{"gene":"KBTBD4","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2021,"finding":"KBTBD4 acts as the substrate-recognition subunit of a CRL3 (CULLIN3-RING E3 ubiquitin ligase) complex; UM171 potentiates CRL3-KBTBD4 activity to target components of the LSD1/RCOR1 (CoREST) corepressor complex for proteasomal degradation, thereby re-establishing H3K4me2 and H3K27ac epigenetic marks in HSCs.","method":"Co-immunoprecipitation, proteasome inhibitor assays, ubiquitylation assays, epigenetic mark quantification (ChIP/mass spec), pharmacological perturbation with UM171","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying complex components, functional degradation assays with proteasome inhibition, independently replicated across multiple subsequent studies","pmids":["33417871"],"is_preprint":false},{"year":2022,"finding":"Indel (in-frame insertion) mutations in the Kelch domain of KBTBD4 found in group 3/4 medulloblastoma confer gain-of-function neomorphic activity, enabling mutant KBTBD4 to recruit CoREST (RCOR1) as a neo-substrate for ubiquitylation and proteasomal degradation, thereby reprogramming epigenetic/transcriptional programs to promote cancer cell stemness.","method":"Ubiquitylation assays in cells expressing mutant KBTBD4, co-immunoprecipitation of CoREST with mutant vs. wild-type KBTBD4, proteasome inhibitor rescue, transcriptional/RNA-seq analysis of >200 medulloblastoma samples","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in-cell ubiquitylation assay, independently replicated by multiple labs","pmids":["35379950"],"is_preprint":false},{"year":2023,"finding":"The CUL3-KBTBD4 E3 ligase complex, activated by UM171, recognizes substrates through a conserved motif within the ELM2 (EGL-27 and MTA1 homology 2) domain; critical amino acid sites in the N-terminus of the ELM2 domain are essential for UM171-mediated degradation. Additional UM171 targets beyond RCOR1/LSD1 include RCOR3, RREB1, ZNF217, and MIER2.","method":"Global proteomics to map UM171-targeted proteome, mutagenesis of conserved ELM2 domain residues, degradation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics plus site-directed mutagenesis in a single lab, mechanistically defines substrate recognition motif","pmids":["36997086"],"is_preprint":false},{"year":2024,"finding":"CRL3-KBTBD4 activated by UM171 also targets chromatin-bound MYC for degradation in HSCs, controlling cell cycle entry; experimental elevation of MYC levels despite CoREST1 degradation specifically compromises reconstitution activity and multipotency of UM171-treated HSCs.","method":"Genetic manipulation to increase MYC levels in UM171-treated HSCs, reconstitution/multipotency assays, comparison with CoREST1 degradation readout","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis experiment (MYC overexpression rescues phenotype despite CoREST degradation), single lab with functional cellular readout","pmids":["38207291"],"is_preprint":false},{"year":2024,"finding":"KBTBD4 cancer hotspot mutations promote CoREST degradation by directly engaging HDAC1/2 as the neomorphic target of the mutant substrate receptor; cryo-EM of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST shows a KBTBD4 homodimer asymmetrically engaging HDAC1 via two KELCH-repeat β-propeller domains, with the mutation inserting a bulky side chain into the HDAC1 active site pocket to stabilize the interface. HDAC1/2 inhibitors block the mutant KBTBD4-HDAC1 interface and proliferation of KBTBD4-mutant medulloblastoma cells.","method":"Cryo-EM structure determination, deep mutational scanning of KBTBD4 cancer hotspot, co-immunoprecipitation, in-cell ubiquitylation assays, HDAC inhibitor proliferation assays","journal":"bioRxiv (preprint)","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus deep mutational scanning plus functional assays; results subsequently published in Nature (PMID:39939763)","pmids":["38798357"],"is_preprint":true},{"year":2025,"finding":"Cryo-EM structures of wild-type and mutant KBTBD4 bound to HDAC1/2 and CoREST reveal that UM171 acts as a bona fide molecular glue binding across the ternary KBTBD4-HDAC1-CoREST interface; both UM171-induced and mutation-induced complexes share the same asymmetric 2:1/2:2 KBTBD4-HDAC architecture requiring both Kelch domains of the KBTBD4 dimer. The indel cancer mutation reshapes the same KBTBD4 surface as UM171, representing a natural mimic of a molecular glue. An endogenous cofactor inositol hexakisphosphate (IP6) makes direct contacts with KBTBD4 and acts as a second molecular glue to buttress the interaction.","method":"Cryo-EM at 2.7–3.3 Å resolution of multiple KBTBD4-HDAC2 and KBTBD4-HDAC2-CoREST1 complexes, structure-activity relationship (SAR) analysis of UM171 analogs, in situ base-editor scanning of KBTBD4 and HDAC1","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple cryo-EM structures at high resolution with functional validation by base-editor scanning and SAR; two independent papers (PMID:40175372 and PMID:39939763) with convergent structural findings","pmids":["40175372","39939763"],"is_preprint":false},{"year":2024,"finding":"UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2; cryo-EM of dimeric KBTBD4 with UM171 and LSD1-HDAC1-CoREST shows an asymmetric assembly where a single UM171 molecule enables both KBTBD4 KELCH propeller domains to clamp the HDAC1 catalytic domain, with one propeller partially masking the HDAC1 active-site rim used by UM171 to extend the interface, and the other propeller cooperatively strengthening HDAC1 binding at a distinct interface. IP6 acts as a second molecular glue making direct contacts with KBTBD4.","method":"Cryo-EM structure determination, proteomics, chemical inhibitor studies, in situ base-editor scanning of KBTBD4 and HDAC1","journal":"bioRxiv (preprint)","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure of ternary complex with functional validation; convergent with published peer-reviewed structures","pmids":["38798619"],"is_preprint":true}],"current_model":"KBTBD4 is the substrate-recognition subunit of a CUL3-RING E3 ubiquitin ligase complex (CRL3-KBTBD4) that, in its wild-type form, uses its KELCH-repeat β-propeller domain to recruit HDAC1/2-containing CoREST corepressor complexes for proteasomal degradation in response to the molecular glue UM171, which bridges the KBTBD4 dimer asymmetrically onto the HDAC1/2 catalytic domain (with IP6 as a cooperative second glue); recurrent in-frame insertion mutations at a hotspot in the KELCH domain found in medulloblastoma confer neomorphic gain-of-function by reshaping the same KBTBD4 surface to constitutively engage HDAC1/2 and drive aberrant CoREST degradation and epigenetic reprogramming, while in normal HSC biology CRL3-KBTBD4 also targets MYC to prevent excessive HSC activation."},"narrative":{"mechanistic_narrative":"KBTBD4 is the substrate-recognition subunit of a CULLIN3-RING E3 ubiquitin ligase (CRL3-KBTBD4) that controls epigenetic state by directing CoREST corepressor components for proteasomal degradation [PMID:33417871]. Through its KELCH-repeat β-propeller domain, KBTBD4 engages the HDAC1/2-LSD1-CoREST complex, recognizing substrates via a conserved motif in the ELM2 domain whose N-terminal residues are required for degradation; targets include RCOR1/LSD1 as well as RCOR3, RREB1, ZNF217, and MIER2 [PMID:33417871, PMID:36997086]. The small molecule UM171 functions as a molecular glue that bridges a KBTBD4 homodimer asymmetrically onto the HDAC1/2 catalytic domain, with both KELCH propellers clamping HDAC1 and the endogenous cofactor inositol hexakisphosphate (IP6) acting as a cooperative second glue that buttresses the interface [PMID:40175372, PMID:39939763, PMID:38798619]. Recurrent in-frame insertion (indel) mutations at a KELCH-domain hotspot in group 3/4 medulloblastoma confer neomorphic gain-of-function: the mutation reshapes the same KBTBD4 surface engaged by UM171, inserting a bulky side chain into the HDAC1 active-site pocket to stabilize the interface and constitutively recruit HDAC1/2 and CoREST as neo-substrates, driving aberrant CoREST degradation, epigenetic reprogramming, and cancer cell stemness; HDAC1/2 inhibitors block this interface and the proliferation of mutant cells [PMID:35379950, PMID:38798357, PMID:40175372, PMID:39939763]. Beyond chromatin regulators, UM171-activated CRL3-KBTBD4 also targets chromatin-bound MYC for degradation in hematopoietic stem cells, restraining cell cycle entry to preserve reconstitution capacity and multipotency [PMID:38207291].","teleology":[{"year":2021,"claim":"Established that KBTBD4 is the substrate receptor of a CRL3 E3 ligase and links it to a pharmacological mechanism by showing UM171 potentiates degradation of CoREST corepressor components, explaining how a small molecule reprograms HSC epigenetics.","evidence":"Co-IP, ubiquitylation and proteasome-inhibitor assays with UM171 perturbation and epigenetic