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Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

Abstract

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined ‘functional hotspots’. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

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Fig. 1: Quantitative and qualitative differences in degrader resistance.
Fig. 2: Deep mutational scanning locates functional hotspots of general relevance in the degrader binding pocket.
Fig. 3: Functional VHL hotspots identified by DMS show neo-substrate dependent resistance and sensitivity to PROTAC treatment.
Fig. 4: VHLP71 is a functional hotspot for degrader specific resistance.
Fig. 5: Functional CRBN hotspots show degrader selectivity and are mutated in refractory multiple myeloma patients.

Data availability

Raw and analysed mutational scanning and hybrid-capture datasets (Figs. 15 and Supplementary Figs 1,35) are available in the Gene Expression Omnibus database under accession code GSE198280. For their analysis the human reference genome (hg38/GRCh38 assembly, GenBank accession 883148) was used. Atomic coordinates and structure factors for the new protein structure VCB–AT7–Brd4BD2 is available at the PDB under accession 7ZNT. All data generated and analysed in this study are included in this published article, its Supplementary Information, the mentioned databases or are available from the corresponding authors upon request. Source data are provided with this paper.

Code availability

All code used for analysis of the experimental data is available at https://github.com/GWinterLab/TPDR.

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Acknowledgements

We thank the Biomedical Sequencing Facility at CeMM for assistance with next-generation sequencing, C. Crowe (Ciulli laboratory) for the gift of purified BET-bromodomain protein, the Diamond Light Source for beamtime (BAG proposal MX14980-13) and J. Bigenzahn (Superti-Furga laboratory) for the gift of plasmids. CeMM and the Winter laboratory are supported by the Austrian Academy of Sciences. The Winter laboratory is further supported by funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement 851478), as well as by funding from the Austrian Science Fund (FWF, projects P32125, P31690 and P7909). The work of the Ciulli laboratory on PROTACs has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007-2013) as a Starting Grant to A.C. (grant agreement ERC-2012-StG-311460 DrugE3CRLs). R.C. is funded by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC) under the EastBio doctoral training programme (BB/M010996/1). Biophysics and drug-discovery activities at Dundee were supported by Wellcome Trust strategic awards 100476/Z/12/Z and 094090/Z/10/Z, respectively. For the purpose of open access, the authors have applied a Creative Commons Attribution (CC-BY) licence to any author-accepted manuscript version arising.

Author information

Authors and Affiliations

Authors

Contributions

A.H., M.B. and G.E.W. conceptualized this study. A.H. and M.B. designed and conducted hybrid-capture assays. A.H., S.B. and M.B. designed and conducted deep mutational scanning assays. A.H., S.B. and E.B. generated cell lines and conducted cellular mutant validation including immunoblotting and drug sensitivity assays. M.B. and H.I. analyzed and visualized hybrid capture and deep mutational scanning data. A.C. and A.T. designed AT7 compound and A.T. synthesized the compound. R.C. expressed and purified recombinant proteins, performed fluorescence polarization measurements and compound synthesis. S.J.H. solved co-crystal structure. J.W. performed degradation and cell viability assays for AT7. A.C. and G.E.W. supervised the work. H.I., A.H. and R.C. generated figures with input from all authors. A.H., R.C., A.C. and G.E.W. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Alessio Ciulli or Georg E. Winter.

Ethics declarations

Competing interests

S.B. is an employee at Proxygen, a company that is developing molecular glue degraders. M.B. is scientific founder, shareholder and employee at Proxygen. G.E.W. is scientific founder and shareholder at Proxygen and Solgate and the Winter laboratory receives research funding from Pfizer. A.C. is a scientific founder, shareholder and advisor of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. S.J.H. and A.T. are currently employees of Amphista Therapeutics. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Merck KaaG, Nurix Therapeutics, Ono Pharmaceutical and Tocris-Biotechne. The other authors are not aware of any affiliations, memberships, funding or financial holdings that might be perceived as affecting the objectivity of this work.

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Extended data

Extended Data Fig. 1 Quantitative and Qualitative Differences in Degrader Resistance.

