Abstract

It is well established that both p53 and MDM2 are short-lived proteins whose stabilities are tightly controlled through ubiquitination-mediated degradation. Although numerous studies indicate that the MDM2 E3 ligase activity, as well as the protein–protein interaction between p53 and MDM2, is the major focus for this regulation, emerging evidence suggests that the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7) plays a critical role. Furthermore, HAUSP inhibition elevates p53 stability and might be beneficial for therapeutic purposes. In this review, we discuss the advances of this dynamic pathway and the contributions of positive and negative regulators affecting HAUSP activity. We also highlight the roles of HAUSP in cancer justifying the production of the first generation of HAUSP inhibitors.

Introduction

Protein ubiquitination is a post-translational modification, where E3 ubiquitin ligases attach ubiquitin moieties to a target protein substrate. In many cases, protein ubiquitination results in protein degradation. This degradation signalling cascade is tightly controlled, yet reversible through the enzymatic activity of deubiquitinating enzymes (DUB) that remove the ubiquitin chains (Komander et al., 2009; Reyes-Turcu et al., 2009). Therefore, modulation of deubiquitinases can specifically regulate the stability of target proteins. Recently, the effort has been focused on identifying specific deubiquitinases that can regulate oncogenes since the removal or inhibition of these DUB results in hyper-ubiquitination and subsequent proteolysis of that oncogene. In this review, we focus on one ubiquitin-specific protease (USP), herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and highlight the complex regulation of the HAUSP/MDM2/p53 axis.

HAUSP protein structure

Phylogenetically, HAUSP is categorized into the largest of the five deubiquitinase subclasses, the USP family, due to a highly conserved catalytic core domain (Everett et al., 1997; Hu et al., 2002; Nijman et al., 2005; Reyes-Turcu et al., 2009). HAUSP is a 135 kDa protein located on chromosome 16p13.2. In fact, the 1102 amino acid residues can be subdivided into three major regions: an N-terminal tumour necrosis factor receptor-associated factor (TRAF)-like domain (residues 53–206), a central catalytic core (residues 208–560), and C-terminal HAUSP ubiquitin-like domains (HUBL1-5) (residues 564–1084) (Figure 1A). The first crystal structure of a USP catalytic domain was characterized both in isolation and in a complex with ubiquitin aldehyde using HAUSP (Hu et al., 2002). This pivotal study revealed that the catalytic core resembled an extended right hand composed of three unique regions: Thumb, Palm, and Fingers. Researchers identified that the Palm and Thumb regions come together to form an ideal ubiquitin binding pocket enriched with acidic amino acids. Within the Palm and Thumb regions are three key amino acids termed the ‘catalytic triad’ (C233, H464, and D481) (Figure 1B). The catalytic core exists in a non-reactive conformation; yet upon ubiquitin binding, the catalytic core undergoes a conformational change, which realigns the catalytic triad residues into proximity, allowing for ubiquitin catalysis (Hu et al., 2002). Further in vitro analysis identified a ‘switch loop’ (residues 285–291) region that aids in the rearrangement of the catalytic triad upon activation; indeed, mutations in this region greatly reduced HAUSP activation (Faesen et al., 2011). Interestingly, the catalytic core in isolation has weak catalytic activity, suggesting that other regions help increase the efficiency of the ubiquitin catalysis reaction.

Figure 1

Overview of HAUSP domains and structure. (A) Functional domains of HAUSP including TRAF-like motif, catalytic core, and five HUBL regions. (B) Functional domain of the catalytic core highlighting the catalytic triad, switch loop, and underlining regions that compose the Thumb, Palm, and Fingers of HAUSP. (C) Rendering of the conformational change HAUSP undergoes from an inactive to an active state upon substrate binding.

Figure 1

Overview of HAUSP domains and structure. (A) Functional domains of HAUSP including TRAF-like motif, catalytic core, and five HUBL regions. (B) Functional domain of the catalytic core highlighting the catalytic triad, switch loop, and underlining regions that compose the Thumb, Palm, and Fingers of HAUSP. (C) Rendering of the conformational change HAUSP undergoes from an inactive to an active state upon substrate binding.

