{"gene":"NADK","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2021,"finding":"Oncogenic KRAS promotes protein kinase C (PKC)-mediated phosphorylation of NADK, leading to its hyperactivation and sustaining both NADP+ and NADPH levels in pancreatic ductal adenocarcinoma (PDAC) cells.","method":"Phosphorylation assays, PKC inhibition, metabolomics in PDAC cell lines with oncogenic KRAS","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation shown in cancer cell context with functional metabolite readout; single lab, two orthogonal approaches (kinase inhibition + metabolomics)","pmids":["34133937"],"is_preprint":false},{"year":2024,"finding":"NUAK1 directly interacts with and phosphorylates NADK at serine 64 (S64), which reduces osimertinib-induced ROS accumulation and confers osimertinib resistance in NSCLC.","method":"Co-IP, in vitro kinase assay, site-directed mutagenesis (S64), genetic/pharmacological NUAK1 blockade in vitro and in vivo","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct phosphorylation site identified by mutagenesis, reciprocal interaction shown by Co-IP, functional rescue experiments in vitro and in vivo","pmids":["39159134"],"is_preprint":false},{"year":2023,"finding":"Metastatic signals drive histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, increasing NADK expression in metastatic breast cancer cells, thereby expanding NADP(H) pools and enabling adaptation to metastatic stress.","method":"Histone variant ChIP, NADK promoter analysis, NADK overexpression/knockdown, metabolomics in metastatic vs. non-metastatic breast cancer cells","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and promoter analysis for epigenetic mechanism, metabolomics for NADP(H) pools; single lab with two orthogonal methods","pmids":["36841051"],"is_preprint":false},{"year":2025,"finding":"Cytosolic NADK is required for DHFR-mediated folate activation under low folic acid conditions; NADK deletion impairs cytosolic NADPH-driven dihydrofolate reductase (DHFR) activity, thereby blocking folate-dependent nucleotide synthesis.","method":"CRISPR deletion of NADK in cancer cell lines, growth in plasma-like medium with varying folic acid, metabolic tracing, DHFR activity assays","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — CRISPR KO with defined metabolic phenotype, direct enzyme activity measurement, multiple cancer lines tested, mechanistic link to DHFR confirmed","pmids":["40316835"],"is_preprint":false},{"year":2025,"finding":"Human NADK forms a tetramer, and its N-terminal region (absent in bacterial NADKs) modulates tetramer conformation to regulate catalytic activity. An R45H mutation in the N-terminal region increases NADK activity and confers chemotherapy resistance; other cancer-associated mutations disrupt tetramer conformation, inactivate NADK, and sensitize lung cancer cells to chemotherapy.","method":"Cryo-EM structure of human tetrameric NADK, NADK mutant activity assays, cell-based chemotherapy sensitivity assays","journal":"Genes & diseases","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with functional mutagenesis and cell-based validation in a single study","pmids":["40330153"],"is_preprint":false},{"year":2026,"finding":"NMRK2 activates NADK transcription through YAP: NMRK2 overexpression disrupts the integrin β-YAP complex (shown by Co-IP), causing YAP nuclear translocation; YAP then directly binds the NADK promoter (-1500 to -1000 bp) to drive NADK transcription (shown by ChIP-qPCR and luciferase assay). NADK knockdown abolished NMRK2-mediated redox protection in myocardial I/R injury.","method":"Co-IP (NMRK2-YAP-integrin β), nucleocytoplasmic fractionation, immunofluorescence, ChIP-qPCR, luciferase reporter, siRNA knockdown of NADK in cardiomyocytes and mouse I/R model","journal":"Redox biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (Co-IP, ChIP-qPCR, luciferase, fractionation, functional KD) in a single rigorous study","pmids":["41762891"],"is_preprint":false},{"year":2025,"finding":"NADK is S-palmitoylated on three cysteine residues (Cys22, Cys23, Cys26) in its amino-terminal domain by the protein-acyl transferase ZDHHC5; palmitoylation relieves autoinhibitory function of the N-terminus and stimulates NADK kinase activity and NADP+ synthesis. ZDHHC5 knockout mice show defective NADK palmitoylation and reduced NADP+ production.","method":"Palmitoylation site mapping by mutagenesis (Cys22/23/26), ZDHHC5 co-expression/KO studies, in vitro kinase activity assay, ZDHHC5-/- mouse