{"gene":"PKMYT1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2017,"finding":"PKMYT1 is a dual-specificity kinase that phosphorylates CDK1 at both Thr14 and Tyr15 (whereas WEE1 exclusively phosphorylates Tyr15), thereby acting as a negative regulator of CDK1/Cyclin B and restraining G2/M transition.","method":"Biochemical characterization and review of cell cycle regulation mechanisms; in vitro kinase assays with Thr14/Tyr15 phospho-specific readouts","journal":"Molecules (Basel, Switzerland)","confidence":"High","confidence_rationale":"Tier 1 / Strong — established by in vitro kinase assays replicated across multiple labs and consistent across the entire corpus","pmids":["29168755"],"is_preprint":false},{"year":2011,"finding":"PKMYT1 (human Myt1 kinase) phosphorylates only protein substrates, not peptide substrates, indicating a requirement for protein-level substrate recognition for both its Thr and Tyr kinase activities. PKMYT1 is insensitive to staurosporine in a kinase binding assay.","method":"Fluorescence polarization immunoassay, immunoblotting, in vitro kinase assays with peptide and protein substrates, molecular dynamics simulations","journal":"Bioorganic & medicinal chemistry letters","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro assay with protein vs peptide substrates, single lab, no independent replication noted in corpus","pmids":["22189141"],"is_preprint":false},{"year":2015,"finding":"PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in normal neural stem cells (NSCs). In glioblastoma stem-like cells (GSCs), this redundancy is lost, likely due to oncogenic signaling, causing GBM-specific lethality upon PKMYT1 loss.","method":"Genome-wide CRISPR-Cas9 knockout screens in GSCs vs NSCs, in vitro and in vivo validation, CDK1-Y15 phosphorylation measurement","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-scale CRISPR screens with in vitro and in vivo validation, mechanistic epistasis with WEE1 established","pmids":["26673326"],"is_preprint":false},{"year":2022,"finding":"CCNE1 amplification creates a synthetic lethal dependency on PKMYT1 kinase activity. PKMYT1 inhibition (with RP-6306) causes unscheduled CDK1 activation selectively in CCNE1-overexpressing cells, promoting early mitosis during DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through early activation of the MMB-FOXM1 mitotic transcriptional program.","method":"Genome-scale CRISPR-Cas9 synthetic lethality screens in CCNE1-amplified cell models, selective PKMYT1 inhibitor RP-6306 (orally bioavailable), CDK1 phosphorylation assays, xenograft models, transcriptional program analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — genome-wide CRISPR screen, selective chemical tool compound, multiple orthogonal methods, in vivo validation, replicated across models","pmids":["35444283"],"is_preprint":false},{"year":2022,"finding":"RP-6306 is an orally bioavailable and selective PKMYT1 inhibitor identified through structure-based drug design. Crystal structure analysis guided optimization of inhibitor interactions with PKMYT1 active site residues, achieving selectivity over the closely related WEE1 kinase.","method":"Structure-based drug design, in vitro PKMYT1 kinase assays, ADME profiling, xenograft tumor models","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structure-based design with in vitro enzymatic assays and in vivo validation, single lab with multiple orthogonal methods","pmids":["35880755"],"is_preprint":false},{"year":2017,"finding":"PKMYT1 promotes hepatocellular carcinoma cell growth and motility by binding to and inactivating GSK3β, thereby activating β-catenin/TCF signaling.","method":"Co-immunoprecipitation, western blotting, cell viability and migration assays, gene knockdown/overexpression in HCC cell lines","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with functional phenotype, single lab, no independent replication","pmids":["28648520"],"is_preprint":false},{"year":2019,"finding":"PKMYT1 interacts with MCRS1 protein; immunoprecipitation confirmed the interaction, and immunofluorescence showed co-localization of MCRS1 and PKMYT1 in the cytoplasm of gastric cancer cells. MCRS1 overexpression suppresses PKMYT1 expression and inhibits GC cell proliferation, invasion, and migration.","method":"Co-immunoprecipitation, immunofluorescence co-localization, yeast two-hybrid (previous study), WEE1 inhibitor rescue experiments","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and co-localization experiment, single lab, no structural or reconstitution data","pmids":["30953699"],"is_preprint":false},{"year":2020,"finding":"PKMYT1 silencing inhibits G2/M checkpoint-mediated radiation resistance in lung adenocarcinoma: knockdown of PKMYT1 abrogates IR-induced G2/M phase arrest and increases sensitivity of cancer cells to radiation.","method":"siRNA knockdown of PKMYT1, colony survival assay, cell cycle analysis by flow cytometry after ionizing radiation","journal":"Frontiers in genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean knockdown with defined functional readout (G2/M arrest abrogation), single lab","pmids":["32411179"],"is_preprint":false},{"year":2021,"finding":"PKMYT1 binds to cyclin A2 (CCNA2) as demonstrated by co-immunoprecipitation; knockdown of PKMYT1 reduces CCNA2 expression and inhibits proliferation, EMT, migration, and invasion in oral squamous cell carcinoma cells.","method":"Co-immunoprecipitation, shRNA knockdown, CCK-8, colony formation, wound healing, Transwell assays, western blot","journal":"Oncology letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP, single lab, no enzymatic or structural validation of the interaction","pmids":["35069872"],"is_preprint":false},{"year":2022,"finding":"PKMYT1 demethylation of PKMYT1 mRNA m6A sites is regulated by ALKBH5 (m6A demethylase); ALKBH5 knockdown or demethylase activity mutation upregulates PKMYT1 expression. IGF2BP3 acts as the m6A reader that stabilizes PKMYT1 mRNA via its m6A modification site.","method":"MeRIP-seq, MeRIP-qRT-PCR, RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), RNA-seq, in vivo lung metastasis model","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA pulldown with MS identification of reader protein, multiple orthogonal methods, single lab","pmids":["35114989"],"is_preprint":false},{"year":2022,"finding":"PKMYT1 directly binds AKT1 and abrogates AKT1 kinase activation in lung adenocarcinoma cells; this tumor-suppressive function depends on PKMYT1's tyrosine and threonine kinase activity.","method":"Co-immunoprecipitation (direct binding assay), RNA-seq, AKT pathway inhibitors, knockdown/overexpression with proliferation and invasion assays","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP without reciprocal pull-down or in vitro reconstitution, single lab","pmids":["36350496"],"is_preprint":false},{"year":2023,"finding":"Combined WEE1 and PKMYT1 inhibition synergistically promotes CDK activation, exacerbates DNA replication stress and replication catastrophe, and activates inflammatory STAT1 signaling in ovarian cancer cells, demonstrating that PKMYT1 and WEE1 are synthetic lethal partners.","method":"Small-molecule inhibitor combinations in ovarian cancer cells and organoid models, CDK activity assays, DNA damage markers, STAT1 pathway analysis","journal":"NAR cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis with organoid models, multiple pathway readouts, single lab","pmids":["37325550"],"is_preprint":false},{"year":2023,"finding":"ELF3 transcription factor upregulates PKMYT1 expression by binding the PKMYT1 promoter region in gallbladder cancer cells, thereby promoting phosphorylation of CDK1 and inducing gemcitabine resistance via the ELF3/PKMYT1/CDK1 axis.","method":"Luciferase reporter assay, ChIP assay, western blot for CDK1 phosphorylation, shRNA knockdown, in vivo xenograft