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.
Highlights
(DUBs) regulate virtually all physiological and pathophysiological processes[1,8,9,10]
We recently reported the structure-guided development of XL188, a noncovalent inhibitor of Ubiquitin-specific peptidase 7 (USP7) that binds in the thumb-palm cleft that guides the ubiquitin C-terminus into the active site
To test USP7 target engagement in a cellular context, we used competitive activity-based protein profiling (ABPP) with MCF7 crude cell extracts and the deubiquitinating enzyme (DUB) activity-based probe (ABP) hemagglutinin (HA)-ubiquitin vinylmethylsulfone (HA-Ub-VS): XL177A inhibited HA-Ub-VS labeling with IC50s of 85 and 8 nM following 30 min and 4 hr compound preincubations, respectively (Fig. 1f,g)
Summary
(DUBs) regulate virtually all physiological and pathophysiological processes[1,8,9,10]. There have been a number of small molecule USP7 inhibitors reported to date[26,27], and these compounds have consistently demonstrated the ability to stabilize p53 protein levels in cyto, they do not exhibit p53-dependent growth suppression[26,28] These findings have largely been credited to USP7’s role in other cellular pathways, they could be due to unidentified off-targets of these compounds. We recently reported XL18829, a potent and selective USP7 inhibitor that, in contrast to other reported small molecules, induces p53-dependent growth suppression in TP53-WT Ewing Sarcoma cell lines[30] This somewhat surprising finding suggested to us that the functional significance of the USP7-p53 axis in Ewing Sarcoma, and perhaps cancer more broadly, is not completely understood and that improved research tools are needed to enable reliable on-target analysis of USP7 biology. Our findings demonstrate that covalent targeting of the active-site is a viable strategy for development of DUB inhibitors, provide a well-validated chemical tool for investigation of USP7 biology, and demonstrate that p53 is a relevant biomarker for cellular responses to USP7 inhibition
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