model","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — site-directed mutagenesis identifying palmitoylation sites, KO mouse model; preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2024,"finding":"PRMT6 methylates NADK at residues R39, R41, and R45, suppressing NADK kinase activity and NADP+ synthesis. PRMT6-mediated methylation at R45 coordinates with Akt-mediated phosphorylation to regulate NADK: phosphorylation by Akt stimulates NADK activity through relief of amino-terminal autoinhibition, while PRMT6 methylation antagonizes this activation. PRMT6 can also inhibit NADK in a phosphorylation-independent manner.","method":"In vitro methylation assay, site-directed mutagenesis of R39/R41/R45, kinase activity assays, Akt phosphorylation assays, cancer cell proliferation assays","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro methylation and kinase assays with mutagenesis; preprint not yet peer-reviewed, single lab","pmids":[],"is_preprint":true},{"year":2025,"finding":"NADK knockdown reduces NADPH levels and FSP1 expression, promoting ferroptosis in LUAD cells induced by Erastin/RSL3; NADK governs ferroptosis resistance via the NADPH/FSP1 axis.","method":"siRNA knockdown of NADK in LUAD cell lines, ROS/MDA/Fe2+ measurements, FSP1 protein quantification, in vivo xenograft proliferation","journal":"Journal of cancer research and clinical oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KD with defined molecular readouts (NADPH, FSP1) and in vivo validation; single lab","pmids":["38700533"],"is_preprint":false},{"year":2025,"finding":"NADK is the metabolic hub connecting NMN-driven NAD+ salvage to NADPH synthesis; NADK is required upstream of G6PD and ME1 for NADPH production and ferroptosis resistance. ThioNAM (NADK inhibitor) or NADK knockdown abolishes the ferroptosis-rescuing effects of NMN supplementation.","method":"Pharmacological inhibition (thioNAM), siRNA knockdown, NADK overexpression, G6PD/ME1 intervention, NADPH/GSH/ROS/MDA quantification in HT1080 cells","journal":"Antioxidants (Basel)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and pharmacological interventions with metabolite readouts; single lab, cell-line model","pmids":["41462596"],"is_preprint":false}],"current_model":"NADK is the sole cytosolic enzyme that phosphorylates NAD+ to NADP+, forming a tetrameric structure whose activity is regulated at multiple post-translational levels: activated by PKC-mediated phosphorylation (driven by oncogenic KRAS), by NUAK1-mediated phosphorylation at S64, and by ZDHHC5-mediated S-palmitoylation at Cys22/23/26 (all relieving N-terminal autoinhibition), while suppressed by PRMT6-mediated arginine methylation at R39/R41/R45; its transcription is directly activated by YAP binding the NADK promoter downstream of NMRK2-mediated disruption of the integrin β-YAP complex; cytosolic NADK-derived NADPH is conditionally essential for DHFR-mediated folate activation and nucleotide synthesis, and also sustains ferroptosis resistance via the NADPH/GSH/GPX4 and NADPH/FSP1 axes."},"narrative":{"mechanistic_narrative":"NADK is the cytosolic kinase that phosphorylates NAD+ to generate NADP+, the metabolic node that sets the cellular NADP(H) pool and thereby supplies reductive biosynthetic and antioxidant capacity [PMID:40316835, PMID:41462596]. The human enzyme assembles into a tetramer whose catalytic output is governed by an N-terminal region absent in bacterial orthologs; this segment imposes autoinhibition that is read out through tetramer conformation, such that the activating R45H mutation raises activity and confers chemotherapy resistance while other mutations distort the tetramer and inactivate the enzyme [PMID:40330153]. Multiple post-translational modifications converge on this N-terminal autoinhibitory switch: NUAK1 phosphorylates Ser64 [PMID:39159134], and these activating inputs are antagonized by PRMT6-mediated arginine methylation at R39/R41/R45. In cancer, oncogenic KRAS drives PKC-mediated phosphorylation of NADK to sustain NADP+/NADPH in pancreatic cells [PMID:34133937], and NADK transcription is induced both by H3.3 variant-dependent promoter regulation in metastatic breast cancer [PMID:36841051] and by YAP binding the NADK promoter downstream of NMRK2-driven disruption of the integrin-β/YAP complex [PMID:41762891]. The NADPH produced sustains DHFR-dependent folate activation and nucleotide synthesis under low-folate conditions [PMID:40316835] and underwrites ferroptosis resistance through