{"gene":"TNKS2","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2001,"finding":"TNKS2 (TANK2) was identified as a second human tankyrase with poly(ADP-ribose) polymerase (PARP) activity that directly interacts with the telomere-binding protein TRF1 in yeast two-hybrid and in vitro binding assays. TNKS2 shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif (SAM), and PARP catalytic domains but possesses a unique N-terminal domain.","method":"Yeast two-hybrid, in vitro binding assay, domain analysis, overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal binding assay (yeast + in vitro), foundational paper with 191 citations, replicated domain architecture","pmids":["11454873"],"is_preprint":false},{"year":2001,"finding":"TNKS2 localizes predominantly to a perinuclear region. When highly overexpressed, TNKS2 causes rapid cell death by necrosis (loss of mitochondrial membrane potential without PARP1 cleavage), and this cell death is prevented by the PARP inhibitor 3-aminobenzamide, establishing that TNKS2 PARP activity mediates the cytotoxic effect.","method":"Subcellular localization by imaging, overexpression, mitochondrial membrane potential assay, PARP inhibitor rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (imaging, functional rescue with inhibitor), high-citation foundational study","pmids":["11454873"],"is_preprint":false},{"year":2001,"finding":"TNKS2 (referred to as TNKL) was cloned and found to encode a 1166-aa protein with ankyrin repeat and PARP catalytic domains essentially identical in organization to tankyrase (TNKS1), establishing TNKS1 and TNKS2 as a distinct gene family; TNKS2 maps to chromosome 10.","method":"Serological cDNA library screening, sequence and domain analysis","journal":"Genes and immunity","confidence":"Medium","confidence_rationale":"Tier 3 — cloning and bioinformatic domain analysis, no direct functional assay beyond sequence","pmids":["11294570"],"is_preprint":false},{"year":2020,"finding":"TNKS2 (Tankyrase-2) poly-ADP-ribosylates VEGF in the Golgi compartment. PARP-16 (ER-localized) first catalyzes mono-ADP-ribosylation of VEGF, which is required as a primer for subsequent poly-ADP-ribosylation by Golgi-associated TNKS2, thereby reducing VEGF biological activity.","method":"In vitro PARP assay, subcellular fractionation, co-immunoprecipitation, sequential modification assay","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro enzymatic assay with defined substrate and ordered mechanism, single lab","pmids":["32472322"],"is_preprint":false},{"year":2024,"finding":"A genome-wide CRISPRi screen revealed that TNKS2 (and TNKS1) bind the peroxisomal membrane protein PEX14 and regulate peroxisome protein import efficiency through PARsylation of peroxisomal membrane proteins. Loss of RNF146, an E3 ligase activated by poly(ADP-ribose), stabilizes TNKS/TNKS2 activity, leading to increased AXIN1 degradation and β-catenin transcriptional activation.","method":"Genome-wide CRISPRi screen, Co-IP/pulldown for PEX14 interaction, peroxisome import assay, Wnt reporter assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — genome-wide screen combined with direct interaction assay and functional epistasis, multiple orthogonal methods","pmids":["38967608"],"is_preprint":false},{"year":2023,"finding":"SASH1 physically binds TNKS2 through a bona fide tankyrase-binding motif (TBM) containing S519. The SASH1 S519N disease variant alters TNKS2 binding kinetics and affinity, and TNKS2 binding is required for SASH1-mediated promotion of stem-like characteristics in human melanocytes.","method":"Multiple binding assays (co-IP, pulldown, kinetics), TBM motif identification, functional melanocyte stem cell assays with SASH1 S519N variant","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal binding assays with mutagenesis and functional readout, but preprint single lab","pmids":["37808724"],"is_preprint":true},{"year":2021,"finding":"Loss of TNKS2 (PARP5B) expression in a carcinogen-induced head and neck squamous cell carcinoma model caused loss of 53BP1+ double-strand break foci and ATM activation, while inducing ATR activation and a shift to homologous recombination (HR)-based repair, indicating TNKS2 is required for nonhomologous end joining (NHEJ) at double-strand breaks in vivo.","method":"PARP5B null mouse model, immunofluorescence for 53BP1/ATM/ATR, multiprotein complex identification by co-immunoprecipitation, PARP inhibitor (XAV939) combination treatment","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KO with mechanistic readouts (foci, pathway markers), single lab","pmids":["34710250"],"is_preprint":false},{"year":2016,"finding":"TNKS2 is