Although the catalytic cleft is responsible for ubiquitin binding and subsequent catalysis, domains outside the catalytic core are required for substrate binding. The TRAF-like domain, which closely resembles the domains of TRAF family proteins, was identified as the minimal region for binding of many HAUSP-dependent substrates (Hu et al., 2002, 2006; Saridakis et al., 2005; Sheng et al., 2006). Crystallography studies of the TRAF-like domain revealed a unique shallow groove necessary for substrate recruitment and binding (Saridakis et al., 2005; Hu et al., 2006; Sheng et al., 2006). Interestingly, through the generation of HAUSP domain deletion mutants, the nuclear localization of HAUSP has been suggested to be in part dependent on the TRAF-like domain (Zapata et al., 2001; Fernandez-Montalvan et al., 2007). To assess the importance of each domain on HAUSP enzymatic activity, different domain deletion mutants were tested in vitro. While deletion of the TRAF-like domain only partially reduced HAUSP activity, deletion of the C-terminus strongly inhibited HAUSP deubiquitination activity in vitro (Fernandez-Montalvan et al., 2007; Ma et al., 2010; Faesen et al., 2011).

The C-terminus of HAUSP is composed of five HUBL domains (ordered in a 2-1-2 pattern), which are widely divergent in sequence and charge distribution (Faesen et al., 2011). HUBL1/2/3 have been demonstrated, similar to the TRAF-like domain, to bind to specific substrates, but the addition of HUBL1/2/3 to the catalytic core scarcely enhanced HAUSP activity (Faesen et al., 2011; Kim et al., 2016). In contrast, by specifically adding just HUBL4/5 and the 19 amino acid C-terminal tail, HAUSP catalytic activity was mostly reconstituted, suggesting an important role for this specific region (Faesen et al., 2011). Mechanistically, crystallography and biochemical experiments demonstrate that HUBL4/5 directly interact and cooperate with the switch loop in the catalytic domain facilitating the conformational change, subsequently increasing HAUSP affinity for ubiquitin (Faesen et al., 2011). Recently, it was demonstrated that the 19 amino acid C-terminal tail has the ability to markedly reconstitute the enzymatic activity of the catalytic domain in vitro; specifically, researchers show that this region can bind to and stabilize the catalytic core in an active conformation allowing for more efficient ubiquitin catalysis (Rouge et al., 2016).

In summary, these extensive biochemical and crystallography studies using HAUSP uncovered domain-specific functions important for HAUSP target protein recognition and subsequent ubiquitin removal: (i) the TRAF-like domain is necessary for recruitment of substrates; (ii) the catalytic core, normally inactive, undergoes conformational changes upon ubiquitin binding that reorganize the catalytic triad to allow for efficient ubiquitin catalysis; and (iii) the HUBL domains are essential for full enzymatic activity through binding to the catalytic domain (HUBL4/5 to the switch region) and enhancing the affinity towards removing substrate ubiquitination (Figure 1C).