mark quantification in HSCs","pmids":["33417871"],"confidence":"High","gaps":["Structural basis of UM171-induced substrate engagement not defined","Full substrate repertoire not mapped"]},{"year":2022,"claim":"Showed that medulloblastoma KELCH-domain indel mutations are neomorphic gain-of-function, converting CoREST/RCOR1 into a neo-substrate and linking KBTBD4 directly to oncogenic epigenetic reprogramming.","evidence":"In-cell ubiquitylation, mutant vs. wild-type Co-IP, proteasome-inhibitor rescue, and RNA-seq across >200 medulloblastoma samples","pmids":["35379950"],"confidence":"High","gaps":["Molecular basis by which the mutation reshapes substrate binding unresolved","Whether HDAC1/2 rather than CoREST is the direct neo-target not yet distinguished"]},{"year":2023,"claim":"Defined the substrate recognition determinant by mapping the targeted proteome and identifying a conserved ELM2-domain motif required for degradation, broadening the known target set beyond RCOR1/LSD1.","evidence":"Global proteomics of the UM171-targeted proteome with site-directed mutagenesis of ELM2 residues and degradation assays in a single lab","pmids":["36997086"],"confidence":"Medium","gaps":["Single-lab proteomics not independently replicated","Functional significance of each newly named target not individually validated"]},{"year":2024,"claim":"Extended CRL3-KBTBD4 function to MYC turnover, showing UM171-activated degradation of chromatin-bound MYC controls HSC cell-cycle entry independently of CoREST degradation.","evidence":"MYC overexpression epistasis in UM171-treated HSCs with reconstitution and multipotency assays, single lab","pmids":["38207291"],"confidence":"Medium","gaps":["Direct ubiquitylation of MYC by CRL3-KBTBD4 not structurally demonstrated","Relationship between MYC and CoREST target selection unclear"]},{"year":2024,"claim":"Resolved the structural neomorphic mechanism: cryo-EM and deep mutational scanning showed mutant KBTBD4 homodimers asymmetrically clamp HDAC1 via two KELCH propellers, with the inserted side chain entering the HDAC1 active-site pocket, identifying HDAC1/2 as the direct neo-target and HDAC inhibitors as a therapeutic vulnerability.","evidence":"Cryo-EM of two cancer mutants bound to LSD1-HDAC1-CoREST, deep mutational scanning, in-cell ubiquitylation, and HDAC-inhibitor proliferation assays (preprint, later published in Nature)","pmids":["38798357"],"confidence":"High","gaps":["In vivo efficacy of HDAC inhibition against KBTBD4-mutant tumors not established","Generality across all hotspot indel variants not fully mapped"]},{"year":2025,"claim":"Unified the small-molecule and genetic mechanisms by demonstrating UM171 is a bona fide molecular glue acting across the KBTBD4-HDAC1-CoREST interface and that the indel mutation is a natural molecular-glue mimic reshaping the same surface, with IP6 as a cooperative endogenous second glue.","evidence":"High-resolution cryo-EM (2.7–3.3 Å) of multiple wild-type and mutant KBTBD4-HDAC2(-CoREST1) complexes, UM171 analog SAR, and in situ base-editor scanning of KBTBD4 and HDAC1","pmids":["40175372","39939763","38798619"],"confidence":"High","gaps":["Physiological role and regulation of IP6 cofactor occupancy not defined","How the same surface discriminates among the multiple ELM2-motif substrates remains open"]},{"year":null,"claim":"It remains unknown how substrate selectivity is governed across the full KBTBD4 target set and whether endogenous signals other than UM171 or IP6 modulate CRL3-KBTBD4 activity in vivo.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No mechanism connecting ELM2-motif recognition to substrate prioritization","Endogenous physiological trigger for wild-type CRL3-KBTBD4 activation undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,4]}],"complexes":["CRL3-KBTBD4 (CUL3-RING E3 ubiquitin ligase)"],"partners":["CUL3","HDAC1","HDAC2","RCOR1","LSD1","MYC"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NVX7","full_name":"Kelch repeat and BTB domain-containing protein 4","aliases":["BTB and kelch domain-containing protein 4"],"length_aa":534,"mass_kda":59.9,"function":"Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex which targets CoREST corepressor complex components RCOR1, KDM1A/LSD1 and HDAC2 for proteasomal degradation (PubMed:33417871). RCOR1 is likely to be the primary target while degradation of KDM1A and HDAC2 is likely due to their association with RCOR1 (PubMed:33417871). Also targets