a direct target of miR-490-3p, which binds the TNKS2 3'-UTR and negatively regulates TNKS2 protein expression, thereby blocking β-catenin signaling activation and suppressing TNBC cell proliferation and invasion.","method":"Dual-luciferase 3'-UTR reporter assay, Western blot, siRNA knockdown, rescue with miR-490-3p-resistant TNKS2","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'-UTR binding validated by luciferase assay with rescue experiment, single lab","pmids":["27506313"],"is_preprint":false},{"year":2025,"finding":"TNKS2 is uniquely required for WNT/β-catenin signaling in tumor cells that have lost chromosome 8p (which depletes TNKS1 expression), creating a collateral vulnerability. A structure-guided, first-in-class TNKS2-selective inhibitor was developed that selectively suppresses WNT signaling only in TNKS1-deficient cancer cells and organoids.","method":"Structure-guided drug design, cancer cell line and organoid models, genetic depletion of TNKS1, WNT reporter assays","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1–2 — structure-based inhibitor + genetic epistasis + functional WNT assay, preprint single lab","pmids":["40093088"],"is_preprint":true},{"year":2024,"finding":"X-ray crystallography of TNKS2 catalytic domain with quinazolin-4-one inhibitors revealed a novel binding subsite between a mobile active site loop and the canonical nicotinamide-binding site. Nitro- and diol-substituents at C-8 engage new interactions with TNKS2, improving affinity (best IC50 = 14 nM) and selectivity, and attenuate Wnt/β-catenin signaling in cells.","method":"X-ray crystallography, enzymatic IC50 assay, cell-based Wnt reporter assay","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure with functional validation in cells, but preprint","pmids":["bio_10.1101_2024.06.23.600314"],"is_preprint":true},{"year":2025,"finding":"The TNKS2 ARC4 domain mediates protein-substrate binding and can be specifically targeted by a pyrrolone-based compound (ARCher-142/S8, 8 µM potency). NMR and X-ray crystallography identified the ARC4 binding site and a unique hydrophobic sub-pocket. Targeting ARC4 is sufficient to attenuate WNT/β-catenin signaling in cells, demonstrating that the scaffolding (ARC-domain) function of TNKS2 is required for Wnt pathway regulation.","method":"FRET-based high-throughput screening, NMR, X-ray crystallography, cell-based WNT reporter assay","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 — structural validation (NMR + crystal structure) plus cell-based functional assay, preprint single lab","pmids":["bio_10.1101_2025.03.31.646301"],"is_preprint":true}],"current_model":"TNKS2 is a telomere- and peroxisome-associated PARP-family enzyme that uses its ankyrin repeat (ARC) domains to scaffold substrates including TRF1, PEX14, AXIN1, and SASH1, and its catalytic domain to poly-ADP-ribosylate these and other substrates (including VEGF in the Golgi), thereby regulating telomere homeostasis, peroxisome import, Wnt/β-catenin signaling via AXIN1 destabilization, NHEJ-based DNA repair, and melanocyte stem cell maintenance; its activity is functionally redundant with TNKS1, and selective inhibition of TNKS2 alone is sufficient to suppress WNT signaling in TNKS1-deficient tumor cells."},"narrative":{"teleology":[{"year":2001,"claim":"Identification of TNKS2 as a second tankyrase with PARP activity and TRF1-binding capacity established that tankyrase function is encoded by a two-gene family with overlapping domain architecture but distinct N-termini.","evidence":"Yeast two-hybrid, in vitro binding, domain analysis, and sequence cloning from two independent groups","pmids":["11454873","11294570"],"confidence":"High","gaps":["Endogenous TNKS2–TRF1 interaction at telomeres not demonstrated","Functional redundancy versus specialization with TNKS1 not addressed","No in vivo loss-of-function data"]},{"year":2001,"claim":"Demonstrating that TNKS2 overexpression causes PARP-dependent necrosis established that its catalytic activity has potent cellular consequences and that TNKS2 localizes to a perinuclear compartment.","evidence":"Overexpression imaging, mitochondrial membrane potential assay, rescue by PARP inhibitor 3-aminobenzamide","pmids":["11454873"],"confidence":"High","gaps":["Perinuclear compartment not identified as a specific organelle","Relevance of overexpression-induced necrosis to endogenous TNKS2 function unclear"]},{"year":2016,"claim":"Showing that miR-490-3p directly targets TNKS2 3′-UTR to suppress β-catenin signaling provided the first evidence linking endogenous TNKS2 expression levels to Wnt pathway output in cancer cells.","evidence":"Dual-luciferase 3′-UTR