HAUSP regulates the MDM2/p53 axis

One of the first examples of a deubiquitinase exhibiting a specific role in regulating protein stability was the interaction between HAUSP and p53. We identified HAUSP as a novel p53-interacting protein through mass spectrometry of purified p53 (Li et al., 2002). The TRAF-like domain of HAUSP was mapped as the minimal region necessary to bind to p53 (Hu et al., 2002; Li et al., 2002). Indeed, overexpression of HAUSP was able to stabilize p53 even in the presence of excess MDM2, the major E3 ubiquitin ligase antagonizing p53 stability. Notably, wild-type HAUSP removed the ubiquitin moieties from p53, in contrast to the catalytically inactive HAUSP (C233S), demonstrating that the enzymatic activity of HAUSP was required for p53 deubiquitination. Subsequent p53 stabilization induced p53-mediated responses such as apoptosis and cell cycle arrest (Li et al., 2002). Typically, overexpression of a deubiquitinase removes substrate-specific ubiquitin resulting in protein stabilization; in contrast, ablation of a deubiquitinase leads to an increase in substrate-specific ubiquitin causing protein destabilization. In the case of p53, partially reducing HAUSP levels destabilized endogenous p53 (Li et al., 2004); interestingly, nearly complete ablation or genetic disruption of HAUSP stabilized p53 levels (Cummins et al., 2004; Li et al., 2004). Mechanistically, we uncovered that HAUSP interacts with MDM2 both in vivo and in vitro (Li et al., 2004). Crystal structure analyses demonstrate that although MDM2 interacts with HAUSP at a much higher affinity than p53, they both bind to the same shallow groove in the TRAF-like domain of HAUSP in a mutually exclusive manner (Hu et al., 2006; Sheng et al., 2006). Further studies found additional MDM2-binding regions in the C-terminus of HAUSP required for MDM2 regulation (Ma et al., 2010; Faesen et al., 2011; Rouge et al., 2016). Notably, we demonstrated HAUSP as a bona fide deubiquitinase of MDM2 where overexpression of HAUSP drives MDM2 protein stabilization (Li et al., 2004). Although HAUSP interacts with both p53 and MDM2 and exhibits deubiquitinase activities towards both proteins in vitro, HAUSP-mediated deubiquitination of MDM2 is required to maintain a sufficient level of the protein to act as an E3 ligase for p53. When HAUSP levels are reduced to a point where MDM2 becomes destabilized, the pool of MDM2 available to ubiquitinate p53 is not sufficient to degrade the protein, and the net outcome results in p53 stabilization (Li et al., 2004) (Figure 2A). Shortly after these findings, the MDM2 homology and p53 antagonist MDMX was identified as a direct interactor of HAUSP (Meulmeester et al., 2005a). Like p53 and MDM2, MDMX was identified to bind to the TRAF-like domain of HAUSP (Sarkari et al., 2010). Indeed, HAUSP deubiquitinated MDMX and partially rescued the MDM2-mediated degradation of MDMX. Furthermore, knocking down HAUSP led to MDMX degradation and indirectly activated p53 (Meulmeester et al., 2005a). Under stressed conditions, HAUSP binding to MDMX and MDM2 is less efficient; indeed, overexpression of HAUSP cannot rescue the DNA damage-induced degradation of MDMX (Meulmeester et al., 2005b; Tang et al., 2006). Interestingly, ATM-dependent phosphorylation of MDM2 and MDMX has been suggested to interrupt the binding with HAUSP after DNA damage (Meulmeester et al., 2005b).

Figure 2

Regulators and HAUSP function. (A) HAUSP deubiquitinates MDM2. Ubiquitinated MDM2 is degraded and subsequently p53 is stabilized to induce cell death or inhibit cell proliferation. (B) HAUSP activity is positively regulated by GMPS. ABRO1 positively regulates HAUSP/p53 binding, while TSPYL5 negatively regulates HAUSP/p53 binding and deubiquitination. (C) HAUSP/MDM2 binding and deubiquitination is positively regulated by DAXX and negatively regulated by RASSF1A. (D) Transcriptional activation either directly with FoxO6 or indirectly through PHF8. Ub, ubiquitin. Green triangles represent positive regulators; red triangles represent negative regulators.

Figure 2

Regulators and HAUSP function. (A) HAUSP deubiquitinates MDM2. Ubiquitinated MDM2 is degraded and subsequently p53 is stabilized to induce cell death or inhibit cell proliferation. (B) HAUSP activity is positively regulated by GMPS. ABRO1 positively regulates HAUSP/p53 binding, while TSPYL5 negatively regulates HAUSP/p53 binding and deubiquitination. (C) HAUSP/MDM2 binding and deubiquitination is positively regulated by DAXX and negatively regulated by RASSF1A. (D) Transcriptional activation either directly with FoxO6 or indirectly through PHF8. Ub, ubiquitin. Green triangles represent positive regulators; red triangles represent negative regulators.