NADPH/FSP1 and NADPH/GSH axes, positioning NADK upstream of G6PD and ME1 in coupling NAD+ salvage to antioxidant defense [PMID:38700533, PMID:41462596].","teleology":[{"year":2021,"claim":"Established that NADK activity is not constitutive but is amplified by oncogenic signaling, linking KRAS-driven PKC phosphorylation to NADP(H) supply in tumor cells.","evidence":"Phosphorylation assays, PKC inhibition, and metabolomics in KRAS-mutant PDAC lines","pmids":["34133937"],"confidence":"Medium","gaps":["PKC phosphosite(s) on NADK not mapped","single-lab cancer-cell context without structural mechanism"]},{"year":2023,"claim":"Showed that NADK output is set transcriptionally as well as enzymatically, with H3.3 variant-mediated promoter regulation expanding NADP(H) pools to support metastatic adaptation.","evidence":"Histone variant ChIP, promoter analysis, overexpression/knockdown and metabolomics in breast cancer cells","pmids":["36841051"],"confidence":"Medium","gaps":["Transcription factors recruiting H3.3 not identified","single-lab, breast-cancer specific"]},{"year":2024,"claim":"Identified a defined activating phosphosite (S64) and a direct upstream kinase, explaining how NADK is tuned to limit ROS and drive drug resistance.","evidence":"Co-IP, in vitro kinase assay, S64 mutagenesis, NUAK1 blockade in vitro and in vivo in NSCLC","pmids":["39159134"],"confidence":"High","gaps":["Structural basis of how S64 phosphorylation relieves autoinhibition not resolved","interplay with other modifications untested"]},{"year":2024,"claim":"Defined arginine methylation as a negative regulatory input that antagonizes activating phosphorylation at the N-terminal autoinhibitory region.","evidence":"In vitro methylation and kinase assays, R39/R41/R45 mutagenesis, Akt phosphorylation crosstalk (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","phosphorylation-independent inhibition mechanism unclear","in vivo relevance untested"]},{"year":2025,"claim":"Resolved the human tetrameric architecture and showed the N-terminal region (lacking in bacteria) controls activity via tetramer conformation, providing a structural framework for the regulatory modifications.","evidence":"Cryo-EM of human NADK tetramer with mutant activity and chemotherapy-sensitivity assays","pmids":["40330153"],"confidence":"High","gaps":["How phosphorylation/methylation/palmitoylation physically reshape the tetramer not directly visualized","no structure of modified enzyme"]},{"year":2025,"claim":"Added a lipid-based activating modification, showing ZDHHC5 palmitoylates the N-terminal cysteines to relieve autoinhibition and sustain NADP+ synthesis.","evidence":"Cys22/23/26 mutagenesis, ZDHHC5 co-expression and KO mouse, in vitro kinase assays (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","tissue/cell contexts requiring palmitoylation not defined","coordination with phospho/methyl inputs unknown"]},{"year":2025,"claim":"Connected NADK output to a specific anabolic requirement, showing cytosolic NADPH is needed for DHFR activity and folate-dependent nucleotide synthesis under low folate.","evidence":"CRISPR NADK deletion, plasma-like medium folate titration, metabolic tracing and DHFR activity assays across cancer lines","pmids":["40316835"],"confidence":"High","gaps":["Conditional nature limits generalization to folate-replete settings","other NADPH-dependent pathways not comprehensively mapped"]},{"year":2025,"claim":"Placed NADK upstream of ferroptosis defense, demonstrating its NADPH output sustains FSP1 and GSH/GPX4 axes and bridges NAD+ salvage (NMN) to antioxidant protection.","evidence":"siRNA/thioNAM inhibition, overexpression, G6PD/ME1 intervention with NADPH/GSH/ROS/FSP1 readouts in LUAD and HT1080 cells, plus xenografts","pmids":["38700533","41462596"],"confidence":"Medium","gaps":["Direct contribution of NADK-derived vs G6PD/ME1-derived NADPH not partitioned","cell-line restricted"]},{"year":2026,"claim":"Defined a transcriptional activation circuit, showing NMRK2 frees YAP from the integrin-β/YAP complex to drive NADK promoter transcription and redox protection in cardiac ischemia-reperfusion.","evidence":"Co-IP, fractionation, ChIP-qPCR, luciferase reporter and NADK knockdown in cardiomyocytes and mouse I/R model","pmids":["41762891"],"confidence":"High","gaps":["Mechanism by which NMRK2 disrupts the integrin-β/YAP complex not detailed","generality beyond cardiac I/R untested"]},{"year":null,"claim":"How the distinct N-terminal modifications (S64 phosphorylation, R39/41/45 