reporter, Western blot, knockdown and rescue in TNBC cells","pmids":["27506313"],"confidence":"Medium","gaps":["TNKS2 PARsylation of AXIN1 not directly measured in this study","Contribution of TNKS1 not controlled for"]},{"year":2020,"claim":"Revealing that TNKS2 poly-ADP-ribosylates VEGF in the Golgi, extending mono-ADP-ribose primers placed by ER-localized PARP-16, defined a sequential inter-PARP modification cascade on a secreted growth factor.","evidence":"In vitro PARP assay, subcellular fractionation, co-immunoprecipitation, sequential modification assay","pmids":["32472322"],"confidence":"Medium","gaps":["In vivo relevance of VEGF PARsylation to angiogenesis not tested","No structural data on TNKS2–VEGF interface","Single-lab observation"]},{"year":2021,"claim":"Loss of TNKS2 in vivo eliminated 53BP1 foci and ATM signaling while activating ATR, establishing TNKS2 as a determinant of NHEJ versus HR pathway choice at DNA double-strand breaks.","evidence":"PARP5B-null mouse HNSCC model, immunofluorescence for repair factors, PARP inhibitor combination","pmids":["34710250"],"confidence":"Medium","gaps":["Direct PARsylation substrate at DSBs not identified","Whether TNKS1 compensates in wild-type context not resolved","Single carcinogen-induced tumor model"]},{"year":2024,"claim":"A genome-wide CRISPRi screen uncovered TNKS2 binding to the peroxisomal membrane protein PEX14, showing that tankyrase PARsylation of peroxisomal substrates regulates protein import and linking the RNF146–tankyrase axis to both peroxisome biology and AXIN1 degradation–driven Wnt activation.","evidence":"CRISPRi screen, Co-IP/pulldown for PEX14, peroxisome import assay, Wnt reporter assay","pmids":["38967608"],"confidence":"High","gaps":["Identity of PARsylated peroxisomal membrane proteins beyond PEX14 not determined","Relative contributions of TNKS1 vs TNKS2 to peroxisome import not dissected"]},{"year":2025,"claim":"Genetic and pharmacological studies demonstrated that TNKS2 becomes uniquely essential for Wnt signaling when TNKS1 is lost through chromosome 8p deletion, defining a paralog-based collateral vulnerability in cancer; structural targeting of both the PARP catalytic site and the ARC4 substrate-binding domain independently attenuates Wnt output.","evidence":"Structure-guided TNKS2-selective inhibitor in organoids and cell lines (preprint); NMR/X-ray of ARC4 with pyrrolone compound plus Wnt reporter (preprint); TNKS1 genetic depletion","pmids":["40093088","bio_10.1101_2025.03.31.646301"],"confidence":"Medium","gaps":["Preprint data; not yet peer-reviewed","In vivo therapeutic efficacy of TNKS2-selective inhibitors not demonstrated","Selectivity over other ARC-domain-containing proteins not fully profiled"]},{"year":null,"claim":"The full substrate repertoire of TNKS2 PARsylation, the structural basis of TNKS2-specific versus TNKS1-redundant functions, and the endogenous contexts in which TNKS2 scaffolding (ARC domains) versus catalytic activity are rate-limiting remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No proteome-wide PARsylation substrate catalog specific to TNKS2","No full-length TNKS2 structure or cryo-EM model","Relative contribution of TNKS2 versus TNKS1 in normal physiology unclear beyond double-KO studies"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,4]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[3]},{"term_id":"GO:0005777","term_label":"peroxisome","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,7,8]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[6]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[4]}],"complexes":[],"partners":["TRF1","PEX14","AXIN1","SASH1","RNF146","VEGF","TNKS1"],"other_free_text":[]},"mechanistic_narrative":"TNKS2 is a poly(ADP-ribose) polymerase (PARP) family member that uses ankyrin repeat cluster (ARC) domains to scaffold substrates and a catalytic PARP domain to poly-ADP-ribosylate them, thereby regulating telomere homeostasis, Wnt/β-catenin signaling, peroxisome protein import, and DNA double-strand break repair pathway choice. TNKS2 directly binds TRF1 at telomeres and PEX14 at peroxisomes, and PARsylates substrates such as AXIN1 to promote their RNF146-dependent degradation, activating β-catenin transcription [PMID:11454873, PMID:38967608]. In the Golgi, TNKS2 extends mono-ADP-ribose primers deposited by PARP-16 on VEGF, reducing VEGF biological activity [PMID:32472322]. Loss of TNKS2 abolishes 53BP1 foci and ATM activation at double-strand breaks, shifting repair from NHEJ toward homologous recombination, and TNKS2 becomes uniquely essential for Wnt signaling in tumors that have lost its paralog TNKS1 through chromosome 8p deletion [PMID:34710250, PMID:40093088]."