To illustrate the physiological function of HAUSP in vivo, we previously generated a conventional Hausp knockout mouse showing early embryonic lethality between days E6.5 and E7.5, which was partially rescued through concomitant p53 depletion (Kon et al., 2010). Subsequently, we created a conditional allele of Hausp, which enabled Hausp deletion specifically in the neural progenitors when crossed to a nestin promoter-driven Cre recombinase. Hausp deletion dramatically decreased cortex thickness, inhibited neuronal cell development, and caused perinatal lethality, which was significantly improved in the p53-null background (Kon et al., 2011). Nevertheless, in contrast to the case of Mdm2 and Mdmx mutant mice (both conventional and conditional) (Marine and Lozano, 2010), inactivation of p53 failed to completely rescue the neonatal lethality of these Hausp mutant mice. Taken together, these in vivo results implicate that inactivation of HAUSP can (i) induce destabilization of MDM2, which is effective in activating p53 responses, and (ii) highlight a p53-independent network regulated through HAUSP. Although out of the scope of this review, the latter notion is further supported by many recent studies demonstrating that HAUSP is involved in modulating the stability of proteins regulating the immune response, epigenetic regulation, DNA replication, metabolism, cell proliferation, and DNA damage response (van der Horst et al., 2006; Song et al., 2008a; Huang et al., 2011; Ma et al., 2012; Colleran et al., 2013; Gao et al., 2013; van Loosdregt et al., 2013; Hao et al., 2015; Lecona et al., 2016; Mungamuri et al., 2016).

Regulators and co-factors of the HAUSP/MDM2/p53 axis

Considering the importance of the dynamic relationship between HAUSP and the MDM2/p53 axis, it is not surprising that HAUSP function/activity is also tightly regulated. To date, three separate mechanisms have been outlined that both negatively and positively regulate this pathway: (i) proteins that regulate the affinity of HAUSP/p53 binding (van der Knaap et al., 2005; Epping et al., 2011; Faesen et al., 2011; Reddy et al., 2014; Zhang et al., 2014); (ii) proteins that regulate the affinity of HAUSP/MDM2 binding (Tang et al., 2006; Song et al., 2008b; Tang et al., 2010); and (iii) transcriptional regulators of the HAUSP promoter (Hu et al., 2015; Wang et al., 2016) (Figure 2B–D).

TSPY-like 5 (TSPYL5) was identified as a novel binding partner for HAUSP through mass spectrometry. Indeed, TSPYL5 interacts with the TRAF-like domain of HAUSP, antagonizing the binding affinity of HAUSP and p53 (Epping et al., 2011). Overexpressing TSPYL5 limits the interaction between p53 and HAUSP thereby increasing ubiquitinated p53 levels, subsequently driving p53 proteolysis and diminishing downstream p53 networks. Conversely, knocking down TSPYL5 allows for a more uninhibited HAUSP/p53 complex with a net result of p53 stabilization (Epping et al., 2011). In contrast to the negative regulation from TSPYL5, Abraxas brother 1 (ABRO1) was shown to enhance the binding between HAUSP and p53 (Zhang et al., 2014). In fact, ABRO1 binds to the C-terminal HUBL domains that are known to control HAUSP activity. Therefore, elevating ABRO1 expression increases p53 stability by facilitating HAUSP-dependent p53 deubiquitination; conversely, decreasing ABRO1 levels inhibits HAUSP-mediated p53 deubiquitination driving p53 degradation (Zhang et al., 2014). Interestingly, both TSPYL5 and ABRO1 are specific for the HAUSP/p53 interaction without modulating HAUSP levels or MDM2 activity (Figure 2B).