models","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase reporter for transcriptional regulation, CDK1 phosphorylation readout, single lab with multiple orthogonal methods","pmids":["36988891"],"is_preprint":false},{"year":2023,"finding":"TEAD4 transcriptionally activates PKMYT1 by binding its promoter; TEAD4-driven PKMYT1 upregulation facilitates glycolysis and proliferation in gastric cancer cells.","method":"Dual-luciferase assay, chromatin immunoprecipitation (ChIP), JASPAR database prediction, CCK-8, colony formation, extracellular acidification rate measurement","journal":"Molecular and cellular probes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase reporter confirm transcriptional regulation, single lab with two orthogonal methods","pmids":["37729973"],"is_preprint":false},{"year":2024,"finding":"PKMYT1 inhibition in CCNE1-amplified cancers functions in addition to its canonical CDK1-phosphorylating role by regulating PLK1 expression and phosphorylation, promoting PDAC tumorigenesis. TP53 function and PRKDC activation modulate sensitivity to PKMYT1 inhibition.","method":"Genome-wide CRISPR screens, PLK1 expression/phosphorylation analysis, in vitro cell line models, xenograft models (CDX and PDX), pharmacological PKMYT1 inhibition","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide CRISPR screen plus mechanistic follow-up with PLK1 analysis, in vivo validation, single lab","pmids":["38570712"],"is_preprint":false},{"year":2024,"finding":"Low-molecular weight cyclin E (LMW-E) specifically upregulates PKMYT1 expression and protein stability compared to full-length cyclin E, increasing CDK1 phosphorylation. PKMYT1 inhibition in this context accelerates premature mitotic entry, inhibits replication fork restart, and enhances DNA damage and apoptosis in an LMW-E-dependent manner.","method":"Tumor sample proteogenomics, cell line LMW-E expression, PKMYT1 inhibitor RP-6306, CDK1 phosphorylation assays, replication fork assays, DNA damage markers, LMW-E transgenic murine mammary tumor models, PDX models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal models (cell lines, transgenic mice, PDX), LMW-E-specific mechanistic axis defined, single lab with replicated findings across model systems","pmids":["39186665"],"is_preprint":false},{"year":2024,"finding":"X-ray co-crystal structure of PKMYT1 with inhibitor compounds confirmed that binding interactions with residues Asp251 and Tyr121 in the PKMYT1 active site are critical for potency and selectivity over WEE1.","method":"X-ray crystallography (co-crystal structure of PKMYT1 with inhibitors), structure-activity relationship analysis","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation (potency/selectivity readouts), single lab","pmids":["39163619"],"is_preprint":false},{"year":2023,"finding":"Molecular dynamics simulations and binding free energy calculations identified that the non-conserved gatekeeper residue Thr187 in PKMYT1 (vs Asn376 in WEE1) is a critical determinant of binding selectivity for RP-6306 toward PKMYT1 over WEE1. A water-mediated hydrogen bond between RP-6306 and Gly191 at the PKMYT1 hinge domain is absent in the WEE1 complex.","method":"Molecular docking, multiple microsecond-length molecular dynamics simulations, end-point free energy calculations (MM-PBSA/GBSA), per-residue free energy decomposition","journal":"Journal of biomolecular structure & dynamics","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational analysis only, no experimental mutagenesis validation reported in this paper","pmids":["37345529"],"is_preprint":false},{"year":2025,"finding":"PKMYT1 phosphorylates nucleophosmin 1 (NPM1) at the S260 site in osteosarcoma cells. This phosphorylation competitively impairs NPM1 SUMOylation, which in turn interferes with recruitment of BRCA1, RAP80, and RAD51 to double-strand break sites, thereby impairing DSB repair and promoting chemoresistance to cisplatin.","method":"Kinome-wide CRISPR screen, transcriptome sequencing, phosphorylation site mapping (NPM1-S260), SUMOylation assay, co-immunoprecipitation for BRCA1/RAP80/RAD51 recruitment, in vitro functional assays, patient-derived clinical specimens","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus multiple mechanistic assays (phosphorylation, SUMOylation, DNA repair factor recruitment), single lab","pmids":["40393983"],"is_preprint":false},{"year":2025,"finding":"Meisoindigo (Mei) forms a selective and reversible covalent bond with the Cys301 residue of PKMYT1, acting as a molecular glue that enhances the interaction between PKMYT1 and the E3 ubiquitin ligase TRIM25 by approximately 30-fold. This triggers K48-linked polyubiquitination and proteasomal degradation of PKMYT1.","method":"Activity-based protein profiling (ABPP), covalent binding characterization, ubiquitination assays (K48-linkage), co-immunoprecipitation of PKMYT1-TRIM25 interaction, proteasome inhibitor rescue experiments, xenograft model","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ABPP target identification, covalent bond characterization, E3 ligase interaction quantification, multiple orthogonal methods, single lab","pmids":["40279509"],"is_preprint":false},{"year":2025,"finding":"USP49 deubiquitinase interacts with PKMYT1 and limits its ubiquitination and subsequent proteasomal degradation, thereby stabilizing PKMYT1 protein levels in triple-negative breast cancer cells. Overexpression of PKMYT1 rescues malignant phenotypes in USP49-deficient TNBC cells.","method":"Co-immunoprecipitation of USP49-PKMYT1, ubiquitination assays, siRNA knockdown, rescue overexpression, cell viability and cell cycle assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with functional rescue experiment, single lab, no in vitro deubiquitinase reconstitution","pmids":["41443044"],"is_preprint":false},{"year":2025,"finding":"PKMYT1 inhibition (RP-6306) activates the cGAS-STING pathway and potentiates type I and II interferon signaling, upregulating chemokines CCL5 and CXCL10, thereby enhancing CD8+ T cell infiltration and synergizing with PD-L1 blockade in castration-resistant prostate cancer models.","method":"PCa cell lines and syngeneic mouse models, single-cell RNA sequencing, pharmacological PKMYT1 inhibition (RP-6306), flow cytometry for T cell infiltration, cGAS-STING pathway assays, combination with anti-PD-L1","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway activation (cGAS-STING) confirmed in cell lines and syngeneic in vivo models, single lab","pmids":["41617394"],"is_preprint":false},{"year":2026,"finding":"PKMYT1 kinase activity increases during anaphase and plays a novel mitotic role distinct from WEE1: chemical inhibition of PKMYT1 induces premature anaphase leading to chromosome segregation errors (chromatin bridges and micronuclei), which activate the cGAS-STING pathway. PKMYT1 also contributes to maintaining spindle assembly checkpoint integrity, as its inhibition promotes mitotic slippage in the presence of anti-microtubule drugs. PKMYT1 thus acts alongside Cyclin B1 degradation to control CDK1-Cyclin B1 activity during metaphase-to-anaphase transition.","method":"Chemical inhibition of PKMYT1 with live-cell imaging, chromosome segregation error analysis, micronuclei and chromatin bridge quantification, cGAS-STING pathway activation assays, spindle assembly checkpoint assays with anti-microtubule drugs","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays (live imaging, chromosome segregation, SAC, cGAS-STING), novel mechanistic function established beyond canonical CDK1 phosphorylation","pmids":["42243523"],"is_preprint":false},{"year":2026,"finding":"PKMYT1 directly activates Snail transcription in clear cell renal cell carcinoma by upregulating FoxM1, which represses E-cadherin and activates vimentin expression, thereby inducing EMT. Inhibition of the FoxM1/Snail/EMT pathway reversed