RCOR3, MIER2 and MIER3 for proteasomal degradation as well as associated proteins ZNF217 and RREB1 (PubMed:36997086). Degradation is dependent on the presence of an ELM2 domain in the target proteins (PubMed:36997086)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9NVX7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KBTBD4","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KBTBD4","total_profiled":1310},"omim":[{"mim_id":"617645","title":"KELCH REPEAT- AND BTB/POZ DOMAIN-CONTAINING PROTEIN 4; KBTBD4","url":"https://www.omim.org/entry/617645"},{"mim_id":"155255","title":"MEDULLOBLASTOMA; MDB","url":"https://www.omim.org/entry/155255"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KBTBD4"},"hgnc":{"alias_symbol":["FLJ10450","HSPC252"],"prev_symbol":["BKLHD4"]},"alphafold":{"accession":"Q9NVX7","domains":[{"cath_id":"3.30.710.10","chopping":"26-141","consensus_level":"high","plddt":89.7566,"start":26,"end":141},{"cath_id":"1.25.40.420","chopping":"163-235","consensus_level":"high","plddt":88.5482,"start":163,"end":235},{"cath_id":"2.120.10.80","chopping":"240-258_280-518","consensus_level":"high","plddt":76.9051,"start":240,"end":518}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NVX7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NVX7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NVX7-F1-predicted_aligned_error_v6.png","plddt_mean":77.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KBTBD4","jax_strain_url":"https://www.jax.org/strain/search?query=KBTBD4"},"sequence":{"accession":"Q9NVX7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NVX7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NVX7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NVX7"}},"corpus_meta":[{"pmid":"33417871","id":"PMC_33417871","title":"UM171 Preserves Epigenetic Marks that Are Reduced in Ex Vivo Culture of Human HSCs via Potentiation of the CLR3-KBTBD4 Complex.","date":"2021","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/33417871","citation_count":78,"is_preprint":false},{"pmid":"39939763","id":"PMC_39939763","title":"Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.","date":"2025","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/39939763","citation_count":30,"is_preprint":false},{"pmid":"35379950","id":"PMC_35379950","title":"Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation.","date":"2022","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/35379950","citation_count":25,"is_preprint":false},{"pmid":"38207291","id":"PMC_38207291","title":"KBTBD4-mediated reduction of MYC is critical for hematopoietic stem cell expansion upon UM171 treatment.","date":"2024","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/38207291","citation_count":11,"is_preprint":false},{"pmid":"40175372","id":"PMC_40175372","title":"Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40175372","citation_count":8,"is_preprint":false},{"pmid":"36997086","id":"PMC_36997086","title":"The stem cell-supporting small molecule UM171 triggers Cul3-KBTBD4-mediated degradation of ELM2 domain-harboring proteins.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36997086","citation_count":8,"is_preprint":false},{"pmid":"35000018","id":"PMC_35000018","title":"Role of proliferative marker index and KBTBD4 mutation in the pathological diagnosis of pineal parenchymal tumors.","date":"2022","source":"Brain tumor pathology","url":"https://pubmed.ncbi.nlm.nih.gov/35000018","citation_count":3,"is_preprint":false},{"pmid":"31403685","id":"PMC_31403685","title":"Lack of KBTBD4 Mutations in Molecularly Classified Brazilian Medulloblastomas.","date":"2019","source":"Journal of neuropathology and experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/31403685","citation_count":3,"is_preprint":false},{"pmid":"38798357","id":"PMC_38798357","title":"KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38798357","citation_count":3,"is_preprint":false},{"pmid":"38798619","id":"PMC_38798619","title":"Asymmetric Engagement of Dimeric CRL3 KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38798619","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.23.630090","title":"YDS-GlueFold: Surpassing AlphaFold 3-Type Models for Molecular Glue-Induced Ternary Complex Prediction","date":"2024-12-23","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.23.630090","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8208,"output_tokens":2424,"usd":0.030492,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9452,"output_tokens":2983,"usd":0.060917,"stage2_stop_reason":"end_turn"},"total_usd":0.091409,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2021,\n      \"finding\": \"KBTBD4 acts as the substrate-recognition subunit of a CRL3 (CULLIN3-RING E3 ubiquitin ligase) complex; UM171 potentiates CRL3-KBTBD4 activity to target components of the LSD1/RCOR1 (CoREST) corepressor complex for proteasomal degradation, thereby re-establishing H3K4me2 and H3K27ac epigenetic marks in HSCs.