Similar to ABRO1 binding, guanosine 5′-monophosphate synthase (GMPS) binds to HUBL domains in the C-terminus of HAUSP (Faesen et al., 2011). Mechanistically, GMPS has the unique ability to allosterically increase HAUSP catalytic activity by promoting the catalytic triad into the active conformation through stabilization of HUBL4/5 (Faesen et al., 2011). Although GMPS strongly facilitates HAUSP-mediated p53 deubiquitination, these effects apply to many HAUSP-specific substrates, as GMPS modulates HAUSP activity but not substrate-specific binding (van der Knaap et al., 2005, 2010; Reddy et al., 2014). Essentially, GMPS maintains the ability to switch HAUSP from an active to inactive state. Adding a second layer of regulation, GMPS is typically found mono-ubiquitinated by TRIM21 and sequestered in the cytoplasm away from HAUSP. Under normal conditions, HAUSP, having a higher affinity to MDM2 than p53, stabilizes MDM2 thereby maintaining a low level of p53. Upon stress, GMPS untethers from TRIM21 and shuttles into the nucleus to facilitate HAUSP/p53 deubiquitination and activate p53 signalling (Reddy et al., 2014).

The second class of regulators affects the HAUSP/MDM2 complex, thereby indirectly modulating p53 levels. Death domain-associated protein (DAXX) was identified as a positive regulator of the HAUSP/MDM2 complex, where DAXX levels enhance the interaction between HAUSP and MDM2 (Tang et al., 2006). Overexpression of DAXX increases HAUSP-mediated MDM2 deubiquitination, thereby stabilizing MDM2 and increasing p53 degradation; conversely, inhibiting DAXX stabilizes p53, since the binding and deubiquitination of MDM2 are compromised (Tang et al., 2006). In actuality, the relationship among DAXX, HAUSP, and MDM2 is complicated and dynamic: MDM2 can ubiquitinate and degrade DAXX, while HAUSP can reverse this process through deubiquitination (Tang et al., 2010). Combating the positive regulation of DAXX on HAUSP/MDM2 is the tumour suppressor Ras association domain family 1 protein (RASSF1A) (Song et al., 2008b). Mechanistically, RASSF1A disrupts the binding between individual proteins within the HAUSP/MDM2/DAXX complex. Overexpression of RASSF1A promotes MDM2 self-ubiquitination by blocking HAUSP-mediated deubiquitination thereby indirectly stabilizing p53. Conversely, inhibiting RASSF1A increases MDM2 stability, which in turn destabilizes p53 (Song et al., 2008b) (Figure 2C).

The third mode of HAUSP regulation is at the transcriptional level. Forkhead box O6 (FoxO6) motifs were identified in the HAUSP promoter region. Indeed, FoxO6 was able to bind to the HAUSP promoter and induce HAUSP transcription (Hu et al., 2015). Not surprisingly, overexpression of FoxO6 led to p53 stabilization and activation (Hu et al., 2015). Recently, the histone demethylase plant homeodomain finger-containing protein 8 (PHF8 or KDM7B) was identified as a novel binding partner and substrate for HAUSP-mediated deubiquitination (Wang et al., 2016). Interestingly, PHF8, which can act as a transcriptional co-activator indirectly regulating many target gene promoters, was also shown to regulate HAUSP transcription. A positive feedback interaction was recently observed between these two proteins. Indeed, HAUSP can stabilize PHF8 through deubiquitination and in turn, PHF8 modulates HAUSP transcription (Wang et al., 2016) (Figure 2D).