PKMYT1-induced metastasis in mice.","method":"Exogenous PKMYT1 modulation, in vitro migration/invasion assays, EMT marker western blot, in vivo mouse metastasis model, FoxM1/Snail pathway analysis","journal":"Cell biology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway placement via gain/loss-of-function with in vivo validation, but mechanistic link between PKMYT1 kinase activity and FoxM1 not directly established at molecular level","pmids":["41693360"],"is_preprint":false},{"year":2026,"finding":"Allosteric inhibitor P29 binds a previously unknown cryptic allosteric site on PKMYT1, inducing conformational rearrangement of the P-loop and inhibiting PKMYT1 through a mixed ATP-competitive and non-competitive mechanism. A closely related analogue P32 engages PKMYT1 in the ATP binding pocket despite similar structure.","method":"X-ray crystallography, kinetic mechanistic assays (ATP competition), cell-based engagement assays, computational structural modeling (AlphaFold2/3, MD simulations)","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of allosteric site plus kinetic assays establishing mechanism of inhibition, single lab with multiple orthogonal structural and biochemical methods","pmids":["42227652"],"is_preprint":false},{"year":2025,"finding":"Rb1 loss in breast cancer creates synthetic lethality with combined ATR and PKMYT1 coinhibition. Rb1 deficiency impairs double-strand DNA repair by attenuating homologous recombination and non-homologous end joining, leading to replication fork collapse. Coinhibition of ATR (S-G2 checkpoint) and PKMYT1 (G2-M checkpoint) leads to replication stress, premature mitotic entry, and DNA damage accumulation, with JNK/p38 stress response pathway activation driving apoptosis.","method":"In vitro coinhibition in Rb1-deficient vs proficient cell lines, genetic Rb1 knockdown/re-expression, PDX in vivo models, γH2AX/Ki67 biomarker analysis, HR and NHEJ repair assays, proteogenomic pathway analysis","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic manipulation (KD/rescue), multiple orthogonal cellular and in vivo models, mechanistic pathway identified (HR/NHEJ impairment, JNK/p38 activation), single lab with comprehensive validation","pmids":["41442499"],"is_preprint":false},{"year":2024,"finding":"E2F7 transcription factor directly activates PKMYT1 transcription by binding to the PKMYT1 promoter in gastric cancer cells; the E2F7/PKMYT1 axis promotes proliferation and reduces adriamycin (ADM) sensitivity through MAPK pathway activation.","method":"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), western blot (ERK/p-ERK), CCK-8 assay, IC50 determination, rescue experiments","journal":"Revista de investigacion clinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase reporter confirming transcriptional regulation, MAPK pathway validated by western blot, single lab","pmids":["38253021"],"is_preprint":false},{"year":2024,"finding":"PKMYT1 promotes SREBP2-mediated expression of cholesterol biosynthesis enzymes through activating the TNF/TRAF1/AKT pathway in triple-negative breast cancer cells.","method":"PKMYT1 knockdown, SREBP2 activity assays, cholesterol biosynthesis enzyme expression analysis, TNF/TRAF1/AKT pathway western blot, atorvastatin sensitivity assays","journal":"PeerJ","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pathway placement by knockdown with western blot readouts, no direct kinase-substrate relationship established, single lab","pmids":["39011373"],"is_preprint":false},{"year":2025,"finding":"PKMYT1 inhibition with RP-6306 induces premature mitotic entry, causing DNA damage and mitotic catastrophe that triggers PANoptosis (concurrent apoptosis, pyroptosis, and necroptosis) in pancreatic cancer cells.","method":"PKMYT1 inhibitor RP-6306, in vitro cell death assays, PANoptosis marker analysis (apoptosis/pyroptosis/necroptosis), in vivo xenograft models, combination with gemcitabine","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell death pathway markers characterized, in vivo validation, mechanistic link from CDK1 hyperactivation to PANoptosis established, single lab","pmids":["40664640"],"is_preprint":false}],"current_model":"PKMYT1 is a membrane-associated dual-specificity kinase (Thr14 and Tyr15) that phosphorylates CDK1 to maintain the inhibitory state of CDK1/Cyclin B, thereby restraining G2/M cell cycle entry; beyond this canonical role, PKMYT1 also acts during anaphase to regulate chromosome segregation fidelity and spindle assembly checkpoint integrity, interacts with substrates including NPM1 (phosphorylating S260 to impair DSB repair), binds partners including GSK3β, AKT1, MCRS1, and cyclin A2, is transcriptionally regulated by E2F7, ELF3, and TEAD4, is post-translationally stabilized by USP49 and degraded via TRIM25-mediated ubiquitination, and its inhibition (which causes unscheduled CDK1 activation) is synthetically lethal with CCNE1 amplification and several other oncogenic alterations, while also activating cGAS-STING-dependent innate immune responses."},"narrative":{"mechanistic_narrative":"PKMYT1 is a membrane-associated dual-specificity kinase that phosphorylates CDK1 at both Thr14 and Tyr15 to maintain CDK1/Cyclin B in its inhibitory state and restrain the G2/M transition, distinguishing it from WEE1, which phosphorylates only Tyr15 [PMID:29168755]. Its kinase activities require intact protein substrates rather than peptides, indicating recognition at the protein level [PMID:22189141]. In normal cells PKMYT1 acts redundantly with WEE1 to enforce mitotic timing, but this redundancy is lost in oncogenic contexts, creating selective dependencies: PKMYT1 loss is lethal in glioblastoma stem cells [PMID:26673326], and PKMYT1 kinase activity is synthetically lethal with CCNE1 amplification, where its inhibition drives unscheduled CDK1 activation and premature mitosis during DNA synthesis [PMID:35444283]. This synthetic-lethal logic extends to low-molecular-weight cyclin E, which stabilizes PKMYT1 to raise CDK1 phosphorylation [PMID:39186665], to Rb1 loss combined with ATR inhibition [PMID:41442499], and to combined WEE1/PKMYT1 inhibition, all converging on replication catastrophe and DNA damage [PMID:37325550]. Beyond CDK1, PKMYT1 has a distinct mitotic role: its activity rises during anaphase to safeguard chromosome segregation and spindle assembly checkpoint integrity, and its inhibition produces segregation errors that activate cGAS-STING innate immune signaling [PMID:42243523, PMID:41617394]. The selective inhibitor RP-6306 exploits these dependencies, achieving selectivity over WEE1 through active-site contacts at Asp251 and Tyr121, while an allosteric chemotype engages a cryptic P-loop site [PMID:35880755, PMID:39163619, PMID:42227652]. PKMYT1 also phosphorylates NPM1 at S260 to impair SUMOylation-dependent recruitment of BRCA1, RAP80, and RAD51 to double-strand breaks, linking it to DNA repair and chemoresistance [PMID:40393983]. PKMYT1 is transcriptionally upregulated by E2F7, ELF3, and TEAD4 [PMID:38253021, PMID:36988891, PMID:37729973], its mRNA is stabilized via m6A reader IGF2BP3 under ALKBH5 control [PMID:35114989], and its protein level is set by opposing USP49 stabilization and TRIM25-mediated K48 ubiquitination [PMID:41443044, PMID:40279509].","teleology":[{"year":2017,"claim":"Established PKMYT1's core biochemical identity, distinguishing it from WEE1 by its dual Thr14/Tyr15 phosphorylation of CDK1 and defining it as a G2/M-restraining kinase.","evidence":"In vitro kinase assays with Thr14/Tyr15 phospho-specific readouts","pmids":["29168755"],"confidence":"High","gaps":["Does not address membrane targeting or substrate-recognition determinants","No structural basis for dual specificity provided"]},{"year":2011,"claim":"Showed PKMYT1 requires protein-level substrates rather than peptides, indicating that substrate context governs