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibitor assays, ubiquitylation assays, epigenetic mark quantification (ChIP/mass spec), pharmacological perturbation with UM171\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying complex components, functional degradation assays with proteasome inhibition, independently replicated across multiple subsequent studies\",\n      \"pmids\": [\"33417871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Indel (in-frame insertion) mutations in the Kelch domain of KBTBD4 found in group 3/4 medulloblastoma confer gain-of-function neomorphic activity, enabling mutant KBTBD4 to recruit CoREST (RCOR1) as a neo-substrate for ubiquitylation and proteasomal degradation, thereby reprogramming epigenetic/transcriptional programs to promote cancer cell stemness.\",\n      \"method\": \"Ubiquitylation assays in cells expressing mutant KBTBD4, co-immunoprecipitation of CoREST with mutant vs. wild-type KBTBD4, proteasome inhibitor rescue, transcriptional/RNA-seq analysis of >200 medulloblastoma samples\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in-cell ubiquitylation assay, independently replicated by multiple labs\",\n      \"pmids\": [\"35379950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The CUL3-KBTBD4 E3 ligase complex, activated by UM171, recognizes substrates through a conserved motif within the ELM2 (EGL-27 and MTA1 homology 2) domain; critical amino acid sites in the N-terminus of the ELM2 domain are essential for UM171-mediated degradation. Additional UM171 targets beyond RCOR1/LSD1 include RCOR3, RREB1, ZNF217, and MIER2.\",\n      \"method\": \"Global proteomics to map UM171-targeted proteome, mutagenesis of conserved ELM2 domain residues, degradation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics plus site-directed mutagenesis in a single lab, mechanistically defines substrate recognition motif\",\n      \"pmids\": [\"36997086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CRL3-KBTBD4 activated by UM171 also targets chromatin-bound MYC for degradation in HSCs, controlling cell cycle entry; experimental elevation of MYC levels despite CoREST1 degradation specifically compromises reconstitution activity and multipotency of UM171-treated HSCs.\",\n      \"method\": \"Genetic manipulation to increase MYC levels in UM171-treated HSCs, reconstitution/multipotency assays, comparison with CoREST1 degradation readout\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis experiment (MYC overexpression rescues phenotype despite CoREST degradation), single lab with functional cellular readout\",\n      \"pmids\": [\"38207291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KBTBD4 cancer hotspot mutations promote CoREST degradation by directly engaging HDAC1/2 as the neomorphic target of the mutant substrate receptor; cryo-EM of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST shows a KBTBD4 homodimer asymmetrically engaging HDAC1 via two KELCH-repeat β-propeller domains, with the mutation inserting a bulky side chain into the HDAC1 active site pocket to stabilize the interface. HDAC1/2 inhibitors block the mutant KBTBD4-HDAC1 interface and proliferation of KBTBD4-mutant medulloblastoma cells.\",\n      \"method\": \"Cryo-EM structure determination, deep mutational scanning of KBTBD4 cancer hotspot, co-immunoprecipitation, in-cell ubiquitylation assays, HDAC inhibitor proliferation assays\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus deep mutational scanning plus functional assays; results subsequently published in Nature (PMID:39939763)\",\n      \"pmids\": [\"38798357\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of wild-type and mutant KBTBD4 bound to HDAC1/2 and CoREST reveal that UM171 acts as a bona fide molecular glue binding across the ternary KBTBD4-HDAC1-CoREST interface; both UM171-induced and mutation-induced complexes share the same asymmetric 2:1/2:2 KBTBD4-HDAC architecture requiring both Kelch domains of the KBTBD4 dimer. The indel cancer mutation reshapes the same KBTBD4 surface as UM171, representing a natural mimic of a molecular glue. An endogenous cofactor inositol hexakisphosphate (IP6) makes direct contacts with KBTBD4 and acts as a second molecular glue to buttress the interaction.