},"prefetch_data":{"uniprot":{"accession":"Q9H2K2","full_name":"Poly [ADP-ribose] polymerase tankyrase-2","aliases":["ADP-ribosyltransferase diphtheria toxin-like 6","ARTD6","Poly [ADP-ribose] polymerase 5B","Protein poly-ADP-ribosyltransferase tankyrase-2","TNKS-2","TRF1-interacting ankyrin-related ADP-ribose polymerase 2","Tankyrase II","Tankyrase-2","TANK2","Tankyrase-like protein","Tankyrase-related protein"],"length_aa":1166,"mass_kda":126.9,"function":"Poly-ADP-ribosyltransferase involved in various processes such as Wnt signaling pathway, telomere length and vesicle trafficking (PubMed:11739745, PubMed:11802774, PubMed:19759537, PubMed:21478859, PubMed:23622245, PubMed:25043379). Acts as an activator of the Wnt signaling pathway by mediating poly-ADP-ribosylation of AXIN1 and AXIN2, 2 key components of the beta-catenin destruction complex: poly-ADP-ribosylated target proteins are recognized by RNF146, which mediates their ubiquitination and subsequent degradation (PubMed:19759537, PubMed:21478859). Also mediates poly-ADP-ribosylation of BLZF1 and CASC3, followed by recruitment of RNF146 and subsequent ubiquitination (PubMed:21478859). Mediates poly-ADP-ribosylation of TERF1, thereby contributing to the regulation of telomere length (PubMed:11739745). Stimulates 26S proteasome activity (PubMed:23622245)","subcellular_location":"Cytoplasm; Golgi apparatus membrane; Nucleus; Chromosome, telomere","url":"https://www.uniprot.org/uniprotkb/Q9H2K2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TNKS2","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TNKS2","total_profiled":1310},"omim":[{"mim_id":"620871","title":"DNA DAMAGE-INDUCIBLE 1 HOMOLOG 2; DDI2","url":"https://www.omim.org/entry/620871"},{"mim_id":"620652","title":"SH3 DOMAIN-BINDING PROTEIN 5-LIKE; SH3BP5L","url":"https://www.omim.org/entry/620652"},{"mim_id":"607128","title":"TANKYRASE 2; TNKS2","url":"https://www.omim.org/entry/607128"},{"mim_id":"605612","title":"SH3 DOMAIN-BINDING PROTEIN 5; SH3BP5","url":"https://www.omim.org/entry/605612"},{"mim_id":"300410","title":"ANGIOMOTIN; AMOT","url":"https://www.omim.org/entry/300410"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TNKS2"},"hgnc":{"alias_symbol":["TNKL","TANK2","PARP-5b","PARP-5c","PARP5B","PARP5C","pART6","ARTD6"],"prev_symbol":[]},"alphafold":{"accession":"Q9H2K2","domains":[{"cath_id":"1.25.40.20","chopping":"22-172","consensus_level":"high","plddt":93.2993,"start":22,"end":172},{"cath_id":"1.25.40.20","chopping":"177-318","consensus_level":"high","plddt":96.8878,"start":177,"end":318},{"cath_id":"1.25.40.20","chopping":"336-482","consensus_level":"medium","plddt":92.8896,"start":336,"end":482},{"cath_id":"1.10.150.50","chopping":"877-934","consensus_level":"high","plddt":80.1997,"start":877,"end":934},{"cath_id":"3.90.228.10","chopping":"955-1156","consensus_level":"medium","plddt":68.8884,"start":955,"end":1156}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H2K2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H2K2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H2K2-F1-predicted_aligned_error_v6.png","plddt_mean":83.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TNKS2","jax_strain_url":"https://www.jax.org/strain/search?query=TNKS2"},"sequence":{"accession":"Q9H2K2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H2K2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H2K2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H2K2"}},"corpus_meta":[{"pmid":"11454873","id":"PMC_11454873","title":"TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11454873","citation_count":191,"is_preprint":false},{"pmid":"22449978","id":"PMC_22449978","title":"miR-20a promotes migration and invasion by regulating TNKS2 in human cervical cancer cells.","date":"2012","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/22449978","citation_count":99,"is_preprint":false},{"pmid":"27506313","id":"PMC_27506313","title":"miR-490-3p inhibits the growth and invasiveness in triple-negative breast cancer by repressing the expression of TNKS2.","date":"2016","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/27506313","citation_count":43,"is_preprint":false},{"pmid":"11294570","id":"PMC_11294570","title":"Cloning and characterization of TNKL, a member of tankyrase gene family.","date":"2001","source":"Genes and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/11294570","citation_count":34,"is_preprint":false},{"pmid":"34861798","id":"PMC_34861798","title":"Down-regulation of hsa_circ_0045474 induces macrophage autophagy in tuberculosis via miR-582-5p/TNKS2 axis.","date":"2021","source":"Innate immunity","url":"https://pubmed.ncbi.nlm.nih.gov/34861798","citation_count":21,"is_preprint":false},{"pmid":"24291818","id":"PMC_24291818","title":"Molecular insights on TNKS1/TNKS2 and inhibitor-IWR1 interactions.","date":"2014","source":"Molecular bioSystems","url":"https://pubmed.ncbi.nlm.nih.gov/24291818","citation_count":16,"is_preprint":false},{"pmid":"36185280","id":"PMC_36185280","title":"Icariin attenuates the tumor growth by targeting miR-1-3p/TNKS2/Wnt/β-catenin signaling axis in ovarian cancer.