its catalysis and complicating peptide-based inhibitor screens.","evidence":"In vitro kinase assays comparing peptide vs protein substrates plus MD simulations","pmids":["22189141"],"confidence":"Medium","gaps":["Structural basis for protein-substrate requirement not defined","Single lab, no independent replication noted"]},{"year":2015,"claim":"Demonstrated WEE1/PKMYT1 redundancy in normal cells is broken in tumor cells, establishing the conceptual basis for tumor-selective PKMYT1 dependency.","evidence":"Genome-wide CRISPR knockout screens in GSCs vs NSCs with CDK1-Y15 readouts and in vivo validation","pmids":["26673326"],"confidence":"High","gaps":["Molecular cause of lost redundancy in tumor cells not fully defined","Limited to glioblastoma context"]},{"year":2022,"claim":"Identified CCNE1 amplification as a synthetic-lethal partner and delivered RP-6306 as a selective tool/clinical-grade inhibitor, converting PKMYT1 biology into a therapeutic strategy.","evidence":"Genome-scale CRISPR synthetic-lethality screens, structure-based inhibitor design, CDK1 phosphorylation assays, and xenografts","pmids":["35444283","35880755"],"confidence":"High","gaps":["Full set of co-dependencies beyond CCNE1 not enumerated","MMB-FOXM1 contribution defined only in part"]},{"year":2023,"claim":"Mapped transcriptional and post-transcriptional control of PKMYT1, showing E2F7, ELF3, TEAD4 drive its expression and m6A reader IGF2BP3 stabilizes its mRNA, explaining how tumors elevate PKMYT1.","evidence":"ChIP, luciferase reporters, MeRIP-seq, RNA pulldown with MS, and rescue experiments across cancer models","pmids":["36988891","37729973","38253021","35114989"],"confidence":"Medium","gaps":["Relative contribution of each regulator in normal tissue unknown","Direct kinase-pathway coupling for several axes (e.g., metabolic outputs) not established"]},{"year":2023,"claim":"Defined WEE1 as a pharmacological synthetic-lethal partner, showing combined inhibition drives replication catastrophe and inflammatory STAT1 signaling.","evidence":"Inhibitor combinations in ovarian cancer cells and organoids with CDK activity and DNA damage readouts","pmids":["37325550"],"confidence":"Medium","gaps":["Mechanism linking replication stress to STAT1 not fully resolved","Single lab"]},{"year":2024,"claim":"Extended the dependency landscape to LMW-E, PLK1 regulation, and TP53/PRKDC context, refining biomarkers and revealing non-canonical effectors beyond CDK1 phosphorylation.","evidence":"Genome-wide CRISPR screens, PLK1 phosphorylation analysis, proteogenomics, transgenic and PDX models with RP-6306","pmids":["38570712","39186665"],"confidence":"High","gaps":["Direct kinase-substrate relationship for PLK1 regulation not established","Mechanism of LMW-E-specific PKMYT1 stabilization unresolved"]},{"year":2025,"claim":"Revealed PKMYT1 phosphorylates NPM1-S260 to impair DSB repair, defining a kinase-substrate axis that connects PKMYT1 to homologous-recombination factor recruitment and chemoresistance.","evidence":"Kinome CRISPR screen, phospho-site mapping, SUMOylation and Co-IP assays for BRCA1/RAP80/RAD51 recruitment in osteosarcoma","pmids":["40393983"],"confidence":"Medium","gaps":["In vitro reconstitution of NPM1 phosphorylation not shown","Generality beyond osteosarcoma untested"]},{"year":2025,"claim":"Established the bidirectional control of PKMYT1 protein stability by USP49 (stabilizing) and TRIM25 (degrading), with meisoindigo acting as a molecular glue at Cys301 to drive K48 ubiquitination.","evidence":"Co-IP, ubiquitination assays, ABPP covalent target ID, and proteasome rescue with xenograft validation","pmids":["40279509","41443044"],"confidence":"Medium","gaps":["No in vitro deubiquitinase reconstitution for USP49 (Low-confidence)","Physiological balance of USP49 vs TRIM25 not defined"]},{"year":2025,"claim":"Connected PKMYT1 inhibition to immune activation and cell death, showing RP-6306 triggers cGAS-STING, interferon signaling, CD8+ T-cell infiltration, and PANoptosis, broadening therapeutic rationale.","evidence":"Syngeneic mouse models, scRNA-seq, cGAS-STING and cell-death marker assays, combination with anti-PD-L1 and gemcitabine","pmids":["41617394","40664640"],"confidence":"Medium","gaps":["Whether immune activation requires segregation errors vs replication stress not dissected","Durability of T-cell responses untested"]},{"year":2026,"claim":"Defined a CDK1-restraint-independent anaphase function for PKMYT1 in chromosome segregation and spindle assembly checkpoint integrity, establishing a mitotic role distinct from WEE1.","evidence":"Live-cell imaging, chromosome segregation and micronuclei quantification, SAC assays with anti-microtubule drugs, cGAS-STING activation","pmids":["42243523"],"confidence":"High","gaps":["Anaphase substrate(s) of PKMYT1 not identified","Mechanism of SAC maintenance unresolved"]},{"year":2026,"claim":"Characterized a cryptic allosteric site on PKMYT1, revealing a P-loop conformational mechanism distinct from ATP-competitive engagement and expanding inhibitor design space.","evidence":"X-ray crystallography, ATP-competition kinetics, cellular engagement assays, and computational modeling","pmids":["42227652"],"confidence":"High","gaps":["Cellular potency advantage of allosteric vs orthosteric inhibition not established","Selectivity profile across the kinome not fully mapped"]},{"year":null,"claim":"The substrate repertoire and recruitment mechanism underlying PKMYT1's anaphase and DNA-repair roles remain undefined, as does which contexts depend on kinase activity versus scaffolding interactions.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No anaphase substrate identified","Several reported binding partners (GSK3β, AKT1, MCRS1, cyclin A2) rest on single Co-IPs without reciprocal or reconstitution validation","Membrane-targeting determinants not characterized in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,18]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,18]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[18]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,2,3,22]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[18,25]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[21,22]}],"complexes":[],"partners":["CDK1","NPM1","TRIM25","USP49","GSK3B","AKT1","MCRS1","CCNA2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99640","full_name":"Membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase","aliases":["Myt1 kinase"],"length_aa":499,"mass_kda":54.5,"function":"Acts as a negative regulator of entry into mitosis (G2 to M transition) by phosphorylation of the CDK1 kinase specifically when CDK1 is complexed to cyclins (PubMed:10373560, PubMed:10504341, PubMed:9001210, PubMed:9268380). Mediates phosphorylation of CDK1 predominantly on 'Thr-14'. Also involved in Golgi fragmentation (PubMed:9001210, PubMed:9268380). May be involved in phosphorylation of CDK1 on 'Tyr-15' to a lesser degree, however tyrosine kinase activity is unclear and may be indirect (PubMed:9001210, PubMed:9268380)","subcellular_location":"Endoplasmic reticulum membrane; Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/Q99640/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PKMYT1","classification":"Common 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Therapy","date":"2024-11-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.11.10.622866","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.09.16.613317","title":"Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity","date":"2024-09-20","source":"bioRxiv","url":"https://doi.org/10.1101/2024.09.16.613317","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.12.642754","title":"WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response","date":"2025-03-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.12.642754","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39542,"output_tokens":7355,"usd":0.114475,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16682,"output_tokens":4262,"usd":0.09498,"stage2_stop_reason":"end_turn"},"total_usd":0.209455,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"PKMYT1 is a dual-specificity kinase that phosphorylates CDK1 at both Thr14 and Tyr15 (whereas WEE1 exclusively phosphorylates Tyr15), thereby acting as a negative regulator of CDK1/Cyclin B and restraining G2/M transition.