HAUSP function in cancer

Although HAUSP has been shown to regulate a wide array of substrates, the expression pattern of HAUSP was uncertain in cancers. The first report to demonstrate a role for HAUSP in cancer progression was using prostate cancer samples. Increased levels of HAUSP were directly correlated with tumour aggressiveness, as high-grade prostate cancer samples had elevated HAUSP expression compared with low-grade samples. Interestingly, HAUSP expression was correlated with nuclear exclusion of the tumour suppressor PTEN as a mechanism for enhanced tumorigenesis (Song et al., 2008a). Increased HAUSP expression at both protein and mRNA levels was also observed in multiple myeloma tumours compared with normal peripheral blood mononuclear cells (Chauhan et al., 2012). When multiple myeloma patient samples were stratified based on HAUSP expression, the patients with higher HAUSP levels showed a poorer overall and event-free survival, strongly suggesting a role of HAUSP in the pathogenesis of multiple myeloma (Chauhan et al., 2012). A similar trend was seen in gliomas where increased HAUSP expression was strongly tied with disease severity and was associated with poorer overall patient survival (Cheng et al., 2013). Similarly, high HAUSP expression predicts a poorer outcome in neuroblastoma patients (Fan et al., 2013). HAUSP can also act as an independent prognostic marker for overall survival in epithelial ovarian cancer, as high expression correlated with a worse survival outcome (Ma and Yu, 2016). Furthermore, elevated HAUSP expression strongly correlated with lymphatic invasion. Indeed, overexpressing HAUSP in ovarian cancer cells increased cell invasiveness by over 50%, while knocking down HAUSP in ovarian cancer cell lines led to a decrease in cell viability, suggesting a role for HAUSP in the pathogenesis of ovarian cancer (Ma and Yu, 2016). Additionally, HAUSP expression was elevated in non-small cell lung cancer samples (Zhao et al., 2015), excluding adenocarcinoma patients where HAUSP levels were actually lower than controls for unknown reasons (Masuya et al., 2006). Clinically, HAUSP levels inversely correlated with overall survival and could be used as an independent prognostic marker for NSCLC (Zhao et al., 2015). Furthermore, HAUSP expression correlated with cancer stage, lymph node metastasis, and tumour size (Zhao et al., 2015). In line with this, knocking down HAUSP in lung carcinoma cells resulted in decreased transwell invasion and increased apoptosis in vitro and through an in vivo xenograft model (Zhao et al., 2015). Recently, HAUSP was shown to stabilize PHF8 and thereby elevate PHF8-specific target, namely Cyclin A2, which regulates cell proliferation. Indeed, this HAUSP/PHF8 axis was shown to be dysregulated in breast cancer. HAUSP was overexpressed and strongly correlated with histological grades of breast cancer (Wang et al., 2016). Furthermore, PHF8 and Cyclin A2 were elevated where HAUSP expression was high, underscoring a role for HAUSP in breast carcinogenesis (Wang et al., 2016). In fact, knocking down HAUSP in a breast cancer cell line MCF-7 that was implanted in nude mice significantly inhibited tumour growth, at least in part, in a PHF8-dependent manner (Wang et al., 2016).

The above-mentioned studies demonstrate a specific and direct role for HAUSP in cancer, and many studies have shown strong correlation with specific HAUSP substrates, namely PTEN (Song et al., 2008a) and PHF8 (Wang et al., 2016). Indirectly, the modulators of HAUSP function are also dysregulated in cancers. TSPYL5 overexpression has a causal role in breast cancer presumably through inhibiting HAUSP/p53 binding, leading to downregulated p53 levels (Epping et al., 2011). Downregulation of ABRO1 also inhibits HAUSP/p53 binding; in fact, ABRO1 is downregulated in different cancers including liver and breast (Zhang et al., 2014). Amplification of the oncoprotein MDM2 occurs in many cancers (Wasylishen and Lozano, 2016). Conceivably, loss of the negative regulator of HAUSP/MDM2 binding would drive MDM2 stabilization and p53 degradation. Indeed, RASSF1A is inactivated in a variety of human cancers (Dammann et al., 2000; Agathanggelou et al., 2005). Although a definitive relationship has not been established between RASSF1A and HAUSP in cancer, it is tempting to speculate that diminished RASSF1A levels can stabilize MDM2 in a HAUSP-dependent manner driving p53 degradation.

