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Cited by 128Ubiquitin and proteasome pathwaysCancer-related Molecular PathwaysProtein Degradation and Inhibitors

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Selective USP7 inhibition elicits cancer cell killing through a p53-dependent mechanism.

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Schauer, Nathan J., Liu, Xiaoxi, Magin, Robert S., Doherty, Laura M., Chan, Wai Cheung, Ficarro, Scott B., Hu, Wanyi, Roberts, Rebekka M., Iacob, Roxana E., Stolte, Björn, Giacomelli, Andrew O., Perera, Sumner, McKay, Kyle, Boswell, Sarah A., Weisberg, Ellen L., Ray, Arghya, Chauhan, Dharminder, Dhe-Paganon, Sirano, Anderson, Ken C., Griffin, James D., Li, Jianing, Hahn, William C., Sorger, Peter K., Engen, John R., Stegmaier, Kimberly, Marto, Jarrod A., Buhrlage, Sara J.·2020·Scientific Reports
DOI 10.1038/s41598-020-62076-xPMC PMC7093416
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Abstract

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.

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Figure 1 Design of a potent irreversible USP7 inhibitor. (a) Co-crystal structure of XL188 bound to the USP7 catalytic domain, highlighting the ligand’s solvent accessibility and distance to the catalytic cysteine (PDB: 5VS6). (b) Chemical structures of XL188, XL177A, and XL177B. (c) IC50 plots of full-length USP7 cleavage of Ub-AMC following 6-hour pre-treatment with XL177A or XL177B (n = 3 experimental replicates, error bars = 95% CI). (d) Intact MS spectra of USP7 catalytic domain treated with DMSO (red) or a 2.5-fold molar excess XL177A for 15 minutes (green), showing single labeling of USP7 with a mass shift corresponding to the inhibitor (n = 2 experimental replicates). (e) MS-MS spectra of USP7 catalytic domain treated with 2.5-fold molar excess XL177A for 15 minutes. The XL177A-labeled cysteine (C223) is indicated in the peptide sequence shown. (f) Western blots showing USP7 labeling by the DUB ABPP HA-Ub-VS in whole cell lysate after 30-minute or 4-hour pre-treatment with XL177A or XL177B. Full blots are presented in Fig. S14. (g) Densitometry from F (n = 2 experimental replicates, error bars = SD).
Figure 1 Design of a potent irreversible USP7 inhibitor. (a) Co-crystal structure of XL188 bound to the USP7 catalytic domain, highlighting the ligand’s solvent accessibility and distance to the catalytic cysteine (PDB: 5VS6). (b) Chemical structures of XL188, XL177A, and XL177B. (c) IC50 plots of full-length USP7 cleavage of Ub-AMC following 6-hour pre-treatment with XL177A or XL177B (n = 3 experimental replicates, error bars = 95% CI). (d) Intact MS spectra of USP7 catalytic domain treated with DMSO (red) or a 2.5-fold molar excess XL177A for 15 minutes (green), showing single labeling of USP7 with a mass shift corresponding to the inhibitor (n = 2 experimental replicates). (e) MS-MS spectra of USP7 catalytic domain treated with 2.5-fold molar excess XL177A for 15 minutes. The XL177A-labeled cysteine (C223) is indicated in the peptide sequence shown. (f) Western blots showing USP7 labeling by the DUB ABPP HA-Ub-VS in whole cell lysate after 30-minute or 4-hour pre-treatment with XL177A or XL177B. Full blots are presented in Fig. S14. (g) Densitometry from F (n = 2 experimental replicates, error bars = SD).CC BY · PubMed Central
Figure 2 XL177A is a potent USP7 inhibitor and p53 stabilizer in cyto. (a) Western blots showing USP7 labeling by HA-Ub-VS after 6-hour cell treatment with XL177A or XL177B. (b) Densitometry from A (n = 2 experimental replicates, error bars = SD). (c) Whole cell lysate Western blots of MCF7 cells after 2-hr treatment with XL177A or XL177B (n = 2 experimental replicates). Full blots are presented in Fig. S14. (d) Whole cell lysate Western blots of MCF7 cells after 16-hr treatment with XL177A or XL177B (n = 2). (d) Quantitative real-time PCR of MCF7 cells treated for 24 hours with 1 μM XL177A or XL177B (n = 2 experimental replicates with 3 technical replicates each, error bars = SEM). E) Cell cycle analysis based on propidium iodide staining of MCF7 cells after 24-hr treatment with 1 μM XL177A or XL177B (n = 3 experimental replicates, error bars = SD).
Figure 2 XL177A is a potent USP7 inhibitor and p53 stabilizer in cyto. (a) Western blots showing USP7 labeling by HA-Ub-VS after 6-hour cell treatment with XL177A or XL177B. (b) Densitometry from A (n = 2 experimental replicates, error bars = SD). (c) Whole cell lysate Western blots of MCF7 cells after 2-hr treatment with XL177A or XL177B (n = 2 experimental replicates). Full blots are presented in Fig. S14. (d) Whole cell lysate Western blots of MCF7 cells after 16-hr treatment with XL177A or XL177B (n = 2). (d) Quantitative real-time PCR of MCF7 cells treated for 24 hours with 1 μM XL177A or XL177B (n = 2 experimental replicates with 3 technical replicates each, error bars = SEM). E) Cell cycle analysis based on propidium iodide staining of MCF7 cells after 24-hr treatment with 1 μM XL177A or XL177B (n = 3 experimental replicates, error bars = SD).CC BY · PubMed Central
Figure 3 XL177A has a similar USP7 binding mode to XL188. (a) Structure-activity-relationship of XL177A showing the potency of the indicated compounds against full-length USP7 in Ub-AMC assays (n = 2 or 3 experimental replicates). (b) Comparative Ub-AMC IC50 curves of USP7-WT, USP7-Q351S, and USP7-F291N after 6-hr pre-incubation with XL177A (n = 2 experimental replicates, error bars = 95% CI). (c–e) Structure of USP7 CD highlighting regions with increased (green) or decreased (purple) hydrogen exchange after treatment with XL188 (c), XL041 (d), and XL177A (e) (PDB: 5VS6; n = 2 experimental replicates).