\",\n      \"method\": \"Biochemical characterization and review of cell cycle regulation mechanisms; in vitro kinase assays with Thr14/Tyr15 phospho-specific readouts\",\n      \"journal\": \"Molecules (Basel, Switzerland)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — established by in vitro kinase assays replicated across multiple labs and consistent across the entire corpus\",\n      \"pmids\": [\"29168755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PKMYT1 (human Myt1 kinase) phosphorylates only protein substrates, not peptide substrates, indicating a requirement for protein-level substrate recognition for both its Thr and Tyr kinase activities. PKMYT1 is insensitive to staurosporine in a kinase binding assay.\",\n      \"method\": \"Fluorescence polarization immunoassay, immunoblotting, in vitro kinase assays with peptide and protein substrates, molecular dynamics simulations\",\n      \"journal\": \"Bioorganic & medicinal chemistry letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro assay with protein vs peptide substrates, single lab, no independent replication noted in corpus\",\n      \"pmids\": [\"22189141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in normal neural stem cells (NSCs). In glioblastoma stem-like cells (GSCs), this redundancy is lost, likely due to oncogenic signaling, causing GBM-specific lethality upon PKMYT1 loss.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 knockout screens in GSCs vs NSCs, in vitro and in vivo validation, CDK1-Y15 phosphorylation measurement\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-scale CRISPR screens with in vitro and in vivo validation, mechanistic epistasis with WEE1 established\",\n      \"pmids\": [\"26673326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CCNE1 amplification creates a synthetic lethal dependency on PKMYT1 kinase activity. PKMYT1 inhibition (with RP-6306) causes unscheduled CDK1 activation selectively in CCNE1-overexpressing cells, promoting early mitosis during DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through early activation of the MMB-FOXM1 mitotic transcriptional program.\",\n      \"method\": \"Genome-scale CRISPR-Cas9 synthetic lethality screens in CCNE1-amplified cell models, selective PKMYT1 inhibitor RP-6306 (orally bioavailable), CDK1 phosphorylation assays, xenograft models, transcriptional program analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — genome-wide CRISPR screen, selective chemical tool compound, multiple orthogonal methods, in vivo validation, replicated across models\",\n      \"pmids\": [\"35444283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RP-6306 is an orally bioavailable and selective PKMYT1 inhibitor identified through structure-based drug design. Crystal structure analysis guided optimization of inhibitor interactions with PKMYT1 active site residues, achieving selectivity over the closely related WEE1 kinase.\",\n      \"method\": \"Structure-based drug design, in vitro PKMYT1 kinase assays, ADME profiling, xenograft tumor models\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structure-based design with in vitro enzymatic assays and in vivo validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35880755\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKMYT1 promotes hepatocellular carcinoma cell growth and motility by binding to and inactivating GSK3β, thereby activating β-catenin/TCF signaling.\",\n      \"method\": \"Co-immunoprecipitation, western blotting, cell viability and migration assays, gene knockdown/overexpression in HCC cell lines\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with functional phenotype, single lab, no independent replication\",\n      \"pmids\": [\"28648520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PKMYT1 interacts with MCRS1 protein; immunoprecipitation confirmed the interaction, and immunofluorescence showed co-localization of MCRS1 and PKMYT1 in the cytoplasm of gastric cancer cells. MCRS1 overexpression suppresses PKMYT1 expression and inhibits GC cell proliferation, invasion, and migration.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, yeast two-hybrid (previous study), WEE1 inhibitor rescue experiments\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and co-localization experiment, single lab, no structural or reconstitution data\",\n      \"pmids\": [\"30953699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PKMYT1 silencing inhibits G2/M checkpoint-mediated radiation resistance in lung adenocarcinoma: knockdown of PKMYT1 abrogates IR-induced G2/M phase arrest and increases sensitivity of cancer cells to radiation.\",\n      \"method\": \"siRNA knockdown of PKMYT1, colony survival assay, cell cycle analysis by flow cytometry after ionizing radiation\",\n      \"journal\": \"Frontiers in genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean knockdown with defined functional readout (G2/M arrest abrogation), single lab\",\n      \"pmids\": [\"32411179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PKMYT1 binds to cyclin A2 (CCNA2) as demonstrated by co-immunoprecipitation; knockdown of PKMYT1 reduces CCNA2 expression and inhibits proliferation, EMT, migration, and invasion in oral squamous cell carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, shRNA knockdown, CCK-8, colony formation, wound healing, Transwell assays, western blot\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP, single lab, no enzymatic or structural validation of the interaction\",\n      \"pmids\": [\"35069872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PKMYT1 demethylation of PKMYT1 mRNA m6A sites is regulated by ALKBH5 (m6A demethylase); ALKBH5 knockdown or demethylase activity mutation upregulates PKMYT1 expression. IGF2BP3 acts as the m6A reader that stabilizes PKMYT1 mRNA via its m6A modification site.\",\n      \"method\": \"MeRIP-seq, MeRIP-qRT-PCR, RNA pulldown, mass spectrometry, RNA immunoprecipitation (RIP), RNA-seq, in vivo lung metastasis model\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA pulldown with MS identification of reader protein, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"35114989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PKMYT1 directly binds AKT1 and abrogates AKT1 kinase activation in lung adenocarcinoma cells; this tumor-suppressive function depends on PKMYT1's tyrosine and threonine kinase activity.\",\n      \"method\": \"Co-immunoprecipitation (direct binding assay), RNA-seq, AKT pathway inhibitors, knockdown/overexpression with proliferation and invasion assays\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP without reciprocal pull-down or in vitro reconstitution, single lab\",\n      \"pmids\": [\"36350496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Combined WEE1 and PKMYT1 inhibition synergistically promotes CDK activation, exacerbates DNA replication stress and replication catastrophe, and activates inflammatory STAT1 signaling in ovarian cancer cells, demonstrating that PKMYT1 and WEE1 are synthetic lethal partners.\",\n      \"method\": \"Small-molecule inhibitor combinations in ovarian cancer cells and organoid models, CDK activity assays, DNA damage markers, STAT1 pathway analysis\",\n      \"journal\": \"NAR cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis with organoid models, multiple pathway readouts, single lab\",\n      \"pmids\": [\"37325550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ELF3 transcription factor upregulates PKMYT1 expression by binding the PKMYT1 promoter region in gallbladder cancer cells, thereby promoting phosphorylation of CDK1 and inducing gemcitabine resistance via the ELF3/PKMYT1/CDK1 axis.