\",\n      \"method\": \"Cryo-EM at 2.7–3.3 Å resolution of multiple KBTBD4-HDAC2 and KBTBD4-HDAC2-CoREST1 complexes, structure-activity relationship (SAR) analysis of UM171 analogs, in situ base-editor scanning of KBTBD4 and HDAC1\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple cryo-EM structures at high resolution with functional validation by base-editor scanning and SAR; two independent papers (PMID:40175372 and PMID:39939763) with convergent structural findings\",\n      \"pmids\": [\"40175372\", \"39939763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2; cryo-EM of dimeric KBTBD4 with UM171 and LSD1-HDAC1-CoREST shows an asymmetric assembly where a single UM171 molecule enables both KBTBD4 KELCH propeller domains to clamp the HDAC1 catalytic domain, with one propeller partially masking the HDAC1 active-site rim used by UM171 to extend the interface, and the other propeller cooperatively strengthening HDAC1 binding at a distinct interface. IP6 acts as a second molecular glue making direct contacts with KBTBD4.\",\n      \"method\": \"Cryo-EM structure determination, proteomics, chemical inhibitor studies, in situ base-editor scanning of KBTBD4 and HDAC1\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure of ternary complex with functional validation; convergent with published peer-reviewed structures\",\n      \"pmids\": [\"38798619\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"KBTBD4 is the substrate-recognition subunit of a CUL3-RING E3 ubiquitin ligase complex (CRL3-KBTBD4) that, in its wild-type form, uses its KELCH-repeat β-propeller domain to recruit HDAC1/2-containing CoREST corepressor complexes for proteasomal degradation in response to the molecular glue UM171, which bridges the KBTBD4 dimer asymmetrically onto the HDAC1/2 catalytic domain (with IP6 as a cooperative second glue); recurrent in-frame insertion mutations at a hotspot in the KELCH domain found in medulloblastoma confer neomorphic gain-of-function by reshaping the same KBTBD4 surface to constitutively engage HDAC1/2 and drive aberrant CoREST degradation and epigenetic reprogramming, while in normal HSC biology CRL3-KBTBD4 also targets MYC to prevent excessive HSC activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KBTBD4 is the substrate-recognition subunit of a CULLIN3-RING E3 ubiquitin ligase (CRL3-KBTBD4) that controls epigenetic state by directing CoREST corepressor components for proteasomal degradation [#0]. Through its KELCH-repeat \\u03b2-propeller domain, KBTBD4 engages the HDAC1/2-LSD1-CoREST complex, recognizing substrates via a conserved motif in the ELM2 domain whose N-terminal residues are required for degradation; targets include RCOR1/LSD1 as well as RCOR3, RREB1, ZNF217, and MIER2 [#0, #2]. The small molecule UM171 functions as a molecular glue that bridges a KBTBD4 homodimer asymmetrically onto the HDAC1/2 catalytic domain, with both KELCH propellers clamping HDAC1 and the endogenous cofactor inositol hexakisphosphate (IP6) acting as a cooperative second glue that buttresses the interface [#5, #6]. Recurrent in-frame insertion (indel) mutations at a KELCH-domain hotspot in group 3/4 medulloblastoma confer neomorphic gain-of-function: the mutation reshapes the same KBTBD4 surface engaged by UM171, inserting a bulky side chain into the HDAC1 active-site pocket to stabilize the interface and constitutively recruit HDAC1/2 and CoREST as neo-substrates, driving aberrant CoREST degradation, epigenetic reprogramming, and cancer cell stemness; HDAC1/2 inhibitors block this interface and the proliferation of mutant cells [#1, #4, #5]. Beyond chromatin regulators, UM171-activated CRL3-KBTBD4 also targets chromatin-bound MYC for degradation in hematopoietic stem cells, restraining cell cycle entry to preserve reconstitution capacity and multipotency [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Established that KBTBD4 is the substrate receptor of a CRL3 E3 ligase and links it to a pharmacological mechanism by showing UM171 potentiates degradation of CoREST corepressor components, explaining how a small molecule reprograms HSC epigenetics.