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36185280","citation_count":14,"is_preprint":false},{"pmid":"32435397","id":"PMC_32435397","title":"From PARP1 to TNKS2 Inhibition: A Structure-Based Approach.","date":"2020","source":"ACS medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/32435397","citation_count":13,"is_preprint":false},{"pmid":"33565341","id":"PMC_33565341","title":"Association of relative leukocyte telomere length and genetic variants in telomere-related genes (TERT, TERT-CLPTM1, TRF1, TNKS2, TRF2) with atrophic age-related macular degeneration.","date":"2021","source":"Ophthalmic genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33565341","citation_count":10,"is_preprint":false},{"pmid":"33849554","id":"PMC_33849554","title":"MicroRNA-490-3p inhibits migration and chemoresistance of colorectal cancer cells via targeting TNKS2.","date":"2021","source":"World journal of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33849554","citation_count":9,"is_preprint":false},{"pmid":"32576588","id":"PMC_32576588","title":"Association of Relative Leucocyte Telomere Length and Gene Single Nucleotide Polymorphisms (TERT, TRF1, TNKS2) in Laryngeal Squamous Cell Carcinoma.","date":"2020","source":"Cancer genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/32576588","citation_count":9,"is_preprint":false},{"pmid":"34357507","id":"PMC_34357507","title":"miR-582-5p inhibits migration and chemo-resistant capabilities of colorectal cancer cells by targeting TNKS2.","date":"2021","source":"Genes & genomics","url":"https://pubmed.ncbi.nlm.nih.gov/34357507","citation_count":8,"is_preprint":false},{"pmid":"26293798","id":"PMC_26293798","title":"Tankyrase 2 (TNKS2) polymorphism associated with risk in developing non-small cell lung cancer in a Chinese population.","date":"2015","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/26293798","citation_count":6,"is_preprint":false},{"pmid":"38967608","id":"PMC_38967608","title":"A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.","date":"2024","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/38967608","citation_count":5,"is_preprint":false},{"pmid":"37581526","id":"PMC_37581526","title":"Therapeutic Path to Triple Knockout: Investigating the Pan-inhibitory Mechanisms of AKT, CDK9, and TNKS2 by a Novel 2-phenylquinazolinone Derivative in Cancer Therapy- An In-silico Investigation Therapy.","date":"2024","source":"Current pharmaceutical biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/37581526","citation_count":4,"is_preprint":false},{"pmid":"34710250","id":"PMC_34710250","title":"PARP5B is required for nonhomologous end joining during tumorigenesis in vivo.","date":"2021","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/34710250","citation_count":3,"is_preprint":false},{"pmid":"32472322","id":"PMC_32472322","title":"Regulation of poly ADP-ribosylation of VEGF by an interplay between PARP-16 and TNKS-2.","date":"2020","source":"Molecular and cellular 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domain","date":"2025-03-31","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.31.646301","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12719,"output_tokens":2880,"usd":0.040678},"stage2":{"model":"claude-opus-4-6","input_tokens":6236,"output_tokens":2345,"usd":0.134708},"total_usd":0.175386,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"TNKS2 (TANK2) was identified as a second human tankyrase with poly(ADP-ribose) polymerase (PARP) activity that directly interacts with the telomere-binding protein TRF1 in yeast two-hybrid and in vitro binding assays. TNKS2 shares 85% amino acid identity with TANK1 in the ankyrin repeat, sterile alpha-motif (SAM), and PARP catalytic domains but possesses a unique N-terminal domain.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, domain analysis, overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding assay (yeast + in vitro), foundational paper with 191 citations, replicated domain architecture\",\n      \"pmids\": [\"11454873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TNKS2 localizes predominantly to a perinuclear region. When highly overexpressed, TNKS2 causes rapid cell death by necrosis (loss of mitochondrial membrane potential without PARP1 cleavage), and this cell death is prevented by the PARP inhibitor 3-aminobenzamide, establishing that TNKS2 PARP activity mediates the cytotoxic effect.