methylation, Cys22/23/26 palmitoylation) are integrated structurally and competitively to set net tetramer activity in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of a modified NADK tetramer","combinatorial regulation not reconstituted","tissue-specific dominance of each input unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[3,4,6,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[3,9]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,9]}],"complexes":[],"partners":["NUAK1","PRMT6","ZDHHC5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95544","full_name":"NAD kinase","aliases":["Poly(P)/ATP NAD kinase"],"length_aa":446,"mass_kda":49.2,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/O95544/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NADK","classification":"Not Classified","n_dependent_lines":12,"n_total_lines":1208,"dependency_fraction":0.009933774834437087},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MTMR1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/NADK","total_profiled":1310},"omim":[{"mim_id":"615787","title":"NAD KINASE 2, MITOCHONDRIAL; NADK2","url":"https://www.omim.org/entry/615787"},{"mim_id":"611616","title":"NAD KINASE; NADK","url":"https://www.omim.org/entry/611616"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NADK"},"hgnc":{"alias_symbol":["FLJ13052","NADK1"],"prev_symbol":[]},"alphafold":{"accession":"O95544","domains":[{"cath_id":"3.40.50.10330","chopping":"105-230","consensus_level":"high","plddt":93.844,"start":105,"end":230},{"cath_id":"2.60.200.30","chopping":"239-248_274-402","consensus_level":"medium","plddt":95.4392,"start":239,"end":402}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95544","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95544-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95544-F1-predicted_aligned_error_v6.png","plddt_mean":80.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NADK","jax_strain_url":"https://www.jax.org/strain/search?query=NADK"},"sequence":{"accession":"O95544","fasta_url":"https://rest.uniprot.org/uniprotkb/O95544.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95544/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95544"}},"corpus_meta":[{"pmid":"36512915","id":"PMC_36512915","title":"Molecular properties and regulation of NAD+ kinase (NADK).","date":"2022","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/36512915","citation_count":60,"is_preprint":false},{"pmid":"21517775","id":"PMC_21517775","title":"NMN/NaMN adenylyltransferase (NMNAT) and NAD kinase (NADK) inhibitors: chemistry and potential therapeutic applications.","date":"2011","source":"Current medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21517775","citation_count":35,"is_preprint":false},{"pmid":"34133937","id":"PMC_34133937","title":"NADK is activated by oncogenic signaling to sustain pancreatic ductal adenocarcinoma.","date":"2021","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34133937","citation_count":35,"is_preprint":false},{"pmid":"36841051","id":"PMC_36841051","title":"NADK-mediated de novo NADP(H) synthesis is a metabolic adaptation essential for breast cancer metastasis.","date":"2023","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/36841051","citation_count":30,"is_preprint":false},{"pmid":"24968225","id":"PMC_24968225","title":"Genome-wide analysis of the NADK gene family in plants.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24968225","citation_count":27,"is_preprint":false},{"pmid":"40088743","id":"PMC_40088743","title":"Schisandra total lignans ameliorate neuronal ferroptosis in 3xTg-AD mice via regulating NADK/NADPH/GSH pathway.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40088743","citation_count":15,"is_preprint":false},{"pmid":"39159134","id":"PMC_39159134","title":"NUAK1-Mediated Phosphorylation of NADK Mitigates ROS Accumulation to Promote Osimertinib Resistance in Non-Small Cell Lung Carcinoma.","date":"2024","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/39159134","citation_count":14,"is_preprint":false},{"pmid":"31275331","id":"PMC_31275331","title":"Calmodulin Is the Fundamental Regulator of NADK-Mediated NAD Signaling in Plants.","date":"2019","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/31275331","citation_count":14,"is_preprint":false},{"pmid":"38700533","id":"PMC_38700533","title":"Knockdown of NADK promotes LUAD ferroptosis via NADPH/FSP1 axis.","date":"2024","source":"Journal of cancer research and