A degron-mimicking molecular glue drives CRBN homo-dimerization and degradation
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): T Cell, Jurkat Cell
SUBMITTER: 
Despoina Kapsitidou
LAB HEAD: Debora Bonenfant
PROVIDER: PXD060544 | Pride | 2025-09-08
REPOSITORIES: Pride
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Project description:In the 1950s the drug thalidomide administered as a sedative to pregnant women led ot the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and ist IMiD derivatives recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4(CRBN)) ubiquitin ligase. Through an unbiased screen we identify the homeobox trranscription factor MEIS2 as an endogenous substrate of CRL4(CRBN). By definition, a specific target of CRL4(CRBN) is expected to have a very low intensity on negative control arrays (E1_E2), (E1_CRBN), (E1_E2_Cdt2), (E1_E2_Cdt2_revlimid), (E1_E2_Cdt2_CSN) or with CRL4(CRBN) in presence of inhibitor (E1_E2_CRBN_revlimid) and high intensity on arrays with CRL4(CRBN) (E1_E2_CRBN) or CRL4(CRBN) in presence of CSN (E1_E2_CRBN_CSN) Accordingly 16 protein microarrays were subjected to in vitro ubiquitylation using the following enzyme combinations: 3x Uba1+UbcH5a; 2x Uba1+UbcH5a+CRL4(DDB2); 3x Uba1+UbcH5a+CRL4(CRBN); 2x Uba1+UbcH5a+CRL4(CRBN)+lenalidomide; 2x Uba1+UbcH5a+CRL4(CRBN)+CSN; 2x Uba1+UbcH5a+CRL4(Cdt2); 1x Uba1+UbcH5a+CRL4(Cdt2)+CSN; 1x Uba1+UbcH5a+CRL4(Cdt2)+lenalidomide
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Project description:Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. Mass spectrometry illuminates a patch of favourably ubiquitinable lysines on one face of in vitro ubiquitinated Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack highlighted by cryo-EM. In vitro ubiquitination coupled with cellular degradation and ubiquitinomics confirm the importance of Lys456 and the ‘ubiquitination zone’ lysines. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination bu UBE2R1, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization.
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Project description:By simultaneously capturing thousands of ubiquitination sites, mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA) mass spectrometry and neural network-based data processing, specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples the number of quantified ubiquitinated peptides, to 70,000 identifications in single LC-MS runs. At the same time, it significantly improves robustness and quantification precision, thus for the first time enabling large-scale ubiquitinomics screens. We further report a novel strategy for capturing targets of deubiquitinases (DUBs), by simultaneously recording the dynamics of the ubiquitinome and the proteome and at high temporal resolution upon enzyme inhibition. Applying it to the anticancer target USP7, we accurately time ubiquitination and consequent changes in abundance of more than 8,000 proteins, thereby mapping novel putative USP7 targets. We show a strikingly fast modulation of the ubiquitinome when inhibiting USP7 and discover hundreds of ubiquitination sites that are regulated within minutes. Yet only a small fraction of those are degraded, thus dissecting the degradative and non-degradative scope of USP7 action. Mapping the impact of different USP7 inhibitors on the ubiquitinome, we observe substantial overlap of their signatures, but also marked differences. Our method enables for the first time to rapidly profile the mode-of-action of candidate drugs targeting DUBs or ubiquitin ligases at an unprecedented depth.
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Project description:The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their anti-tumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the anti-myeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide. Gene expression changes induced by lenalidomide were dramatically suppressed in the presence of CRBN depletion further demonstrating that CRBN is required for lenalidomide activity. Downstream targets of CRBN include interferon regulatory factor 4 (IRF4) previously reported to also be a target of lenalidomide. Patients exposed to and putatively resistant to lenalidomide had lower CRBN levels in paired samples before and after therapy. In summary, CRBN is an essential requirement for IMiD activity, and a possible biomarker for the clinical assessment of anti-myeloma efficacy. We included 15 samples from multiple myeloma cell lines.
2011-09-30 | E-GEOD-31421 | biostudies-arrayexpress
Project description:Lenalidomide is a therapeutically active compound that binds to E3 ubiquitin ligase recruiter cereblon (CRBN) and induces cytotoxicity. We have identified eukaryotic translation initiation factor 2 subunit C2 (EIF2C2) as a new member of CRBN-downstream binding protein that plays an important role in microRNA (miRNA) maturation and function. The treatment of immunomodulatory drug (IMiD)-sensitive multiple myeloma (MM) cells with lenalidomide altered the steady-state levels of CRBN, EIF2C2 and miRNAs and induced apoptosis. However, although the treatment of IMiD-resistant MM cells with lenalidomide altered the steady-state levels of CRBN, EIF2C2 and miRNAs, but did not massively induce apoptosis. In contrast, silencing of EIF2C2 with its small hairpin RNA significantly altered the levels of miRNAs and induced apoptosis regardless of whether those cells are sensitive or resistant to IMiDs. Therefore, EIF2C2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. To find the role of EIF2C2 in MM cell growth, OCI-My5 cell lines My5/LV and My5/CRBN, with low and high CRBN expression, respectively, were treated 12 different ways. The steady-state levels of miRNAs between (1) My5/LV, EIF2C2-shRNA-treated My5/LV and EIF2C2-cDNA-treated My5/LV cells, (2) My5/CRBN, EIF2C2-shRNA-treated My5/CRBN and EIF2C2-cDNA-treated My5/CRBN cells, (3) My5/LV cells, My5/LV cells treated with 10 µM lenalidomide for 72 hours or 120 hours, and EIF2C2-cDNA-treated My5/LV cells treated with 10 µM lenalidomide for 72 hours, and (4) My5/CRBN cells, My5/CRBN cells treated with 10 µM lenalidomide for 72 hours or 120 hours, and EIF2C2-cDNA-treated My5/CRBN cells treated with 10 µM lenalidomide for 72 hours, were compared in this array.
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