\",\n      \"method\": \"Luciferase reporter assay, ChIP assay, western blot for CDK1 phosphorylation, shRNA knockdown, in vivo xenograft models\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase reporter for transcriptional regulation, CDK1 phosphorylation readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36988891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TEAD4 transcriptionally activates PKMYT1 by binding its promoter; TEAD4-driven PKMYT1 upregulation facilitates glycolysis and proliferation in gastric cancer cells.\",\n      \"method\": \"Dual-luciferase assay, chromatin immunoprecipitation (ChIP), JASPAR database prediction, CCK-8, colony formation, extracellular acidification rate measurement\",\n      \"journal\": \"Molecular and cellular probes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase reporter confirm transcriptional regulation, single lab with two orthogonal methods\",\n      \"pmids\": [\"37729973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKMYT1 inhibition in CCNE1-amplified cancers functions in addition to its canonical CDK1-phosphorylating role by regulating PLK1 expression and phosphorylation, promoting PDAC tumorigenesis. TP53 function and PRKDC activation modulate sensitivity to PKMYT1 inhibition.\",\n      \"method\": \"Genome-wide CRISPR screens, PLK1 expression/phosphorylation analysis, in vitro cell line models, xenograft models (CDX and PDX), pharmacological PKMYT1 inhibition\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide CRISPR screen plus mechanistic follow-up with PLK1 analysis, in vivo validation, single lab\",\n      \"pmids\": [\"38570712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Low-molecular weight cyclin E (LMW-E) specifically upregulates PKMYT1 expression and protein stability compared to full-length cyclin E, increasing CDK1 phosphorylation. PKMYT1 inhibition in this context accelerates premature mitotic entry, inhibits replication fork restart, and enhances DNA damage and apoptosis in an LMW-E-dependent manner.\",\n      \"method\": \"Tumor sample proteogenomics, cell line LMW-E expression, PKMYT1 inhibitor RP-6306, CDK1 phosphorylation assays, replication fork assays, DNA damage markers, LMW-E transgenic murine mammary tumor models, PDX models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal models (cell lines, transgenic mice, PDX), LMW-E-specific mechanistic axis defined, single lab with replicated findings across model systems\",\n      \"pmids\": [\"39186665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"X-ray co-crystal structure of PKMYT1 with inhibitor compounds confirmed that binding interactions with residues Asp251 and Tyr121 in the PKMYT1 active site are critical for potency and selectivity over WEE1.\",\n      \"method\": \"X-ray crystallography (co-crystal structure of PKMYT1 with inhibitors), structure-activity relationship analysis\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation (potency/selectivity readouts), single lab\",\n      \"pmids\": [\"39163619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Molecular dynamics simulations and binding free energy calculations identified that the non-conserved gatekeeper residue Thr187 in PKMYT1 (vs Asn376 in WEE1) is a critical determinant of binding selectivity for RP-6306 toward PKMYT1 over WEE1. A water-mediated hydrogen bond between RP-6306 and Gly191 at the PKMYT1 hinge domain is absent in the WEE1 complex.\",\n      \"method\": \"Molecular docking, multiple microsecond-length molecular dynamics simulations, end-point free energy calculations (MM-PBSA/GBSA), per-residue free energy decomposition\",\n      \"journal\": \"Journal of biomolecular structure & dynamics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational analysis only, no experimental mutagenesis validation reported in this paper\",\n      \"pmids\": [\"37345529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PKMYT1 phosphorylates nucleophosmin 1 (NPM1) at the S260 site in osteosarcoma cells. This phosphorylation competitively impairs NPM1 SUMOylation, which in turn interferes with recruitment of BRCA1, RAP80, and RAD51 to double-strand break sites, thereby impairing DSB repair and promoting chemoresistance to cisplatin.\",\n      \"method\": \"Kinome-wide CRISPR screen, transcriptome sequencing, phosphorylation site mapping (NPM1-S260), SUMOylation assay, co-immunoprecipitation for BRCA1/RAP80/RAD51 recruitment, in vitro functional assays, patient-derived clinical specimens\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus multiple mechanistic assays (phosphorylation, SUMOylation, DNA repair factor recruitment), single lab\",\n      \"pmids\": [\"40393983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Meisoindigo (Mei) forms a selective and reversible covalent bond with the Cys301 residue of PKMYT1, acting as a molecular glue that enhances the interaction between PKMYT1 and the E3 ubiquitin ligase TRIM25 by approximately 30-fold. This triggers K48-linked polyubiquitination and proteasomal degradation of PKMYT1.\",\n      \"method\": \"Activity-based protein profiling (ABPP), covalent binding characterization, ubiquitination assays (K48-linkage), co-immunoprecipitation of PKMYT1-TRIM25 interaction, proteasome inhibitor rescue experiments, xenograft model\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ABPP target identification, covalent bond characterization, E3 ligase interaction quantification, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40279509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP49 deubiquitinase interacts with PKMYT1 and limits its ubiquitination and subsequent proteasomal degradation, thereby stabilizing PKMYT1 protein levels in triple-negative breast cancer cells. Overexpression of PKMYT1 rescues malignant phenotypes in USP49-deficient TNBC cells.\",\n      \"method\": \"Co-immunoprecipitation of USP49-PKMYT1, ubiquitination assays, siRNA knockdown, rescue overexpression, cell viability and cell cycle assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with functional rescue experiment, single lab, no in vitro deubiquitinase reconstitution\",\n      \"pmids\": [\"41443044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PKMYT1 inhibition (RP-6306) activates the cGAS-STING pathway and potentiates type I and II interferon signaling, upregulating chemokines CCL5 and CXCL10, thereby enhancing CD8+ T cell infiltration and synergizing with PD-L1 blockade in castration-resistant prostate cancer models.\",\n      \"method\": \"PCa cell lines and syngeneic mouse models, single-cell RNA sequencing, pharmacological PKMYT1 inhibition (RP-6306), flow cytometry for T cell infiltration, cGAS-STING pathway assays, combination with anti-PD-L1\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway activation (cGAS-STING) confirmed in cell lines and syngeneic in vivo models, single lab\",\n      \"pmids\": [\"41617394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PKMYT1 kinase activity increases during anaphase and plays a novel mitotic role distinct from WEE1: chemical inhibition of PKMYT1 induces premature anaphase leading to chromosome segregation errors (chromatin bridges and micronuclei), which activate the cGAS-STING pathway. PKMYT1 also contributes to maintaining spindle assembly checkpoint integrity, as its inhibition promotes mitotic slippage in the presence of anti-microtubule drugs. PKMYT1 thus acts alongside Cyclin B1 degradation to control CDK1-Cyclin B1 activity during metaphase-to-anaphase transition.