clinical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38700533","citation_count":8,"is_preprint":false},{"pmid":"40316835","id":"PMC_40316835","title":"Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.","date":"2025","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/40316835","citation_count":6,"is_preprint":false},{"pmid":"38408517","id":"PMC_38408517","title":"NADK-mediated proline synthesis enhances high-salinity tolerance in the razor clam.","date":"2024","source":"Comparative biochemistry and physiology. Part A, Molecular & integrative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/38408517","citation_count":4,"is_preprint":false},{"pmid":"40330153","id":"PMC_40330153","title":"NADK tetramer defective mutants affect lung cancer response to chemotherapy via controlling NADK activity.","date":"2025","source":"Genes & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/40330153","citation_count":1,"is_preprint":false},{"pmid":"31369599","id":"PMC_31369599","title":"Episodic evolution of a eukaryotic NADK repertoire of ancient provenance.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/31369599","citation_count":1,"is_preprint":false},{"pmid":"40303587","id":"PMC_40303587","title":"NADK as a molecular marker to distinguish between alcohol- and non-alcohol-associated liver cirrhosis: A pilot study.","date":"2025","source":"Clinical and experimental hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/40303587","citation_count":1,"is_preprint":false},{"pmid":"41762891","id":"PMC_41762891","title":"NMRK2-YAP-NADK axis preserves redox protection against myocardial ischemia/reperfusion injury.","date":"2026","source":"Redox biology","url":"https://pubmed.ncbi.nlm.nih.gov/41762891","citation_count":0,"is_preprint":false},{"pmid":"41462596","id":"PMC_41462596","title":"NADK Governs Ferroptosis Susceptibility by Orchestrating NADPH Homeostasis.","date":"2025","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41462596","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.24.666230","title":"Palmitoylation-dependent activation of NADK promotes NADP  <sup>+</sup>  synthesis and tumorigenesis","date":"2025-07-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.24.666230","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.11.14.25340228","title":"Tandem Repeat Polymorphisms Are Associated with Brain Structure: Results of Two Large Population-based Studies","date":"2025-11-19","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.14.25340228","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.26.25334362","title":"Genome-wide meta-analysis and integrative fine-mapping identify novel susceptibility loci and effector genes in psoriatic arthritis","date":"2025-08-27","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.26.25334362","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.11.617635","title":"Arginine methylation coordinates with phosphorylation to regulate NADP  <sup>+</sup>  synthesis","date":"2024-10-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.11.617635","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11851,"output_tokens":2722,"usd":0.038192,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9923,"output_tokens":3251,"usd":0.065445,"stage2_stop_reason":"end_turn"},"total_usd":0.103637,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"Oncogenic KRAS promotes protein kinase C (PKC)-mediated phosphorylation of NADK, leading to its hyperactivation and sustaining both NADP+ and NADPH levels in pancreatic ductal adenocarcinoma (PDAC) cells.\",\n      \"method\": \"Phosphorylation assays, PKC inhibition, metabolomics in PDAC cell lines with oncogenic KRAS\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation shown in cancer cell context with functional metabolite readout; single lab, two orthogonal approaches (kinase inhibition + metabolomics)\",\n      \"pmids\": [\"34133937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 directly interacts with and phosphorylates NADK at serine 64 (S64), which reduces osimertinib-induced ROS accumulation and confers osimertinib resistance in NSCLC.\",\n      \"method\": \"Co-IP, in vitro kinase assay, site-directed mutagenesis (S64), genetic/pharmacological NUAK1 blockade in vitro and in vivo\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct phosphorylation site identified by mutagenesis, reciprocal interaction shown by Co-IP, functional rescue experiments in vitro and in vivo\",\n      \"pmids\": [\"39159134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Metastatic signals drive histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, increasing NADK expression in metastatic breast cancer cells, thereby expanding NADP(H) pools and enabling adaptation to metastatic stress.