Genetic manipulation of HAUSP was demonstrated using an in vivo xenograft model with isogenic colon carcinoma cell lines by either inducing HAUSP knockdown or activating HAUSP overexpression (Becker et al., 2008). Under either condition, due to mechanisms explained above, p53 levels were stabilized and activated, leading to a tumour suppressive phenotype (Becker et al., 2008). Similarly, when HAUSP was genetically deleted from the colorectal cancer cell line DLD1, tumours were significantly smaller in size compared with controls (Du et al., 2010). Collectively, these studies demonstrate that HAUSP can be used as a prognostic marker in certain cancers and underscores the value for generating small molecules against HAUSP to target certain tumour types.

First generation of HAUSP inhibitors

Considering the critical role of HAUSP in regulating the proteolysis of the oncogenes MDM2 and MDMX and indirectly controlling p53 stability, as well as regulating multiple p53-independent substrates, HAUSP has become an attractive target for therapeutic intervention in cancer. Thus, it is not surprising that small molecule inhibitors targeting the deubiquitinase activity of HAUSP have been recently developed. The first compound was characterized in 2009 (Colland et al., 2009). HBX 41108 was shown to block HAUSP-mediated deubiquitination of MDM2, which stabilized p53, independently of genotoxic stress, initiating p53-dependent cell cycle arrest and apoptosis. Mechanistically, it was demonstrated that HBX 41108 ablated HAUSP activity rather than enzyme–substrate interactions (Colland et al., 2009). As HAUSP is highly similar in structure to other cysteine proteases, specificity of these small molecules becomes paramount. Indeed, HBX 41108 can also inhibit USP5, USP8, USP10, and CYLD in vitro (Nicholson and Suresh Kumar, 2011; Reverdy et al., 2012). Therefore, the same research group used a high-throughput screening process and identified HBX 19818 and an analogue HBX 28258 as novel selective HAUSP inhibitors (Reverdy et al., 2012). Even at very high concentrations, neither compound demonstrated cross-reactivity in vitro to a panel of 10 cysteine proteases similar to HAUSP. This specificity is attributed to the fact that these compounds can covalently bind to the active site cysteine in the catalytic domain of HAUSP (Figure 1). Furthermore, both compounds were able to block HAUSP-mediated deubiquitination of MDM2 and led to subsequent p53 activation. Although HBX 19818 was able to induce cell cycle arrest and apoptosis in p53-proficient cells, similar effects were observed in cells where MDM2 cannot regulate p53 and in p53 mutant cells, suggesting that these HAUSP-mediated effects are only in part p53-dependent (Reverdy et al., 2012). Further analysis including in vivo validation is needed to rule out off-target effects and verify clinical promise of using HBX compounds.

Recently, Progenra developed a high-throughput screen, which employs advanced multiplexed enzymatic-based proteomic approaches to determine the potency and selectivity of novel compounds against many deubiquitinases (Nicholson et al., 2008; Tian et al., 2011). Out of these screens, two specific compounds selectively targeting HAUSP were discovered: P22077 (Altun et al., 2011) and P5091 (Chauhan et al., 2012). In-depth characterization of P22077 revealed potent inhibition of HAUSP as well as the closely related USP47 (Altun et al., 2011); indeed, P22077 induced tumour cell death accompanied by MDM2 degradation and subsequent p53 stabilization (Altun et al., 2011; Dar et al., 2013; Fan et al., 2013). In vivo administration of P22077-mediated inhibition of HAUSP deubiquitinase activity significantly ablated tumour cell growth in a xenograft mouse model with no observable weight loss (Fan et al., 2013). Similarly, serial P5091 treatments were well tolerated in mice xenografts (Chauhan et al., 2012). Chauhan et al. (2012) examined the effect of HAUSP inhibition in multiple myeloma and demonstrated that P5091 strikingly inhibited tumour growth and significantly prolonged mouse survival compared with controls in mouse xenografts. Increased apoptosis and blunted cell cycle were observed in the harvested tumours. Mechanistically, P5091 resulted in increased MDM2 ubiquitination and proteolysis, with subsequent p53 and p21 stabilization. Interestingly, the decreased cell viability in multiple myeloma cells was only partially rescued upon MDM2 or p21 deletion, yet the cytotoxic activity of P5091 did not depend on p53 status. Nevertheless, P5091-dependent cytotoxicity was reverted upon HAUSP deletion, and importantly, P5091 did not inhibit other proteases, suggesting that these effects are mediated by HAUSP (Chauhan et al., 2012).