(a) Dose-resolved, normalized viability after 3 d treatment (dBET6 or ARV-771) in KBM7, MV4;11 and MOLM-13 cells. Mean ± s.e.m.; n = 3 independent treatments. (b) Histogram depicting growth competition experiments. WT control KBM7 cells were mixed with mCherry and Cas9 expressing KBM7 cells harboring sgRNAs against the indicated genes. Pools were flow cytometry quantified at days 0, 7, 14 and 21 and mCherry percentages were normalized to day 0 percentage and to a non-targeting control sgRNA (sgMYCdesert). Data represents mean ± s.d. of n = 3 biological replicates. (c) Scheme of targeted hybrid-capture approach coupled to next-generation sequencing to identify mutations in spontaneously resistant cells. (d) Structure depiction of the CUL2-VBC-MZ1-BRD4 complex (PDBs: 5N4W, 5T35). Residues marked in red were identified in hybrid capture analysis. See also Fig. 1 and Supplementary Dataset. (e) Number of spontaneous degrader resistance alterations in the substrate receptor (CRBN, VHL, colored) binned by their distance to the degrader-binding site. See also Fig. 1d and Supplementary Dataset.

Extended Data Fig. 2 Characterization of DMS Libraries and AT7.

(a) Pie charts depicting the distribution of different alterations identified by sequencing the mutational scanning libraries for CRBN (top) and VHL (bottom). (b) Stacked bar graphs and density distributions of residue wise normalized abundance of mutants identified in the DMS libraries for VHL (top) and CRBN (bottom). (c) Chemical structure comparison of the degraders AT1 and AT7. (d) Dose-resolved, normalized viability after 3 d treatment with MZ-1, macroPROTAC-1, cis MZ-1 (a non VHL binding control of MZ-1 or AT7 in MV4;11 cells. Mean ± s.e.m.; n = 3 independent treatments. (e) Dose-resolved, normalized viability after 3 d treatment (AT7) in RKO VHL−/− cells with over-expression of VHLWT. Mean ± s.e.m.; n = 3 independent treatments. (f) Protein levels in HeLa cells treated with MZ-1 or AT7 (18 h, indicated concentration). Representative images of n = 2 independent measurements. (g) Protein levels in RKO VHL−/− cells with over-expression of VHLWT treated with DMSO or AT7 (60 nM, 2 h). Representative images of n = 2 independent measurements. (h) Dose-resolved, normalized viability after 4 d treatment (ACBI1) and 3 d treatment (ARV-771, MZ-1, macroPROTAC-1) in RKO VHL−/− cells with over-expression of VHLWT. Mean ± s.e.m.; n = 3 independent treatments.

Source data

Extended Data Fig. 3 DMS Robustly Locates Functional Hotspots of General Relevance in the Degrader Binding Pocket.

(a) Scatter plot depicting log2 fold-enrichment between different batch mutational scanning resistance measurements of VHL (500 nM ARV-771) or CRBN mutations (500 nM dBET6) normalized to DMSO after 7-day treatment. The rank-based measure of association was estimated via Spearman’s rho statistic and reported P-values were calculated via asymptotic two-sided t approximation without adjustments for multiple comparisons. (b) Stacked bar graphs of log2 fold-enrichment of VHL mutants normalized to DMSO treated with the indicated concentrations of ARV-771 for 7 days. n = 2 independent measurements. (c) Dose-resolved, normalized viability after 3 d treatment with dBET6, CC-90009, dBET57 or CC-885 in RKO CRBN−/− cells with over-expression of CRBNWT. Mean ± s.e.m.; n = 3 independent treatments. (d) Surface structure of CRBN bound by dBET6 (PDB 6BOY). Median log2 fold-enrichment of all CRBN mutations over DMSO across 4 degrader treatments (see Fig. 2d) is mapped in purple to dark grey onto positions mutated in the CRBN library.

Extended Data Fig. 4 Functional VHL Hotspots Show Neo-Substrate Specific Resistance and Sensitivity to PROTACs.