\",\n      \"method\": \"Subcellular localization by imaging, overexpression, mitochondrial membrane potential assay, PARP inhibitor rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (imaging, functional rescue with inhibitor), high-citation foundational study\",\n      \"pmids\": [\"11454873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TNKS2 (referred to as TNKL) was cloned and found to encode a 1166-aa protein with ankyrin repeat and PARP catalytic domains essentially identical in organization to tankyrase (TNKS1), establishing TNKS1 and TNKS2 as a distinct gene family; TNKS2 maps to chromosome 10.\",\n      \"method\": \"Serological cDNA library screening, sequence and domain analysis\",\n      \"journal\": \"Genes and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — cloning and bioinformatic domain analysis, no direct functional assay beyond sequence\",\n      \"pmids\": [\"11294570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TNKS2 (Tankyrase-2) poly-ADP-ribosylates VEGF in the Golgi compartment. PARP-16 (ER-localized) first catalyzes mono-ADP-ribosylation of VEGF, which is required as a primer for subsequent poly-ADP-ribosylation by Golgi-associated TNKS2, thereby reducing VEGF biological activity.\",\n      \"method\": \"In vitro PARP assay, subcellular fractionation, co-immunoprecipitation, sequential modification assay\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro enzymatic assay with defined substrate and ordered mechanism, single lab\",\n      \"pmids\": [\"32472322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A genome-wide CRISPRi screen revealed that TNKS2 (and TNKS1) bind the peroxisomal membrane protein PEX14 and regulate peroxisome protein import efficiency through PARsylation of peroxisomal membrane proteins. Loss of RNF146, an E3 ligase activated by poly(ADP-ribose), stabilizes TNKS/TNKS2 activity, leading to increased AXIN1 degradation and β-catenin transcriptional activation.\",\n      \"method\": \"Genome-wide CRISPRi screen, Co-IP/pulldown for PEX14 interaction, peroxisome import assay, Wnt reporter assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen combined with direct interaction assay and functional epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"38967608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SASH1 physically binds TNKS2 through a bona fide tankyrase-binding motif (TBM) containing S519. The SASH1 S519N disease variant alters TNKS2 binding kinetics and affinity, and TNKS2 binding is required for SASH1-mediated promotion of stem-like characteristics in human melanocytes.\",\n      \"method\": \"Multiple binding assays (co-IP, pulldown, kinetics), TBM motif identification, functional melanocyte stem cell assays with SASH1 S519N variant\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding assays with mutagenesis and functional readout, but preprint single lab\",\n      \"pmids\": [\"37808724\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of TNKS2 (PARP5B) expression in a carcinogen-induced head and neck squamous cell carcinoma model caused loss of 53BP1+ double-strand break foci and ATM activation, while inducing ATR activation and a shift to homologous recombination (HR)-based repair, indicating TNKS2 is required for nonhomologous end joining (NHEJ) at double-strand breaks in vivo.\",\n      \"method\": \"PARP5B null mouse model, immunofluorescence for 53BP1/ATM/ATR, multiprotein complex identification by co-immunoprecipitation, PARP inhibitor (XAV939) combination treatment\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO with mechanistic readouts (foci, pathway markers), single lab\",\n      \"pmids\": [\"34710250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TNKS2 is a direct target of miR-490-3p, which binds the TNKS2 3'-UTR and negatively regulates TNKS2 protein expression, thereby blocking β-catenin signaling activation and suppressing TNBC cell proliferation and invasion.\",\n      \"method\": \"Dual-luciferase 3'-UTR reporter assay, Western blot, siRNA knockdown, rescue with miR-490-3p-resistant TNKS2\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'-UTR binding validated by luciferase assay with rescue experiment, single lab\",\n      \"pmids\": [\"27506313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TNKS2 is uniquely required for WNT/β-catenin signaling in tumor cells that have lost chromosome 8p (which depletes TNKS1 expression), creating a collateral vulnerability. A structure-guided, first-in-class TNKS2-selective inhibitor was developed that selectively suppresses WNT signaling only in TNKS1-deficient cancer cells and organoids.