\",\n      \"method\": \"Histone variant ChIP, NADK promoter analysis, NADK overexpression/knockdown, metabolomics in metastatic vs. non-metastatic breast cancer cells\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and promoter analysis for epigenetic mechanism, metabolomics for NADP(H) pools; single lab with two orthogonal methods\",\n      \"pmids\": [\"36841051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cytosolic NADK is required for DHFR-mediated folate activation under low folic acid conditions; NADK deletion impairs cytosolic NADPH-driven dihydrofolate reductase (DHFR) activity, thereby blocking folate-dependent nucleotide synthesis.\",\n      \"method\": \"CRISPR deletion of NADK in cancer cell lines, growth in plasma-like medium with varying folic acid, metabolic tracing, DHFR activity assays\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — CRISPR KO with defined metabolic phenotype, direct enzyme activity measurement, multiple cancer lines tested, mechanistic link to DHFR confirmed\",\n      \"pmids\": [\"40316835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Human NADK forms a tetramer, and its N-terminal region (absent in bacterial NADKs) modulates tetramer conformation to regulate catalytic activity. An R45H mutation in the N-terminal region increases NADK activity and confers chemotherapy resistance; other cancer-associated mutations disrupt tetramer conformation, inactivate NADK, and sensitize lung cancer cells to chemotherapy.\",\n      \"method\": \"Cryo-EM structure of human tetrameric NADK, NADK mutant activity assays, cell-based chemotherapy sensitivity assays\",\n      \"journal\": \"Genes & diseases\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with functional mutagenesis and cell-based validation in a single study\",\n      \"pmids\": [\"40330153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NMRK2 activates NADK transcription through YAP: NMRK2 overexpression disrupts the integrin β-YAP complex (shown by Co-IP), causing YAP nuclear translocation; YAP then directly binds the NADK promoter (-1500 to -1000 bp) to drive NADK transcription (shown by ChIP-qPCR and luciferase assay). NADK knockdown abolished NMRK2-mediated redox protection in myocardial I/R injury.\",\n      \"method\": \"Co-IP (NMRK2-YAP-integrin β), nucleocytoplasmic fractionation, immunofluorescence, ChIP-qPCR, luciferase reporter, siRNA knockdown of NADK in cardiomyocytes and mouse I/R model\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (Co-IP, ChIP-qPCR, luciferase, fractionation, functional KD) in a single rigorous study\",\n      \"pmids\": [\"41762891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NADK is S-palmitoylated on three cysteine residues (Cys22, Cys23, Cys26) in its amino-terminal domain by the protein-acyl transferase ZDHHC5; palmitoylation relieves autoinhibitory function of the N-terminus and stimulates NADK kinase activity and NADP+ synthesis. ZDHHC5 knockout mice show defective NADK palmitoylation and reduced NADP+ production.\",\n      \"method\": \"Palmitoylation site mapping by mutagenesis (Cys22/23/26), ZDHHC5 co-expression/KO studies, in vitro kinase activity assay, ZDHHC5-/- mouse model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site-directed mutagenesis identifying palmitoylation sites, KO mouse model; preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PRMT6 methylates NADK at residues R39, R41, and R45, suppressing NADK kinase activity and NADP+ synthesis. PRMT6-mediated methylation at R45 coordinates with Akt-mediated phosphorylation to regulate NADK: phosphorylation by Akt stimulates NADK activity through relief of amino-terminal autoinhibition, while PRMT6 methylation antagonizes this activation. PRMT6 can also inhibit NADK in a phosphorylation-independent manner.\",\n      \"method\": \"In vitro methylation assay, site-directed mutagenesis of R39/R41/R45, kinase activity assays, Akt phosphorylation assays, cancer cell proliferation assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro methylation and kinase assays with mutagenesis; preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NADK knockdown reduces NADPH levels and FSP1 expression, promoting ferroptosis in LUAD cells induced by Erastin/RSL3; NADK governs ferroptosis resistance via the NADPH/FSP1 axis.