This first generation of HAUSP inhibitors (summarized in Table 1), along with the development of more suitable screening assays, has created heightened enthusiasm in the field. These assays will allow for the next generation of compounds to yield increased potency while retaining selectivity.

Table 1

First generation of HAUSP inhibitors.

  Validated targets Functional consequence In vivo tumour suppression References 
HBX 41108 graphic ↓MDM2 Cell cycle arrest  N/A Colland et al. (2009), Reverdy et al. (2012) 
↑p53; p21 Apoptosis 
HBX 19818
HBX 25258 
graphic ↓MDM2; claspin Cell cycle arrest N/A Reverdy et al. (2012) 
↑p53; p21 Apoptosis 
P22077 graphic ↓MDM2; claspin; pCHK1; DDB1; TIP60 Cell cycle arrest Neuroblastoma xenograft Altun et al. (2011), Fan et al. (2013), Dar et al. (2013) 
↑p53; p21 Apoptosis 
P5091 graphic ↓MDM2; MDMX Cell cycle arrest Multiple myeloma xenografts Chauhan et al. (2012) 
↑p53; p21 Apoptosis 
Compound 14 graphic ↑p53; p21 N/A N/A Weinstock et al. (2012) 
 
  Validated targets Functional consequence In vivo tumour suppression References 
HBX 41108 graphic ↓MDM2 Cell cycle arrest  N/A Colland et al. (2009), Reverdy et al. (2012) 
↑p53; p21 Apoptosis 
HBX 19818
HBX 25258 
graphic ↓MDM2; claspin Cell cycle arrest N/A Reverdy et al. (2012) 
↑p53; p21 Apoptosis 
P22077 graphic ↓MDM2; claspin; pCHK1; DDB1; TIP60 Cell cycle arrest Neuroblastoma xenograft Altun et al. (2011), Fan et al. (2013), Dar et al. (2013) 
↑p53; p21 Apoptosis 
P5091 graphic ↓MDM2; MDMX Cell cycle arrest Multiple myeloma xenografts Chauhan et al. (2012) 
↑p53; p21 Apoptosis 
Compound 14 graphic ↑p53; p21 N/A N/A Weinstock et al. (2012) 
 

Concluding remarks

The dynamic relationship of the HAUSP/p53 pathway is complex, filled with positive and negative regulators altering the activity or ability of HAUSP to bind to p53 or to regulate MDM2 stability. Nevertheless, the advances made since our initial finding that HAUSP directly deubiquitinates p53 have justified the generation of HAUSP inhibitors with a clinical promise for developing better targeted therapies than already available to activate p53 stability. Many questions still remain regarding HAUSP biology in regulating p53: Are there more regulators that augment or antagonize HAUSP function on p53 or HAUSP function on MDM2? Are there specific transcriptional repressors of HAUSP that can be activated or more HAUSP-specific transcription factors that can be targeted in combination with the small molecules already available?

These inhibitors have shown promise in pre-clinical studies in cancers where the p53 pathway remains intact. Out of the scope of this review, HAUSP has been shown to have many p53-independent targets. It is possible that HAUSP inhibition could act in p53-mutated cancers, or in addition to activating p53, HAUSP inhibition could simultaneously inhibit oncogenic-specific proteins.

Funding

This work was supported by the National Cancer Institute, US National Institutes of Health (NIH) (5R01CA193890, 5RO1CA190477, 5RO1CA085533, and 2P01CA080058 to W.G.) and was partially supported by the NIH Cancer Biology Training Grant T32-CA09503 (O.T.).

Conflict of interest: none declared.

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