\",\n      \"method\": \"Structure-guided drug design, cancer cell line and organoid models, genetic depletion of TNKS1, WNT reporter assays\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — structure-based inhibitor + genetic epistasis + functional WNT assay, preprint single lab\",\n      \"pmids\": [\"40093088\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"X-ray crystallography of TNKS2 catalytic domain with quinazolin-4-one inhibitors revealed a novel binding subsite between a mobile active site loop and the canonical nicotinamide-binding site. Nitro- and diol-substituents at C-8 engage new interactions with TNKS2, improving affinity (best IC50 = 14 nM) and selectivity, and attenuate Wnt/β-catenin signaling in cells.\",\n      \"method\": \"X-ray crystallography, enzymatic IC50 assay, cell-based Wnt reporter assay\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation in cells, but preprint\",\n      \"pmids\": [\"bio_10.1101_2024.06.23.600314\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The TNKS2 ARC4 domain mediates protein-substrate binding and can be specifically targeted by a pyrrolone-based compound (ARCher-142/S8, 8 µM potency). NMR and X-ray crystallography identified the ARC4 binding site and a unique hydrophobic sub-pocket. Targeting ARC4 is sufficient to attenuate WNT/β-catenin signaling in cells, demonstrating that the scaffolding (ARC-domain) function of TNKS2 is required for Wnt pathway regulation.\",\n      \"method\": \"FRET-based high-throughput screening, NMR, X-ray crystallography, cell-based WNT reporter assay\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — structural validation (NMR + crystal structure) plus cell-based functional assay, preprint single lab\",\n      \"pmids\": [\"bio_10.1101_2025.03.31.646301\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TNKS2 is a telomere- and peroxisome-associated PARP-family enzyme that uses its ankyrin repeat (ARC) domains to scaffold substrates including TRF1, PEX14, AXIN1, and SASH1, and its catalytic domain to poly-ADP-ribosylate these and other substrates (including VEGF in the Golgi), thereby regulating telomere homeostasis, peroxisome import, Wnt/β-catenin signaling via AXIN1 destabilization, NHEJ-based DNA repair, and melanocyte stem cell maintenance; its activity is functionally redundant with TNKS1, and selective inhibition of TNKS2 alone is sufficient to suppress WNT signaling in TNKS1-deficient tumor cells.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TNKS2 is a poly(ADP-ribose) polymerase (PARP) family member that uses ankyrin repeat cluster (ARC) domains to scaffold substrates and a catalytic PARP domain to poly-ADP-ribosylate them, thereby regulating telomere homeostasis, Wnt/β-catenin signaling, peroxisome protein import, and DNA double-strand break repair pathway choice. TNKS2 directly binds TRF1 at telomeres and PEX14 at peroxisomes, and PARsylates substrates such as AXIN1 to promote their RNF146-dependent degradation, activating β-catenin transcription [PMID:11454873, PMID:38967608]. In the Golgi, TNKS2 extends mono-ADP-ribose primers deposited by PARP-16 on VEGF, reducing VEGF biological activity [PMID:32472322]. Loss of TNKS2 abolishes 53BP1 foci and ATM activation at double-strand breaks, shifting repair from NHEJ toward homologous recombination, and TNKS2 becomes uniquely essential for Wnt signaling in tumors that have lost its paralog TNKS1 through chromosome 8p deletion [PMID:34710250, PMID:40093088].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of TNKS2 as a second tankyrase with PARP activity and TRF1-binding capacity established that tankyrase function is encoded by a two-gene family with overlapping domain architecture but distinct N-termini.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, domain analysis, and sequence cloning from two independent groups\",\n      \"pmids\": [\"11454873\", \"11294570\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Endogenous TNKS2–TRF1 interaction at telomeres not demonstrated\",\n        \"Functional redundancy versus specialization with TNKS1 not addressed\",\n        \"No in vivo loss-of-function data\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that TNKS2 overexpression causes PARP-dependent necrosis established that its catalytic activity has potent cellular consequences and that TNKS2 localizes to a perinuclear compartment.