Figure 3 XL177A has a similar USP7 binding mode to XL188. (a) Structure-activity-relationship of XL177A showing the potency of the indicated compounds against full-length USP7 in Ub-AMC assays (n = 2 or 3 experimental replicates). (b) Comparative Ub-AMC IC50 curves of USP7-WT, USP7-Q351S, and USP7-F291N after 6-hr pre-incubation with XL177A (n = 2 experimental replicates, error bars = 95% CI). (c–e) Structure of USP7 CD highlighting regions with increased (green) or decreased (purple) hydrogen exchange after treatment with XL188 (c), XL041 (d), and XL177A (e) (PDB: 5VS6; n = 2 experimental replicates).CC BY · PubMed Central
Figure 4 XL177A is selective for USP7. (a) Remaining activity of 41 purified recombinant DUBs against Ub-Rho after 15-minute pre-treatment with XL177A (n = 1). (b) Ratio of Bio-Ub-PA/VME labeling for 60 DUBs in HEK293AD lysate between samples pre-treated for 5 hours with DMSO v. 1 μM XL177A (n = 2 experimental replicates with two technical replicates, reported values are medians of all replicates). (c) Ratio of XL177A-DTB labeling for 566 proteins in HEK293AD lysate between samples pre-treated for 5 hours with DMSO v. 1 μM XL177A (n = 3 experimental replicates with two technical replicates, reported values are medians of all replicates).
Figure 4 XL177A is selective for USP7. (a) Remaining activity of 41 purified recombinant DUBs against Ub-Rho after 15-minute pre-treatment with XL177A (n = 1). (b) Ratio of Bio-Ub-PA/VME labeling for 60 DUBs in HEK293AD lysate between samples pre-treated for 5 hours with DMSO v. 1 μM XL177A (n = 2 experimental replicates with two technical replicates, reported values are medians of all replicates). (c) Ratio of XL177A-DTB labeling for 566 proteins in HEK293AD lysate between samples pre-treated for 5 hours with DMSO v. 1 μM XL177A (n = 3 experimental replicates with two technical replicates, reported values are medians of all replicates).CC BY · PubMed Central
Figure 5 XL177A activates p53 signaling transcriptome-wide. (a) Heat map showing hierarchical clustering of transcriptional profiles from MCF7 cells treated for 24 hours with 0.1 or 1 μM XL177A, 0.1 or 1 μM XL177B, or 1 or 10 μM Nutlin-3A. (b) Volcano plots of genes enriched or depleted after 24-hr treatment with 1 μM XL177A. (c) Expression levels of all genes (clear) or direct p53 target genes (filled) from MCF7 cells treated for 24 hours with 1 μM XL177A (adjusted p < 0.0001), Nutlin-3A (adjusted p < 0.0001), or XL177B (ns). Box: inter-quartile range; Whiskers: gens within 3 SD of mean. (d) Top 5 datasets enriched in the set of genes significantly up- or down-regulated by 1 μM XL177A treatment and their ranking in the set of genes up- or down-regulated by Nutlin-3A. (e,f) Waterfall plot of all detected transcripts from 1 μM XL177A ranked by normalized expression score and compared to DREAM complex targets (e) or direct p53 targets (f).
Figure 5 XL177A activates p53 signaling transcriptome-wide. (a) Heat map showing hierarchical clustering of transcriptional profiles from MCF7 cells treated for 24 hours with 0.1 or 1 μM XL177A, 0.1 or 1 μM XL177B, or 1 or 10 μM Nutlin-3A. (b) Volcano plots of genes enriched or depleted after 24-hr treatment with 1 μM XL177A. (c) Expression levels of all genes (clear) or direct p53 target genes (filled) from MCF7 cells treated for 24 hours with 1 μM XL177A (adjusted p < 0.0001), Nutlin-3A (adjusted p < 0.0001), or XL177B (ns). Box: inter-quartile range; Whiskers: gens within 3 SD of mean. (d) Top 5 datasets enriched in the set of genes significantly up- or down-regulated by 1 μM XL177A treatment and their ranking in the set of genes up- or down-regulated by Nutlin-3A. (e,f) Waterfall plot of all detected transcripts from 1 μM XL177A ranked by normalized expression score and compared to DREAM complex targets (e) or direct p53 targets (f).CC BY · PubMed Central
Figure 6 XL177A acts through a p53-dependent mechanism. (a) Correlation between relative proliferation profiles of 484 cancer cell lines after XL177A treatment for 5 days or CRISPR KO with the indicated sgRNA. (b) Depmap was queried to determine whether mutations in any of >18,000 genes correlated with XL177A AUC. Only TP53 mutant cell lines displayed significantly altered response relative to WT. (c) Lineages enriched as sensitive (left of 0) or resistant (right of 0) to XL177A in the PRISM data set. (d) Relative Cell Titer Glo luminescence of a panel of TP53-WT (red) or TP53-mutant (black) Ewing Sarcoma cell lines after treatment with XL177A for 3 days. (e) Relative Cell Titer Glo luminescence of TC32 cells expressing the indicated sgRNA after treatment with XL177A for 3 days. (f) Correlation between USP7 DEMETER and AVANA dependency scores in the Broad Depmap portal for TP53-WT (red) and TP53-mutant (black) cell lines. (g) Log10 ratio of A549-FF to A549-sgTP53-Renilla cells after treatment of an initial 1:1 mixture of the two cell lines with the indicated sgRNA for the indicated number of days.
Figure 6 XL177A acts through a p53-dependent mechanism. (a) Correlation between relative proliferation profiles of 484 cancer cell lines after XL177A treatment for 5 days or CRISPR KO with the indicated sgRNA. (b) Depmap was queried to determine whether mutations in any of >18,000 genes correlated with XL177A AUC. Only TP53 mutant cell lines displayed significantly altered response relative to WT. (c) Lineages enriched as sensitive (left of 0) or resistant (right of 0) to XL177A in the PRISM data set. (d) Relative Cell Titer Glo luminescence of a panel of TP53-WT (red) or TP53-mutant (black) Ewing Sarcoma cell lines after treatment with XL177A for 3 days. (e) Relative Cell Titer Glo luminescence of TC32 cells expressing the indicated sgRNA after treatment with XL177A for 3 days. (f) Correlation between USP7 DEMETER and AVANA dependency scores in the Broad Depmap portal for TP53-WT (red) and TP53-mutant (black) cell lines. (g) Log10 ratio of A549-FF to A549-sgTP53-Renilla cells after treatment of an initial 1:1 mixture of the two cell lines with the indicated sgRNA for the indicated number of days.CC BY · PubMed Central

