Céline Reverdy

Small-molecule inhibitor of USP7/HAUSP ubiquitin protease stabilizes and activates p53 in cells

Deregulation of the ubiquitin/proteasome system has been implicated in the pathogenesis of many human diseases, including cancer. Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases. Using high-throughput screening, we have discovered HBX 41,108, a small-molecule compound that inhibits USP7 deubiquitinating activity with an IC50 in the submicromolar range. Kinetics data indicate an uncompetitive reversible inhibition mechanism. HBX 41,108 was shown to affect USP7-mediated p53 deubiquitination in vitro and in cells. As RNA interference-mediated USP7 silencing in cancer cells, HBX 41,108 treatment stabilized p53, activated the transcription of a p53 target gene without inducing genotoxic stress, and inhibited cancer cell growth. Finally, HBX 41,108 induced p53-dependent apoptosis as shown in p53 wild-type and null isogenic cancer cell lines. We thus report the identification of the first lead-like inhibitor against USP7, providing a structural basis for the development of new anticancer drugs.[Mol Cancer Ther 2009;8(8):2286–95]

Discovery of Specific Inhibitors of Human USP7/HAUSP Deubiquitinating Enzyme

The human USP7 deubiquitinating enzyme was shown to regulate many proteins involved in the cell cycle, as well as tumor suppressors and oncogenes. Thus, USP7 offers a promising, strategic target for cancer therapy. Using biochemical assays and activity-based protein profiling in living systems, we identified small-molecule antagonists of USP7 and demonstrated USP7 inhibitor occupancy and selectivity in cancer cell lines. These compounds bind USP7 in the active site through a covalent mechanism. In cancer cells, these active-site-targeting inhibitors were shown to regulate the level of several USP7 substrates and thus recapitulated the USP7 knockdown phenotype that leads to G1 arrest in colon cancer cells. The data presented in this report provide proof of principle that USP7 inhibitors may be a valuable therapeutic for cancer. In addition, the discovery of such molecules offers interesting tools for studying deubiquitination.

Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane–cytoskeleton linker ezrin plays a major role in HGF‐induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF‐mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell–cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell–cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell–cell contacts plays an essential role in HGF‐induced cell scattering and implicates Fes in the cross‐talk between cell–cell and cell–matrix adhesion.

Functional interaction between the ubiquitin-specific protease 25 and the SYK tyrosine kinase.

The SYK non-receptor tyrosine kinase is a key effector of immune receptors signaling in hematopoietic cells. Here, we identified and characterized a novel interaction between SYK and the ubiquitin-specific protease 25 (USP25). We report that the second SH2 domain of SYK physically interacts with a tyrosine-rich, C-terminal region of USP25 independently of tyrosine phosphorylation. Moreover, we showed that SYK specifically phosphorylates USP25 and alters its cellular levels. This study thus uncovers a new SYK substrate and reveals a novel SYK function, namely the regulation of USP25 cellular levels.

USP7 inhibition alters homologous recombination repair and targets CLL cells independently of ATM/p53 functional status

The role of deubiquitylase ubiquitin-specific protease 7 (USP7) in the regulation of the p53-dependent DNA damage response (DDR) pathway is well established. Whereas previous studies have mostly focused on the mechanisms underlying how USP7 directly controls p53 stability, we recently showed that USP7 modulates the stability of the DNA damage responsive E3 ubiquitin ligase RAD18. This suggests that targeting USP7 may have therapeutic potential even in tumors with defective p53 or ibrutinib resistance. To test this hypothesis, we studied the effect of USP7 inhibition in chronic lymphocytic leukemia (CLL) where the ataxia telangiectasia mutated (ATM)-p53 pathway is inactivated with relatively high frequency, leading to treatment resistance and poor clinical outcome. We demonstrate that USP7 is upregulated in CLL cells, and its loss or inhibition disrupts homologous recombination repair (HRR). Consequently, USP7 inhibition induces significant tumor-cell killing independently of ATM and p53 through the accumulation of genotoxic levels of DNA damage. Moreover, USP7 inhibition sensitized p53-defective, chemotherapy-resistant CLL cells to clinically achievable doses of HRR-inducing chemotherapeutic agents in vitro and in vivo in a murine xenograft model. Together, these results identify USP7 as a promising therapeutic target for the treatment of hematological malignancies with DDR defects, where ATM/p53-dependent apoptosis is compromised.

erratum: The protein–protein interaction map of Helicobacter pylori

This corrects the article DOI: 35051615

Abstract 2642: Identification and characterization of selective inhibitors of ubiquitin specific protease 7

Regulated protein turnover is primarily controlled by the ubiquitin-proteasome system. The only marketed drug related to the ubiquitin-proteasome system, bortezomib, is acting as a proteasome inhibitor and has been approved for the treatment of some hematological cancers. Targeting the upstream ubiquitin conjugation/deconjugation system carries out promises of therapeutics with increased specificity and selectivity. Ubiquitin-specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a drugable target class due to their thiol-protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome-wide RNAi screen of the catalytically active human USPs in cancer-relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP as promising cancer target. Fluorescence-based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High-throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP7 inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP7-specific series. These will help further validate this novel class of molecular targets and may provide a structural basis for the development of new anticancer drugs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2642.

Abstract B193: Identification of selective inhibitors of ubiquitin specific proteases

Regulated protein turnover is primarily controlled by the ubiquitin‐proteasome system. The therapeutic efficacy of the proteasome inhibitor Bortezomib establishes this system as a valid anticancer therapeutic field. A promising alternative to targeting the proteasome itself lies upstream, at the level of the ubiquitin conjugation/deconjugation step to generate more specific anticancer agents. Ubiquitin‐specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a druggable target class due to their thiol‐protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome‐wide RNAi screen of the catalytically active human USPs in cancer‐relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP and USP8/UBPY as promising cancer targets. Fluorescence‐based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High‐throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP8‐ and USP7‐specific series. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B193.