Abstract
Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase fusion protein.
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Data availability
The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium through the PRIDE39 partner repository with the dataset identifier PXD050270. Protein sequences were retrieved from UniProt (https://www.uniprot.org) with the following accession codes: FBXO22 (human), Q8NEZ5; FBXO22 (mouse), Q78JE5. The predicted protein structure was retrieved from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk) under accession code FBXO22 (human), AF-Q8NEZ5-F1. The data supporting the findings of this study are available within the article and Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We gratefully acknowledge the support of the National Institutes of Health (NIH) R00 CA248715 (X.Z.), NIH T32 GM105538 (A.A.B.), NIH T32 GM149439 (A.M. and M.A.C.), National Science Foundation Graduate Research Fellowship Program (I.A.R.), Damon Runyon Cancer Research Foundation DFS-53-22 (X.Z.) and Illumina Pilot Project Program (X.Z.). We thank the Robert H. Lurie Comprehensive Cancer Center of Northwestern University for the use of the Flow Cytometry Core Facility. We thank H. Li for the helpful discussions regarding CRISPR library cloning and CRISPR screens.
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Contributions
A.A.B. designed and conducted the biochemical and cellular experiments to demonstrate target degradation. C.Z. conducted the modeling study. C.Z. and M.A.C. characterized the compounds. I.A.R. and A.G.C. conducted the experiments to demonstrate the effectiveness of the GFP-based degradation platform. A.M., F.K. and X.Z. conducted the proteomics studies and performed data analysis. X.Z. supervised the project and, with contributions from all authors, wrote the paper.
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Competing interests
A.A.B. and X.Z. are named on a patent application related to TPD, held by Northwestern University (US provisional patent application number 63/538,637). The other authors declare no competing interests.
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Nature Chemical Biology thanks Milka Kostic, Brian Liau, Xiaobao Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Generation of CRISPR-Cas9 transcriptional activation cells for the discovery of E3 ligases supporting targeted protein degradation.
a, The construct of FKBP12-EGFP and a schematic representation of the generation of FKBP12-EGFP expressing HEK293T cells. b, Structures of Len-SLF and SLF. c, Fluorescence quantification of FKBP12-EGFP levels in HEK293T cells treated with 2 μM of Len-SLF or 20 μM of SLF for 24 hours. Data are presented as mean values +/��� SEM (n = 3 biological independent samples). The statistical significance was evaluated through unpaired two-tailed Student’s t-tests, comparing cells treated with Len-SLF or SLF to DMSO. Statistical significance denoted as ***P < 0.001 and ns: not significant. P value is 0.00026. d, The constructs used for the CRISPR-Cas9 transcriptional activation screen. e, Quantitative PCR analysis of IL1B mRNA levels subsequent to the transduction of sgRNAs targeting the promoter regions of the IL1B gene in HEK293T CRISPR-Cas9 transcriptional activation cells. The bar graph (n = 4 technical replicates) is representative of two independent experiments.
Extended Data Fig. 2 Gating strategy and procedure of fluorescence-activated cell sorting for the CRISPR-Cas9 transcriptional activation screen.
Cells were gated for singlets using forward and side scatter. GFP+ cells were gated for the subsequent sorting. Cells from the bottom 15% of the GFP population were sorted and harvested.
Extended Data Fig. 3 Compound screening to identify candidates for the CRIPSR activation screen.
a, HEK293T cell viability after treatment of FKBP12-directed bifunctional compounds (10 µM, 24 hours). Data are presented as mean values +/− SEM (n = 3 biological independent samples). b, Structures of five FKBP12-directed bifunctional compounds that show no significant cytotoxicity (cell viability > 50%) at 10 µM. c, The constructs of FKBP12-EGFP with SFFV and hPGK promoters. d, Fluorescence quantification of FKBP12-EGFP and mCherry levels in HEK293T cells stably expressing FKBP12-EGFP with SFFV or hPGK promoter. The bar graph (n = 8 technical replicates) is representative of two independent experiments with similar results. e, Fluorescence quantification of FKBP12-EGFP/mCherry levels in HEK293T cells stably expressing FKBP12-EGFP with SFFV or hPGK promoter, treated with 2 or 5 μM of candidate bifunctional compounds for 24 hours. Data are presented as mean values +/− SEM (n = 3 biological independent samples for compound treatment, n = 8 biological independent samples for DMSO treatment). The statistical significance was evaluated through unpaired two-tailed Student’s t-tests, comparing cells treated with 22-SLF or Len-SLF to DMSO. Statistical significance denoted as *P < 0.05, ***P < 0.001 and ns: not significant. P values are 0.000011 (2 μM Len-SLF in SFFV), 0.000051 (5 μM Len-SLF in SFFV), 0.039 (2 μM 22-SLF in hPGK), 0.031 (5 μM 22-SLF in hPGK), 0.00011 (2 μM Len-SLF in hPGK) and 0.000014 (5 μM Len-SLF in hPGK). f. Flow cytometry analysis of DMSO-treated cells revealed a silenced GFP population. The gating strategy was the same as described in Extended Data Fig. 2. The result is representative of two independent experiments with similar results.