\",\n      \"method\": \"siRNA knockdown of NADK in LUAD cell lines, ROS/MDA/Fe2+ measurements, FSP1 protein quantification, in vivo xenograft proliferation\",\n      \"journal\": \"Journal of cancer research and clinical oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KD with defined molecular readouts (NADPH, FSP1) and in vivo validation; single lab\",\n      \"pmids\": [\"38700533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NADK is the metabolic hub connecting NMN-driven NAD+ salvage to NADPH synthesis; NADK is required upstream of G6PD and ME1 for NADPH production and ferroptosis resistance. ThioNAM (NADK inhibitor) or NADK knockdown abolishes the ferroptosis-rescuing effects of NMN supplementation.\",\n      \"method\": \"Pharmacological inhibition (thioNAM), siRNA knockdown, NADK overexpression, G6PD/ME1 intervention, NADPH/GSH/ROS/MDA quantification in HT1080 cells\",\n      \"journal\": \"Antioxidants (Basel)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and pharmacological interventions with metabolite readouts; single lab, cell-line model\",\n      \"pmids\": [\"41462596\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NADK is the sole cytosolic enzyme that phosphorylates NAD+ to NADP+, forming a tetrameric structure whose activity is regulated at multiple post-translational levels: activated by PKC-mediated phosphorylation (driven by oncogenic KRAS), by NUAK1-mediated phosphorylation at S64, and by ZDHHC5-mediated S-palmitoylation at Cys22/23/26 (all relieving N-terminal autoinhibition), while suppressed by PRMT6-mediated arginine methylation at R39/R41/R45; its transcription is directly activated by YAP binding the NADK promoter downstream of NMRK2-mediated disruption of the integrin β-YAP complex; cytosolic NADK-derived NADPH is conditionally essential for DHFR-mediated folate activation and nucleotide synthesis, and also sustains ferroptosis resistance via the NADPH/GSH/GPX4 and NADPH/FSP1 axes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NADK is the cytosolic kinase that phosphorylates NAD+ to generate NADP+, the metabolic node that sets the cellular NADP(H) pool and thereby supplies reductive biosynthetic and antioxidant capacity [#3, #9]. The human enzyme assembles into a tetramer whose catalytic output is governed by an N-terminal region absent in bacterial orthologs; this segment imposes autoinhibition that is read out through tetramer conformation, such that the activating R45H mutation raises activity and confers chemotherapy resistance while other mutations distort the tetramer and inactivate the enzyme [#4]. Multiple post-translational modifications converge on this N-terminal autoinhibitory switch: NUAK1 phosphorylates Ser64 [#1], and these activating inputs are antagonized by PRMT6-mediated arginine methylation at R39/R41/R45 [#7]. In cancer, oncogenic KRAS drives PKC-mediated phosphorylation of NADK to sustain NADP+/NADPH in pancreatic cells [#0], and NADK transcription is induced both by H3.3 variant-dependent promoter regulation in metastatic breast cancer [#2] and by YAP binding the NADK promoter downstream of NMRK2-driven disruption of the integrin-\\u03b2/YAP complex [#5]. The NADPH produced sustains DHFR-dependent folate activation and nucleotide synthesis under low-folate conditions [#3] and underwrites ferroptosis resistance through NADPH/FSP1 and NADPH/GSH axes, positioning NADK upstream of G6PD and ME1 in coupling NAD+ salvage to antioxidant defense [#8, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Established that NADK activity is not constitutive but is amplified by oncogenic signaling, linking KRAS-driven PKC phosphorylation to NADP(H) supply in tumor cells.\",\n      \"evidence\": \"Phosphorylation assays, PKC inhibition, and metabolomics in KRAS-mutant PDAC lines\",\n      \"pmids\": [\"34133937\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"PKC phosphosite(s) on NADK not mapped\", \"single-lab cancer-cell context without structural mechanism\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed that NADK output is set transcriptionally as well as enzymatically, with H3.3 variant-mediated promoter regulation expanding NADP(H) pools to support metastatic adaptation.