orthogonal methods\",\n      \"pmids\": [\"34951099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ALKBH4 interacts with small RNA (tRNA) and regulates the formation and metabolism of (R)-5-carboxyhydroxymethyl uridine methyl ester (a modified uridine). This reaction with small RNA enhances protein translation efficiency in an in vitro assay system.\",\n      \"method\": \"RNA binding assay, in vitro translation efficiency assay, identification of tRNA-associated uridine modification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro assay with RNA modification identification, single lab, novel activity claimed but limited orthogonal validation described in abstract\",\n      \"pmids\": [\"37507018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALKBH4 inhibits GSDME (gasdermin E) activation at the transcriptional level by suppressing H3K4me3 histone modification at the GSDME promoter region, thereby reducing 5-FU-induced pyroptosis in gastric cancer cells.\",\n      \"method\": \"ChIP assay (H3K4me3 at GSDME promoter), ALKBH4 knockdown/overexpression, cell proliferation and pyroptosis assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with functional KD/OE assays, single lab, two methods\",\n      \"pmids\": [\"38902235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ALKBH4 maintains GPX4 protein levels in a demethylase activity-dependent manner in breast cancer cells, thereby conferring resistance to ferroptosis. GPX4 overexpression reverses ferroptotic cell death and growth defects caused by ALKBH4 knockdown.\",\n      \"method\": \"ALKBH4 knockdown, GPX4 overexpression rescue, ferroptosis markers (MDA, Fe2+, GSH, ROS), catalytically inactive ALKBH4 mutant, in vivo xenograft\",\n      \"journal\": \"In vitro cellular & developmental biology. Animal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — active-site mutagenesis combined with rescue experiments and in vivo model, single lab\",\n      \"pmids\": [\"42207440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ALKBH4 promotes biogenesis of 5'-tsRNAGlu in NSCLC cells. Overexpression of ALKBH4 inhibits cell proliferation, induces cell cycle arrest, and reduces global translational efficiency; knockdown of 5'-tsRNAGlu attenuates these ALKBH4-mediated tumor-suppressive effects, placing 5'-tsRNAGlu downstream of ALKBH4 in this pathway.\",\n      \"method\": \"CCK-8 and colony formation assays, flow cytometry, polysome profiling, Northern blot, RNA pulldown, RNA immunoprecipitation, dual-luciferase reporter, ALKBH4 overexpression/knockdown\",\n      \"journal\": \"Frontiers in bioscience (Landmark edition)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (tsRNA KD rescues ALKBH4 OE phenotype) plus multiple orthogonal assays, single lab\",\n      \"pmids\": [\"42052838\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ALKBH4 is an Fe(II)/2-oxoglutarate-dependent dioxygenase that demethylates monomethylated lysine-84 on actin (K84me1), promoting non-muscle myosin II binding to actin and thereby regulating actomyosin contractility, cytokinesis, cell migration, and embryonic morphogenesis; additionally, ALKBH4 acts as a 6mA DNA demethylase and tRNA uridine modification enzyme, modulates H3K4me3 levels at gene promoters by competing with WDR5, regulates DNMT1-dependent cytosine methylation, and maintains GPX4 protein levels to confer ferroptosis resistance, with its demethylase catalytic activity required for most of these functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ALKBH4 is an Fe(II)/2-oxoglutarate-dependent dioxygenase that governs actomyosin contractility through site-specific protein demethylation [#0, #1]. It is a catalytically active enzyme whose iron requirement depends on coordination at a histidine-carboxylate site [#1], and its defining substrate is monomethylated lysine-84 on actin (K84me1): by removing this mark, ALKBH4 enables non-muscle myosin II to engage actin, since myosin II binds only unmethylated actin [#0]. Through this activity ALKBH4 localizes to the actomyosin contractile ring and midbody and is required for proper cytokinesis and cell migration, with loss causing multinucleation and cleavage furrow disorganization that is rescued only by catalytically active enzyme [#0]. This function extends to development and reproduction: ALKBH4 controls contractile ring formation during zebrafish epiboly in cooperation with its partner Attractin [#4] and is essential for synaptonemal complex formation during meiotic prophase I in spermatocytes [#3]. Beyond actin, ALKBH4 has been linked to chromatin and RNA regulation, where it competes with WDR5 to lower H3K4me3 at target promoters [#5, #9], modulates DNMT1-dependent cytosine methylation [#7], demethylates 6mA DNA [#6], and influences tRNA-derived small RNA biogenesis and translational efficiency [#8, #11], and it sustains GPX4 levels to confer ferroptosis resistance in a demethylase-dependent manner [#10]. The unifying biochemical principle across these activities is its 2-oxoglutarate/Fe(II) dioxygenase catalysis [#0, #1], though the substrate basis for its nuclear and RNA-associated roles is less defined than its actin demethylase function.\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that ALKBH4 is a genuine catalytically active enzyme before any substrate was known, defining its cofactor requirements.\",\n      \"evidence\": \"EPR/UV-visible spectroscopy, Fe(II)-binding mutagenesis, and in vitro 2-oxoglutarate decarboxylation assay on recombinant protein\",\n      \"pmids\": [\"21166655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified at this stage\", \"Function of the conserved N-terminal cysteine-rich motif unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified actin K84me1 as the physiological substrate and connected ALKBH4 demethylase activity to myosin II engagement and cytokinesis, defining its core cellular function.