Extended Data Fig. 4 An E3 ligase focused CRISPR-Cas9 transcriptional activation screen identifies DCAF16 supporting KB02-SLF-induced degradation of FKBP12-EGFP_NLS.
a, The construct of FKBP12-EGFP_NLS and a schematic representation of the steps in the CRISPR-Cas9 transcriptional activation screen. b, Volcano plot showing the E3 ligase focused CRISPR-Cas9 transcriptional activation screen for FKBP12-EGFP_NLS degradation after treatment of 2 μM KB02-SLF in HEK293T CRISPR-Cas9 transcriptional activation cells for 24 hours (n = 3 biological independent samples). P values were calculated by two-sided t test without adjustment.
Extended Data Fig. 5 22-SLF promotes FBXO22-dependent proteasomal degradation of FKBP12.
a, Gene expression ratio values of FBXO22 and CRBN between tumor and normal samples. Data is obtained from GEPIA (http://gepia.cancer-pku.cn/). Full names of the abbreviations are shown in Supplementary Table 1. b, Genomic PCR confirms FBXO22 knockout in A549, MDA-MB-231 and PC3 cells. The result is representative of two independent experiments with similar results. c, Global proteomic analysis confirms FBXO22 knockout in A549, MDA-MB-231 and PC3 cells. The result is representative of two independent experiments with similar results. d, 22-SLF promoted reduction in FKBP12 levels in MDA-MB-231 and PC3 wildtype, but not FBXO22 knockout cells. The bar graph represents quantification of the FLAG-FKBP12/HSP90 protein content. Data are presented as mean values (n = 2 biological independent samples). e, Global proteomic analysis in A549 wildtype and FBXO22 knockout cells treated with 22-SLF (2 μM, 24 hours) (n = 3 biological independent samples). P values were calculated by two-sided t test and adjusted using Benjamini-Hochberg correction for multiple comparisons. f, Bar graph quantification showing the change in KDM4A and KDM4B upon 22-SLF treatment in A549 wildtype and FBXO22 knockout cells. Data are presented as mean values +/− SEM (n = 2 biological independent samples for DMSO treated samples, n = 3 biological independent samples for 22-SLF samples). The statistical significance was evaluated through unpaired two-tailed Student’s t-tests, comparing cells treated with 22-SLF to DMSO. Statistical significance denoted as *P < 0.05 and ns: not significant. P value is 0.044.
Extended Data Fig. 6 22-SLF rescues Len-SLF-induced FKBP12 degradation in HEK293T FBXO22 knockout cells.
a, 22-biotin-conjugated streptavidin pull-down with lysates of HA-FBXO22-expressing HEK293T cells followed by proteomic analysis revealed FBXO22 as one of the protein targets bound by 22-biotin. Data are presented as mean values (n = 2 biological independent samples). b, HEK293T FBXO22 knockout cells pretreated with 22-SLF (0.1–25 µM, 2 hours) were treated with 0.5 µM of Len-SLF for 4 hours. The graph represents quantification of the FLAG-FKBP12/HSP90 protein content. Data are presented as mean values (n = 2 biological independent samples).
Extended Data Fig. 7 FBXO22 C227 and C228 are involved in 22-SLF-mediated degradation of FKBP12.
a, Interactome studies of FBXO22 wildtype and C227A/C228A double mutant revealed that both FBXO22 wildtype and C227A/C228A double mutant were assembled into the SKP1-CUL1-RBX1 E3 complex. Data are presented as mean values (n = 2 biological independent samples). b, Global proteomic analysis in HEK293T cells expressing HA-FBXO22 wildtype versus C227A/C228A double mutant treated with 22-SLF (0.5 μM, 24 hours) (n = 2 biological independent samples for DMSO treated samples, n = 3 biological independent samples for 22-SLF samples). P values were calculated by two-sided t test and adjusted using Benjamini-Hochberg correction for multiple comparisons.
Extended Data Fig. 8 Evaluation of the impact of FBXO22 K125, V230, and N257 on FKBP12 degradation induced by 22-SLF.
Single mutation of K125, V230 or N257 to alanine in FBXO22 did not block 22-SLF-induced degradation of FKBP12. The bar graph represents quantification of the FLAG-FKBP12/HSP90 protein content. Data are presented as mean values (n = 2 biological independent samples).
Extended Data Fig. 9 Global proteomic analysis in H2228 wildtype and FBXO22 knockout cells.
Proteome in H2228 wildtype and FBXO22 knockout cells was extracted, digested by LysC and trypsin, labeled by TMT tags, and analyzed via MS. Data are presented as mean values (n = 2 biological independent samples).
Extended Data Fig. 10 The acrylamide variant of 22-SLF, 22a-SLF, induced the degradation of FKBP12.
a, Structure of 22a-SLF. b, Comparison of FKBP12 degradation by 22-SLF and 22a-SLF. HEK293T cells expressing HA-FBXO22 were treated with 0.5, 1, or 2 µM of 22-SLF or 22a-SLF for 8 hours. The graph represents quantification of the FLAG-FKBP12/HSP90 protein content. Data are presented as mean values (n = 2 biological independent samples).
Supplementary information
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Basu, A.A., Zhang, C., Riha, I.A. et al. A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01655-9
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DOI: https://doi.org/10.1038/s41589-024-01655-9