\",\n      \"evidence\": \"Overexpression imaging, mitochondrial membrane potential assay, rescue by PARP inhibitor 3-aminobenzamide\",\n      \"pmids\": [\"11454873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Perinuclear compartment not identified as a specific organelle\",\n        \"Relevance of overexpression-induced necrosis to endogenous TNKS2 function unclear\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that miR-490-3p directly targets TNKS2 3′-UTR to suppress β-catenin signaling provided the first evidence linking endogenous TNKS2 expression levels to Wnt pathway output in cancer cells.\",\n      \"evidence\": \"Dual-luciferase 3′-UTR reporter, Western blot, knockdown and rescue in TNBC cells\",\n      \"pmids\": [\"27506313\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"TNKS2 PARsylation of AXIN1 not directly measured in this study\",\n        \"Contribution of TNKS1 not controlled for\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealing that TNKS2 poly-ADP-ribosylates VEGF in the Golgi, extending mono-ADP-ribose primers placed by ER-localized PARP-16, defined a sequential inter-PARP modification cascade on a secreted growth factor.\",\n      \"evidence\": \"In vitro PARP assay, subcellular fractionation, co-immunoprecipitation, sequential modification assay\",\n      \"pmids\": [\"32472322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"In vivo relevance of VEGF PARsylation to angiogenesis not tested\",\n        \"No structural data on TNKS2–VEGF interface\",\n        \"Single-lab observation\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Loss of TNKS2 in vivo eliminated 53BP1 foci and ATM signaling while activating ATR, establishing TNKS2 as a determinant of NHEJ versus HR pathway choice at DNA double-strand breaks.\",\n      \"evidence\": \"PARP5B-null mouse HNSCC model, immunofluorescence for repair factors, PARP inhibitor combination\",\n      \"pmids\": [\"34710250\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct PARsylation substrate at DSBs not identified\",\n        \"Whether TNKS1 compensates in wild-type context not resolved\",\n        \"Single carcinogen-induced tumor model\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"A genome-wide CRISPRi screen uncovered TNKS2 binding to the peroxisomal membrane protein PEX14, showing that tankyrase PARsylation of peroxisomal substrates regulates protein import and linking the RNF146–tankyrase axis to both peroxisome biology and AXIN1 degradation–driven Wnt activation.\",\n      \"evidence\": \"CRISPRi screen, Co-IP/pulldown for PEX14, peroxisome import assay, Wnt reporter assay\",\n      \"pmids\": [\"38967608\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of PARsylated peroxisomal membrane proteins beyond PEX14 not determined\",\n        \"Relative contributions of TNKS1 vs TNKS2 to peroxisome import not dissected\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic and pharmacological studies demonstrated that TNKS2 becomes uniquely essential for Wnt signaling when TNKS1 is lost through chromosome 8p deletion, defining a paralog-based collateral vulnerability in cancer; structural targeting of both the PARP catalytic site and the ARC4 substrate-binding domain independently attenuates Wnt output.\",\n      \"evidence\": \"Structure-guided TNKS2-selective inhibitor in organoids and cell lines (preprint); NMR/X-ray of ARC4 with pyrrolone compound plus Wnt reporter (preprint); TNKS1 genetic depletion\",\n      \"pmids\": [\"40093088\", \"bio_10.1101_2025.03.31.646301\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint data; not yet peer-reviewed\",\n        \"In vivo therapeutic efficacy of TNKS2-selective inhibitors not demonstrated\",\n        \"Selectivity over other ARC-domain-containing proteins not fully profiled\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full substrate repertoire of TNKS2 PARsylation, the structural basis of TNKS2-specific versus TNKS1-redundant functions, and the endogenous contexts in which TNKS2 scaffolding (ARC domains) versus catalytic activity are rate-limiting remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No proteome-wide PARsylation substrate catalog specific to TNKS2\",\n        \"No full-length TNKS2 structure or cryo-EM model\",\n        \"Relative contribution of TNKS2 versus TNKS1 in normal physiology unclear beyond double-KO studies\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005777\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 7, 8]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TRF1\",\n      \"PEX14\",\n      \"AXIN1\",\n      \"SASH1\",\n      \"RNF146\",\n      \"VEGF\",\n      \"TNKS1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}