\",\n      \"evidence\": \"In vitro demethylation assay, active-site mutagenesis, Co-IP, immunofluorescence, reconstitution rescue, and Alkbh4 knockout mice\",\n      \"pmids\": [\"23673617\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of substrate recognition not resolved\", \"Embryonic lethality of full knockout limits in vivo dissection\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Raised the possibility of a nuclear/chromatin-associated role by identifying chromatin and transcription-related interactors.\",\n      \"evidence\": \"Yeast two-hybrid screen with nuclear co-localization by immunofluorescence\",\n      \"pmids\": [\"23145062\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Yeast two-hybrid only, no reciprocal Co-IP or functional validation\", \"Interactions not linked to a demethylation activity\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended ALKBH4 function to meiosis, showing it is required for synaptonemal complex establishment in spermatocytes.\",\n      \"evidence\": \"Inducible Alkbh4 knockout mice, histology of spermatogenesis, immunofluorescence localization to nucleolar structures\",\n      \"pmids\": [\"25153837\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate underlying the meiotic phenotype not identified\", \"Relationship to actin demethylase activity unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated conservation of the actomyosin-regulatory role in vivo and identified Attractin as a functional partner that prefers the active enzyme.\",\n      \"evidence\": \"Zebrafish CRISPR maternal mutants, morpholino knockdown, immunofluorescence, yeast two-hybrid\",\n      \"pmids\": [\"28924386\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Atrn modulates demethylation not defined\", \"Direct biochemical interaction not validated reciprocally\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Introduced a chromatin-regulatory mechanism in which ALKBH4 competes with WDR5 to lower H3K4me3 at promoters, linking it to cancer metastasis.\",\n      \"evidence\": \"Reciprocal Co-IP, ChIP, shRNA knockdown, invasion assays, and in vivo metastasis model in colorectal cancer\",\n      \"pmids\": [\"32478065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether demethylase catalytic activity is required for WDR5 competition unclear\", \"Single lab, single cancer context\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded substrate scope to nucleic acids, proposing ALKBH4 as a 6mA DNA demethylase whose stability is regulated by arsenic-modulated autophagy.\",\n      \"evidence\": \"Western blot, genetic ablation, in vitro transformation, xenograft, and autophagy inhibition\",\n      \"pmids\": [\"37207134\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic demethylation of 6mA not shown biochemically in the abstract\", \"Relationship to actin substrate undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed an indirect route to DNA cytosine methylation control via regulation of DNMT1 protein levels and downstream promoter methylation.\",\n      \"evidence\": \"Quantitative proteomics of knockout cells, Western blot, bisulfite sequencing in HEK293T\",\n      \"pmids\": [\"34951099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which ALKBH4 controls DNMT1 levels not defined\", \"Indirect effect, no direct substrate\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked ALKBH4 to tRNA uridine modification and translational efficiency, broadening its activity to RNA metabolism.\",\n      \"evidence\": \"RNA binding assay, identification of a modified uridine, in vitro translation efficiency assay\",\n      \"pmids\": [\"37507018\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Limited orthogonal validation of the RNA modification activity\", \"In vivo relevance not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected the H3K4me3-lowering activity to suppression of GSDME transcription and pyroptosis resistance in gastric cancer.\",\n      \"evidence\": \"ChIP at GSDME promoter, knockdown/overexpression, proliferation and pyroptosis assays\",\n      \"pmids\": [\"38902235\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct catalytic mechanism on H3K4me3 not shown\", \"Single cancer model\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a demethylase-dependent role in sustaining GPX4 and conferring ferroptosis resistance in breast cancer.\",\n      \"evidence\": \"Knockdown, GPX4 overexpression rescue, ferroptosis markers, catalytically inactive mutant, xenograft\",\n      \"pmids\": [\"42207440\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular substrate connecting ALKBH4 to GPX4 levels unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placed 5'-tsRNAGlu biogenesis downstream of ALKBH4 in a tumor-suppressive, translation-modulating axis in NSCLC.\",\n      \"evidence\": \"Proliferation/colony assays, flow cytometry, polysome profiling, Northern blot, RNA pulldown/IP, luciferase reporter, epistasis with tsRNA knockdown\",\n      \"pmids\": [\"42052838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic step in tsRNA biogenesis not defined\", \"Reconciliation with tumor-promoting roles in other cancers unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how a single 2-oxoglutarate/Fe(II) dioxygenase mechanism accommodates substrates as diverse as actin lysine, 6mA DNA, and tRNA uridine, and which substrate underlies each cellular and disease phenotype.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural or substrate-specificity model across protein, DNA, and RNA activities\", \"Context-dependent tumor-suppressive versus tumor-promoting roles not reconciled\", \"Catalytic requirement not directly tested for several nuclear/RNA functions\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016491\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [8, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [5, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [9, 10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ACTB\", \"MYH9\", \"ATRN\", \"WDR5\", \"DNMT1\", \"GPX4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}