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Asymmetric Engagement of Dimeric CRL3/KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

ABSTRACT: UM171 is a potent agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase (CRL3) complex, UM171 promotes the degradation of the LSD1-CoREST corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2 to promote corepressor degradation. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an asymmetric assembly, in which a single UM171 molecule enables a pair of KELCH-repeat propeller domains to recruit the HDAC1 catalytic domain. One KBTBD4 propeller partially masks the rim of the HDAC1 active site, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a distinct interface. The overall CoREST-HDAC1/2-KBTBD4 interaction is further buttressed by an endogenous cofactor, inositol hexakisphosphate, which acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces is demonstrated by base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, we reveal how the cooperativity offered by a dimeric CRL3 E3 can be leveraged by a small molecule degrader.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000096456 | MassIVE | Mon Nov 18 13:06:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal, a process that is tightly controlled by epigenetic regulation. By co opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2 to promote the degradation of select corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KELCH-repeat propeller domains from the structurally plastic KBTBD4 dimer to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

Project description:Epigenetic regulation of gene expression by histone modification has emerged as a major facet of physiologic and disease processes. As a result, there has been intense interest in developing epigenetic therapies leading to the discovery of small molecule agents that target proteins involved in histone modification. Several histone deacetylase (HDAC) inhibitors are now approved drugs for a specialized group of hematologic malignancies but not yet for a wider range of cancer types including solid tumors. One of the conceptual challenges in targeting HDACs is that even selective class I HDAC inhibitors likely impact these deacetylase activities indiscriminately across a range of distinct HDAC-containing multiprotein complexes. Such broad cellular effects may result in a narrow therapeutic window between disease efficacy and toxicity. Among HDAC complexes, the CoREST complex, which includes HDAC1 or its close paralog HDAC2, the scaffolding protein CoREST, and lysine specific demethylase 1 (LSD1) has attracted special interest. Here we report corin2, designed to dually inhibit the CoREST complex major enzymatic activities, lysine specific demethylase 1 (LSD1) and HDACs 1/2. Corin2 is a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin2 selectively targets the CoREST complex and shows more sustained inhibition of the CoREST complex HDAC activity than entinostat. Cell-based experiments demonstrate that corin2 exhibits a superior anti-proliferative profile against several melanoma lines compared to its parent monofunctional HDAC and LSD1 inhibitors (alone or in combination) but is less toxic to non-cancerous primary human melanocytes. Transcriptomics analysis shows that corin2 is a more powerful inducer of tumor suppressor genes relative to the parent HDAC and LSD1 compounds (alone or in combination). Genetic knockdown of CoREST or LSD1 in cancer cell lines abolishes the differences in potency of corin2 vs. entinostat, suggesting that corin2's favorable pharmacologic effects rely on an intact CoREST complex. Corin2 was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that selectively target particular epigenetic regulatory complexes and offer unique therapeutic opportunities. We characterized the gene expression changes uniquely related to dual inhibition of HDAC1/2 and LSD1 as part of the CoREST complex to identify genes associated with our dual inhibitor's (corin2) antiproliferative mechanism of action in melanoma cells. Transcriptomics analysis shows that corin2 is a more powerful inducer of tumor suppressor genes relative to the parent HDAC and LSD1 compounds (alone or in combination).

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Asymmetric Engagement of Dimeric CRL3/KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

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