\",\n      \"method\": \"Chemical inhibition of PKMYT1 with live-cell imaging, chromosome segregation error analysis, micronuclei and chromatin bridge quantification, cGAS-STING pathway activation assays, spindle assembly checkpoint assays with anti-microtubule drugs\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays (live imaging, chromosome segregation, SAC, cGAS-STING), novel mechanistic function established beyond canonical CDK1 phosphorylation\",\n      \"pmids\": [\"42243523\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PKMYT1 directly activates Snail transcription in clear cell renal cell carcinoma by upregulating FoxM1, which represses E-cadherin and activates vimentin expression, thereby inducing EMT. Inhibition of the FoxM1/Snail/EMT pathway reversed PKMYT1-induced metastasis in mice.\",\n      \"method\": \"Exogenous PKMYT1 modulation, in vitro migration/invasion assays, EMT marker western blot, in vivo mouse metastasis model, FoxM1/Snail pathway analysis\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway placement via gain/loss-of-function with in vivo validation, but mechanistic link between PKMYT1 kinase activity and FoxM1 not directly established at molecular level\",\n      \"pmids\": [\"41693360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Allosteric inhibitor P29 binds a previously unknown cryptic allosteric site on PKMYT1, inducing conformational rearrangement of the P-loop and inhibiting PKMYT1 through a mixed ATP-competitive and non-competitive mechanism. A closely related analogue P32 engages PKMYT1 in the ATP binding pocket despite similar structure.\",\n      \"method\": \"X-ray crystallography, kinetic mechanistic assays (ATP competition), cell-based engagement assays, computational structural modeling (AlphaFold2/3, MD simulations)\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of allosteric site plus kinetic assays establishing mechanism of inhibition, single lab with multiple orthogonal structural and biochemical methods\",\n      \"pmids\": [\"42227652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Rb1 loss in breast cancer creates synthetic lethality with combined ATR and PKMYT1 coinhibition. Rb1 deficiency impairs double-strand DNA repair by attenuating homologous recombination and non-homologous end joining, leading to replication fork collapse. Coinhibition of ATR (S-G2 checkpoint) and PKMYT1 (G2-M checkpoint) leads to replication stress, premature mitotic entry, and DNA damage accumulation, with JNK/p38 stress response pathway activation driving apoptosis.\",\n      \"method\": \"In vitro coinhibition in Rb1-deficient vs proficient cell lines, genetic Rb1 knockdown/re-expression, PDX in vivo models, γH2AX/Ki67 biomarker analysis, HR and NHEJ repair assays, proteogenomic pathway analysis\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic manipulation (KD/rescue), multiple orthogonal cellular and in vivo models, mechanistic pathway identified (HR/NHEJ impairment, JNK/p38 activation), single lab with comprehensive validation\",\n      \"pmids\": [\"41442499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"E2F7 transcription factor directly activates PKMYT1 transcription by binding to the PKMYT1 promoter in gastric cancer cells; the E2F7/PKMYT1 axis promotes proliferation and reduces adriamycin (ADM) sensitivity through MAPK pathway activation.\",\n      \"method\": \"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), western blot (ERK/p-ERK), CCK-8 assay, IC50 determination, rescue experiments\",\n      \"journal\": \"Revista de investigacion clinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase reporter confirming transcriptional regulation, MAPK pathway validated by western blot, single lab\",\n      \"pmids\": [\"38253021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PKMYT1 promotes SREBP2-mediated expression of cholesterol biosynthesis enzymes through activating the TNF/TRAF1/AKT pathway in triple-negative breast cancer cells.\",\n      \"method\": \"PKMYT1 knockdown, SREBP2 activity assays, cholesterol biosynthesis enzyme expression analysis, TNF/TRAF1/AKT pathway western blot, atorvastatin sensitivity assays\",\n      \"journal\": \"PeerJ\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pathway placement by knockdown with western blot readouts, no direct kinase-substrate relationship established, single lab\",\n      \"pmids\": [\"39011373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PKMYT1 inhibition with RP-6306 induces premature mitotic entry, causing DNA damage and mitotic catastrophe that triggers PANoptosis (concurrent apoptosis, pyroptosis, and necroptosis) in pancreatic cancer cells.\",\n      \"method\": \"PKMYT1 inhibitor RP-6306, in vitro cell death assays, PANoptosis marker analysis (apoptosis/pyroptosis/necroptosis), in vivo xenograft models, combination with gemcitabine\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell death pathway markers characterized, in vivo validation, mechanistic link from CDK1 hyperactivation to PANoptosis established, single lab\",\n      \"pmids\": [\"40664640\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PKMYT1 is a membrane-associated dual-specificity kinase (Thr14 and Tyr15) that phosphorylates CDK1 to maintain the inhibitory state of CDK1/Cyclin B, thereby restraining G2/M cell cycle entry; beyond this canonical role, PKMYT1 also acts during anaphase to regulate chromosome segregation fidelity and spindle assembly checkpoint integrity, interacts with substrates including NPM1 (phosphorylating S260 to impair DSB repair), binds partners including GSK3β, AKT1, MCRS1, and cyclin A2, is transcriptionally regulated by E2F7, ELF3, and TEAD4, is post-translationally stabilized by USP49 and degraded via TRIM25-mediated ubiquitination, and its inhibition (which causes unscheduled CDK1 activation) is synthetically lethal with CCNE1 amplification and several other oncogenic alterations, while also activating cGAS-STING-dependent innate immune responses.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PKMYT1 is a membrane-associated dual-specificity kinase that phosphorylates CDK1 at both Thr14 and Tyr15 to maintain CDK1/Cyclin B in its inhibitory state and restrain the G2/M transition, distinguishing it from WEE1, which phosphorylates only Tyr15 [#0]. Its kinase activities require intact protein substrates rather than peptides, indicating recognition at the protein level [#1]. In normal cells PKMYT1 acts redundantly with WEE1 to enforce mitotic timing, but this redundancy is lost in oncogenic contexts, creating selective dependencies: PKMYT1 loss is lethal in glioblastoma stem cells [#2], and PKMYT1 kinase activity is synthetically lethal with CCNE1 amplification, where its inhibition drives unscheduled CDK1 activation and premature mitosis during DNA synthesis [#3]. This synthetic-lethal logic extends to low-molecular-weight cyclin E, which stabilizes PKMYT1 to raise CDK1 phosphorylation [#15], to Rb1 loss combined with ATR inhibition [#25], and to combined WEE1/PKMYT1 inhibition, all converging on replication catastrophe and DNA damage [#11]. Beyond CDK1, PKMYT1 has a distinct mitotic role: its activity rises during anaphase to safeguard chromosome segregation and spindle assembly checkpoint integrity, and its inhibition produces segregation errors that activate cGAS-STING innate immune signaling [#22, #21]. The selective inhibitor RP-6306 exploits these dependencies, achieving selectivity over WEE1 through active-site contacts at Asp251 and Tyr121, while an allosteric chemotype engages a cryptic P-loop site [#4, #16, #24]. PKMYT1 also phosphorylates NPM1 at S260 to impair SUMOylation-dependent recruitment of BRCA1, RAP80, and RAD51 to double-strand breaks, linking it to DNA repair and chemoresistance [#18]. PKMYT1 is transcriptionally upregulated by E2F7, ELF3, and TEAD4 [#26, #12, #13], its mRNA is stabilized via m6A reader IGF2BP3 under ALKBH5 control [#9], and its protein level is set by opposing USP49 stabilization and TRIM25-mediated K48 ubiquitination [#20, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established PKMYT1's core biochemical identity, distinguishing it from WEE1 by its dual Thr14/Tyr15 phosphorylation of CDK1 and defining it as a G2/M-restraining kinase.