\",\n      \"evidence\": \"Histone variant ChIP, promoter analysis, overexpression/knockdown and metabolomics in breast cancer cells\",\n      \"pmids\": [\"36841051\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Transcription factors recruiting H3.3 not identified\", \"single-lab, breast-cancer specific\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a defined activating phosphosite (S64) and a direct upstream kinase, explaining how NADK is tuned to limit ROS and drive drug resistance.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, S64 mutagenesis, NUAK1 blockade in vitro and in vivo in NSCLC\",\n      \"pmids\": [\"39159134\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structural basis of how S64 phosphorylation relieves autoinhibition not resolved\", \"interplay with other modifications untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined arginine methylation as a negative regulatory input that antagonizes activating phosphorylation at the N-terminal autoinhibitory region.\",\n      \"evidence\": \"In vitro methylation and kinase assays, R39/R41/R45 mutagenesis, Akt phosphorylation crosstalk (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"phosphorylation-independent inhibition mechanism unclear\", \"in vivo relevance untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the human tetrameric architecture and showed the N-terminal region (lacking in bacteria) controls activity via tetramer conformation, providing a structural framework for the regulatory modifications.\",\n      \"evidence\": \"Cryo-EM of human NADK tetramer with mutant activity and chemotherapy-sensitivity assays\",\n      \"pmids\": [\"40330153\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How phosphorylation/methylation/palmitoylation physically reshape the tetramer not directly visualized\", \"no structure of modified enzyme\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added a lipid-based activating modification, showing ZDHHC5 palmitoylates the N-terminal cysteines to relieve autoinhibition and sustain NADP+ synthesis.\",\n      \"evidence\": \"Cys22/23/26 mutagenesis, ZDHHC5 co-expression and KO mouse, in vitro kinase assays (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"tissue/cell contexts requiring palmitoylation not defined\", \"coordination with phospho/methyl inputs unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected NADK output to a specific anabolic requirement, showing cytosolic NADPH is needed for DHFR activity and folate-dependent nucleotide synthesis under low folate.\",\n      \"evidence\": \"CRISPR NADK deletion, plasma-like medium folate titration, metabolic tracing and DHFR activity assays across cancer lines\",\n      \"pmids\": [\"40316835\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Conditional nature limits generalization to folate-replete settings\", \"other NADPH-dependent pathways not comprehensively mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed NADK upstream of ferroptosis defense, demonstrating its NADPH output sustains FSP1 and GSH/GPX4 axes and bridges NAD+ salvage (NMN) to antioxidant protection.\",\n      \"evidence\": \"siRNA/thioNAM inhibition, overexpression, G6PD/ME1 intervention with NADPH/GSH/ROS/FSP1 readouts in LUAD and HT1080 cells, plus xenografts\",\n      \"pmids\": [\"38700533\", \"41462596\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct contribution of NADK-derived vs G6PD/ME1-derived NADPH not partitioned\", \"cell-line restricted\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a transcriptional activation circuit, showing NMRK2 frees YAP from the integrin-\\u03b2/YAP complex to drive NADK promoter transcription and redox protection in cardiac ischemia-reperfusion.\",\n      \"evidence\": \"Co-IP, fractionation, ChIP-qPCR, luciferase reporter and NADK knockdown in cardiomyocytes and mouse I/R model\",\n      \"pmids\": [\"41762891\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which NMRK2 disrupts the integrin-\\u03b2/YAP complex not detailed\", \"generality beyond cardiac I/R untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct N-terminal modifications (S64 phosphorylation, R39/41/45 methylation, Cys22/23/26 palmitoylation) are integrated structurally and competitively to set net tetramer activity in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No structure of a modified NADK tetramer\", \"combinatorial regulation not reconstituted\", \"tissue-specific dominance of each input unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [3, 4, 6, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [3, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"NUAK1\",\n      \"PRMT6\",\n      \"ZDHHC5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}