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  1. Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing. Genome Res. 939–946, https://doi.org/10.1101/gr.128124.111.22 (2011). · DOI

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Authors
Schauer, N. J., Liu, X., Magin, R. S., Doherty, L. M., Chan, W. C., Ficarro, S. B., Hu, W., Roberts, R. M., Iacob, R. E., Stolte, B., Giacomelli, A. O., Perera, S., McKay, K., Boswell, S. A., Weisberg, E. L., Ray, A., Chauhan, D., Dhe-Paganon, S., Anderson, K. C., Griffin, J. D., Li, J., Hahn, W. C., Sorger, P. K., Engen, J. R., Stegmaier, K., Marto, J. A., & Buhrlage, S. J.
Affiliations
  • Harvard University (US)
  • Brigham and Women's Hospital (US)
  • Dana-Farber Cancer Institute (US)
  • Northeastern University (US)
  • Broad Institute (US)
  • University of Vermont (US)
Corresponding author
Buhrlage, Sara J.
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-03-24
Volume
10
Issue
1
Pages
5324-5324
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-62076-x
PMC
PMC7093416
OpenAlex
W3013382443
Cited by
125
References
77
Citations by year
2026: 18 · 2025: 26 · 2024: 18 · 2023: 15 · 2022: 22 · 2021: 21 · 2020: 5
FWCI
5.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Open access LMU (Ludwid Maxmilian's Universitat Munchen) (submittedVersion, cc-by)
Funders
  • U.S. Department of Health & Human Services | National Institutes of Health
  • St. Baldrick&apos;s Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
Keywords
USP7, p53, TP53 mutation, predictive biomarkers, structure-based drug design, tumor cell growth inhibition, Ewing sarcoma, malignant rhabdoid tumor, childhood cancer, MDM2, FOXO4, PTEN, MYCN
MeSH terms
Ubiquitin-Specific Peptidase 7, Antineoplastic Agents, Humans, Protease Inhibitors, Tumor Suppressor Protein p53, Apoptosis, Cell Line, Tumor, Cell Proliferation, Ubiquitination, MCF-7 Cells

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