\",\n      \"evidence\": \"In vitro kinase assays with Thr14/Tyr15 phospho-specific readouts\",\n      \"pmids\": [\"29168755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address membrane targeting or substrate-recognition determinants\", \"No structural basis for dual specificity provided\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed PKMYT1 requires protein-level substrates rather than peptides, indicating that substrate context governs its catalysis and complicating peptide-based inhibitor screens.\",\n      \"evidence\": \"In vitro kinase assays comparing peptide vs protein substrates plus MD simulations\",\n      \"pmids\": [\"22189141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for protein-substrate requirement not defined\", \"Single lab, no independent replication noted\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated WEE1/PKMYT1 redundancy in normal cells is broken in tumor cells, establishing the conceptual basis for tumor-selective PKMYT1 dependency.\",\n      \"evidence\": \"Genome-wide CRISPR knockout screens in GSCs vs NSCs with CDK1-Y15 readouts and in vivo validation\",\n      \"pmids\": [\"26673326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular cause of lost redundancy in tumor cells not fully defined\", \"Limited to glioblastoma context\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified CCNE1 amplification as a synthetic-lethal partner and delivered RP-6306 as a selective tool/clinical-grade inhibitor, converting PKMYT1 biology into a therapeutic strategy.\",\n      \"evidence\": \"Genome-scale CRISPR synthetic-lethality screens, structure-based inhibitor design, CDK1 phosphorylation assays, and xenografts\",\n      \"pmids\": [\"35444283\", \"35880755\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full set of co-dependencies beyond CCNE1 not enumerated\", \"MMB-FOXM1 contribution defined only in part\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mapped transcriptional and post-transcriptional control of PKMYT1, showing E2F7, ELF3, TEAD4 drive its expression and m6A reader IGF2BP3 stabilizes its mRNA, explaining how tumors elevate PKMYT1.\",\n      \"evidence\": \"ChIP, luciferase reporters, MeRIP-seq, RNA pulldown with MS, and rescue experiments across cancer models\",\n      \"pmids\": [\"36988891\", \"37729973\", \"38253021\", \"35114989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of each regulator in normal tissue unknown\", \"Direct kinase-pathway coupling for several axes (e.g., metabolic outputs) not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined WEE1 as a pharmacological synthetic-lethal partner, showing combined inhibition drives replication catastrophe and inflammatory STAT1 signaling.\",\n      \"evidence\": \"Inhibitor combinations in ovarian cancer cells and organoids with CDK activity and DNA damage readouts\",\n      \"pmids\": [\"37325550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking replication stress to STAT1 not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the dependency landscape to LMW-E, PLK1 regulation, and TP53/PRKDC context, refining biomarkers and revealing non-canonical effectors beyond CDK1 phosphorylation.\",\n      \"evidence\": \"Genome-wide CRISPR screens, PLK1 phosphorylation analysis, proteogenomics, transgenic and PDX models with RP-6306\",\n      \"pmids\": [\"38570712\", \"39186665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct kinase-substrate relationship for PLK1 regulation not established\", \"Mechanism of LMW-E-specific PKMYT1 stabilization unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed PKMYT1 phosphorylates NPM1-S260 to impair DSB repair, defining a kinase-substrate axis that connects PKMYT1 to homologous-recombination factor recruitment and chemoresistance.\",\n      \"evidence\": \"Kinome CRISPR screen, phospho-site mapping, SUMOylation and Co-IP assays for BRCA1/RAP80/RAD51 recruitment in osteosarcoma\",\n      \"pmids\": [\"40393983\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro reconstitution of NPM1 phosphorylation not shown\", \"Generality beyond osteosarcoma untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established the bidirectional control of PKMYT1 protein stability by USP49 (stabilizing) and TRIM25 (degrading), with meisoindigo acting as a molecular glue at Cys301 to drive K48 ubiquitination.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ABPP covalent target ID, and proteasome rescue with xenograft validation\",\n      \"pmids\": [\"40279509\", \"41443044\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro deubiquitinase reconstitution for USP49 (Low-confidence)\", \"Physiological balance of USP49 vs TRIM25 not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected PKMYT1 inhibition to immune activation and cell death, showing RP-6306 triggers cGAS-STING, interferon signaling, CD8+ T-cell infiltration, and PANoptosis, broadening therapeutic rationale.\",\n      \"evidence\": \"Syngeneic mouse models, scRNA-seq, cGAS-STING and cell-death marker assays, combination with anti-PD-L1 and gemcitabine\",\n      \"pmids\": [\"41617394\", \"40664640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether immune activation requires segregation errors vs replication stress not dissected\", \"Durability of T-cell responses untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a CDK1-restraint-independent anaphase function for PKMYT1 in chromosome segregation and spindle assembly checkpoint integrity, establishing a mitotic role distinct from WEE1.\",\n      \"evidence\": \"Live-cell imaging, chromosome segregation and micronuclei quantification, SAC assays with anti-microtubule drugs, cGAS-STING activation\",\n      \"pmids\": [\"42243523\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Anaphase substrate(s) of PKMYT1 not identified\", \"Mechanism of SAC maintenance unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Characterized a cryptic allosteric site on PKMYT1, revealing a P-loop conformational mechanism distinct from ATP-competitive engagement and expanding inhibitor design space.\",\n      \"evidence\": \"X-ray crystallography, ATP-competition kinetics, cellular engagement assays, and computational modeling\",\n      \"pmids\": [\"42227652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular potency advantage of allosteric vs orthosteric inhibition not established\", \"Selectivity profile across the kinome not fully mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The substrate repertoire and recruitment mechanism underlying PKMYT1's anaphase and DNA-repair roles remain undefined, as does which contexts depend on kinase activity versus scaffolding interactions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No anaphase substrate identified\", \"Several reported binding partners (GSK3\\u03b2, AKT1, MCRS1, cyclin A2) rest on single Co-IPs without reciprocal or reconstitution validation\", \"Membrane-targeting determinants not characterized in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 18]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 18]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 2, 3, 22]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [18, 25]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [21, 22]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDK1\", \"NPM1\", \"TRIM25\", \"USP49\", \"GSK3B\", \"AKT1\", \"MCRS1\", \"CCNA2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":8,"faith_pct":87.5}}