\",\n      \"evidence\": \"Co-IP, ubiquitylation and proteasome-inhibitor assays with UM171 perturbation and epigenetic mark quantification in HSCs\",\n      \"pmids\": [\"33417871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of UM171-induced substrate engagement not defined\", \"Full substrate repertoire not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed that medulloblastoma KELCH-domain indel mutations are neomorphic gain-of-function, converting CoREST/RCOR1 into a neo-substrate and linking KBTBD4 directly to oncogenic epigenetic reprogramming.\",\n      \"evidence\": \"In-cell ubiquitylation, mutant vs. wild-type Co-IP, proteasome-inhibitor rescue, and RNA-seq across >200 medulloblastoma samples\",\n      \"pmids\": [\"35379950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis by which the mutation reshapes substrate binding unresolved\", \"Whether HDAC1/2 rather than CoREST is the direct neo-target not yet distinguished\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined the substrate recognition determinant by mapping the targeted proteome and identifying a conserved ELM2-domain motif required for degradation, broadening the known target set beyond RCOR1/LSD1.\",\n      \"evidence\": \"Global proteomics of the UM171-targeted proteome with site-directed mutagenesis of ELM2 residues and degradation assays in a single lab\",\n      \"pmids\": [\"36997086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab proteomics not independently replicated\", \"Functional significance of each newly named target not individually validated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended CRL3-KBTBD4 function to MYC turnover, showing UM171-activated degradation of chromatin-bound MYC controls HSC cell-cycle entry independently of CoREST degradation.\",\n      \"evidence\": \"MYC overexpression epistasis in UM171-treated HSCs with reconstitution and multipotency assays, single lab\",\n      \"pmids\": [\"38207291\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitylation of MYC by CRL3-KBTBD4 not structurally demonstrated\", \"Relationship between MYC and CoREST target selection unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved the structural neomorphic mechanism: cryo-EM and deep mutational scanning showed mutant KBTBD4 homodimers asymmetrically clamp HDAC1 via two KELCH propellers, with the inserted side chain entering the HDAC1 active-site pocket, identifying HDAC1/2 as the direct neo-target and HDAC inhibitors as a therapeutic vulnerability.\",\n      \"evidence\": \"Cryo-EM of two cancer mutants bound to LSD1-HDAC1-CoREST, deep mutational scanning, in-cell ubiquitylation, and HDAC-inhibitor proliferation assays (preprint, later published in Nature)\",\n      \"pmids\": [\"38798357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy of HDAC inhibition against KBTBD4-mutant tumors not established\", \"Generality across all hotspot indel variants not fully mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Unified the small-molecule and genetic mechanisms by demonstrating UM171 is a bona fide molecular glue acting across the KBTBD4-HDAC1-CoREST interface and that the indel mutation is a natural molecular-glue mimic reshaping the same surface, with IP6 as a cooperative endogenous second glue.\",\n      \"evidence\": \"High-resolution cryo-EM (2.7\\u20133.3 \\u00c5) of multiple wild-type and mutant KBTBD4-HDAC2(-CoREST1) complexes, UM171 analog SAR, and in situ base-editor scanning of KBTBD4 and HDAC1\",\n      \"pmids\": [\"40175372\", \"39939763\", \"38798619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological role and regulation of IP6 cofactor occupancy not defined\", \"How the same surface discriminates among the multiple ELM2-motif substrates remains open\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how substrate selectivity is governed across the full KBTBD4 target set and whether endogenous signals other than UM171 or IP6 modulate CRL3-KBTBD4 activity in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mechanism connecting ELM2-motif recognition to substrate prioritization\", \"Endogenous physiological trigger for wild-type CRL3-KBTBD4 activation undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"complexes\": [\n      \"CRL3-KBTBD4 (CUL3-RING E3 ubiquitin ligase)\"\n    ],\n    \"partners\": [\n      \"CUL3\",\n      \"HDAC1\",\n      \"HDAC2\",\n      \"RCOR1\",\n      \"LSD1\",\n      \"MYC\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}