(a and b) Dose-resolved, normalized viability after 4 d treatment (ACBI1) and 3 d treatment (MZ-1) in RKO VHL−/− cells with over-expression of VHLWT, VHLR69G or VHLN67Q. Mean ± s.e.m.; n = 3 independent treatments. (c and d) Depiction of clonogenic assays via crystal violet staining. RKO VHL−/− cells with over-expression of VHLWT, VHLR69G, VHLN67R or VHLN67Q were treated for 10 days at EC90 of the degrader (2.5 uM ACBI1, 50 nM ARV-771, 75 nM MZ-1). (e) Protein levels in RKO VHL−/− cells with over-expression of VHLWT or VHLN67Q treated with DMSO, MZ-1 (75 nM, 2 h), ARV-771 (50 nM, 2 h) or ACBI1 (2.5 uM, 4 h). Representative images of n = 2 independent measurements. (f) Cocrystal structure of MZ-1 in a ternary complex with VHL-ElonginC-ElonginB and BRD4BD2 (PDB: 5T35). (g) Heatmap depicting differential log2 fold-enrichment of the VHLH110 mutations normalized to DMSO after treatment with ARV-771 (500 nM, 7d). n = 2 independent measurements. (h) Dose-resolved, normalized viability after 3d treatment AT7 (top), macroPROTAC-1 (bottom, left) or ARV-771 (bottom, right) in RKO VHL−/− cells with over-expression of VHLWT or VHLH110L. Mean ± s.e.m.; n = 3 independent treatments. (i) Protein levels in RKO VHL−/− cells with over-expression of VHLWT or VHLH110L treated with DMSO or AT7 (60 nM, 2 h). Representative images of n = 2 independent measurements. (j) Overlay of cocrystal structures of AT2 (grey, purple, blue) and MZ1 (black, PDB:5T35) in a ternary complex with VHL-ElonginC-ElonginB and BRD4BD2 showing a lateral shift of BRD4BD2. (k) Cocrystal structure of AT2 in a ternary complex with VHL-ElonginC-ElonginB and BRD4BD2. See Fig. 3. (l) Dose-resolved, normalized viability after 4 d treatment (ACBI1) and 3 d treatment (MZ-1, ARV-771) in RKO VHL-/- cells with over-expression of VHLWT or VHLY112C. Mean ± s.e.m.; n = 3 independent treatments.

Source data

Extended Data Fig. 5 VHLP71 is a Functional Hotspot for Degrader Specific Resistance.

(a) Heatmap depicting differential log2 fold-enrichment of the VHLP71 mutations normalized to DMSO between treatment with ARV-771 (500 nM, 7d) and macroPROTAC-1 (2 uM, 7d). n = 2 independent measurements. (b) Depiction (left) and quantification (right) of clonogenic assays via crystal violet staining. RKO VHL−/− cells with over-expression of VHLWT or VHLP71I were treated for 10 days at EC90 of the degrader (50 nM ARV-771, 75 nM MZ-1, 1 uM macroPROTAC-1).

Extended Data Fig. 6 Functional CRBN Hotspots Show Degrader Selectivity.

(a, c and f) Depiction of clonogenic assays via crystal violet staining. RKO CRBN−/− cells with over-expression of CRBNWT, CRBNE377K, CRBNN351D or CRBNH397D were treated for 10 days with DMSO, 30 nM dBET6, 60 nM CC-90009, 480 nM dBET57 or the indicated concentration of THAL-SNS-032. (b and g) Dose-resolved, normalized viability after 3 d treatment with THAL-SNS-032, dBET6 or CC-90009 in RKO CRBN−/− cells with over-expression of CRBNWT, CRBNN351D, CRBNH397Y or CRBNH57D. Mean ± s.e.m.; n = 3 independent treatments. (d) Protein levels in RKO CRBN−/− cells with over-expression of CRBNWT or CRBNE377K treated with DMSO, CC-90009 (50 nM, 6 h) or dBET6 (15 nM, 2 h). Representative images of n = 2 independent measurements. (e) Heatmap depicting differential log2 fold-enrichment of CRBNH397 mutations normalized to DMSO with dBET57 treatment (500 nM, 7d). n = 3 independent measurements. (h) Quantification of clonogenic assays via crystal violet extraction and measurement of absorption at 590 nM. RKO CRBN−/− cells with over-expression of CRBNWT or CRBNH57D were treated for 10 days with DMSO, 30 nM dBET6, 60 nM CC-90009, 480 nM dBET57 or 0.6 nM CC-885. See also Fig. 5.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Supplementary Figures 1–3, Supplementary Note

Reporting Summary

Supplementary Data

Hybrid-capture results.

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Source Data Fig. 3

Unprocessed western blot images related to Fig. 3.

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Unprocessed western blot images related to Fig. 4.

Source Data Fig. 5

Unprocessed western blot images related to Fig. 5.

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Source Data Extended Data Fig. 4

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Source Data Extended Data Fig. 6

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Hanzl, A., Casement, R., Imrichova, H. et al. Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01177-2

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