Deubiquitinase Inhibition As a Cancer Therapeutic Strategy

Pharmacology & Therapeutics 147 (2015) 32–54

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Pharmacology & Therapeutics

journal homepage: www.elsevier.com/locate/pharmthera

Associate editor: B. Teicher Deubiquitinase inhibition as a cancer therapeutic strategy

Padraig D'Arcy a,b, Xin Wang a,b, Stig Linder a,b,⁎ a Department of Medical and Health Sciences, Linköping University, SE-58183 Linköping, Sweden b Department of Oncology and Pathology, Karolinska Institute, SE-171 76 Stockholm, Sweden article info abstract

Available online 6 November 2014 The ubiquitin proteasome system (UPS) is the main system for controlled protein degradation and a key regulator of fundamental cellular processes. The dependency of cancer cells on a functioning UPS has made this an at- Keywords: tractive target for development of drugs that show selectivity for tumor cells. Deubiquitinases (DUBs, ubiquitin Cancer therapeutics isopeptidases) are components of the UPS that catalyze the removal of ubiquitin moieties from target proteins Small molecule inhibitors or polyubiquitin chains, resulting in altered signaling or changes in protein stability. A number of DUBs regulate Proteasome processes associated with cell proliferation and apoptosis, and as such represent candidate targets for cancer Deubiquitinase therapeutics. The majority of DUBs are cysteine proteases and are likely to be more “druggable” than E3 ligases. α,β-unsaturated ketones Apoptosis Cysteine residues in the active sites of DUBs are expected to be reactive to various electrophiles. Various compounds containing α,β-unsaturated ketones have indeed been demonstrated to inhibit cellular DUB activity. In- hibition of proteasomal cysteine DUB enzymes (i.e. USP14 and UCHL5) can be predicted to be particularly cytotoxic to cancer cells as it leads to blocking of proteasome function and accumulation of proteasomal substrates. We here provide an overall review of DUBs relevant to cancer and of various small molecules which have been demonstrated to inhibit DUB activity. © 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Contents

1. Introduction...... 32 2. Theproteasome...... 34 3. DUBsasdrugtargetsforcancertherapeutics...... 3435 4. SmallmoleculeDUBinhibitors...... 3443 5. Conclusions...... 3548 Conﬂictsofinterest...... 3648 References...... 3748

1. Introduction cellular metabolism. ~30% of newly synthesized proteins in mammalian cells are rapidly degraded with a half-life of b10 min (Schubert et al., Proteins are vital to the structure and function of cells, and as such 2000). Such a high rate of protein turnover requires a specialized system the regulated control of protein turnover is a fundamental aspect of for the controlled and selective degradation of unwanted proteins. The ubiquitin–proteasome system (UPS) has emerged as a key regulator of protein function and stability. At its most simple level the UPS is com- Abbreviations:19SRP,19Sregulatoryparticle;20SCP,20Scoreparticle;ASK1,apoptosis posed of a tagging factor in the form of the small molecule ubiquitin signaling kinase 1; BRCA1, breast cancer type 1 susceptibility protein; Eer1, eeyarestatin 1; which marks unwanted or damaged proteins for degradation, and the ERAD, ER-associated protein degradation; GA, gambogic acid; JAMM, JAB1/MPN/MOV34 metalloenzyme; MAPK, mitogen activated protein kinase; NFκB, nuclear factor kappa B; proteasome, a large molecular shredder that breaks down proteins PCNA, proliferating cell nuclear antigen; PG, prostaglandin; Rpn11/POH1, regulatory parti- into smaller peptides for use in other anabolic processes. More than clesubunit11/pad onehomolog-1;TLS,trans-lesionssynthesisofDNA;TRAIL,tumornecro- 80% of cellular proteins are degraded by the UPS, high-lighting the im- sis factor-related apoptosis-inducing ligand; UCH, ubiquitin carboxyl-terminal hydrolase; portance of this pathway in the regulation of multiple cellular processes ﬁ UPS, ubiquitin proteasome system; USP, ubiquitin speci c peptidase. (Rock et al., 1994). The multifaceted role of the UPS includes the degra- ⁎ Corresponding author at: Department of Medical and Health Sciences, Linköping University, SE-58183 Linköping, Sweden. Tel.: +46 70 4841275. dation of misfolded and damaged proteins, cell cycle regulators, onco- E-mail addresses: [email protected], [email protected] (S. Linder). gene and tumor suppressor proteins, as well as the regulation of

http://dx.doi.org/10.1016/j.pharmthera.2014.11.002 0163-7258/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 33 antigen processing and control of transcription factor activity (Coux ubiquitin moieties are attached, and iii) poly-ubiquitination where et al., 1996; Hershko & Ciechanover, 1998). Considering the diversity substrates are tagged with polyubiquitin chains (Jentsch & of UPS substrates it is no surprise that this pathway has been implicated Schlenker, 1995; Hicke, 2001; Di Fiore et al., 2003; Haglund et al., in the pathogenesis of many human diseases such as neurodegenerative 2003; Haglund & Dikic, 2005; Ye & Rape, 2009; Lander et al., 2012). disorders, viral diseases and cancer (Ciechanover et al., 2000). In addition to the three different classes of ubiquitination, a ubiqui- The process of ubiquitination is a multi-step process ultimately lead- tin code exists whereby the type of linkages between ubiquitin ing to the covalent modification of a protein substrate with the small mol- monomers determines function. Ubiquitin contains seven lysine res- ecule ubiquitin. Ubiquitin is a highly conserved 76 amino acid protein idues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63), any of that undergoes covalent attachment via an isopeptide bond between which can serve as sites for the covalent attachment of other ubiqui- the carboxy glycine residue (G76) of ubiquitin to the ε-amino groups of tin molecules. The nature of the linkages within the polyubiquitin lysine residues in target proteins. The process of ubiquitination is depen- chain has consequences in determining the fate of the conjoined dent on the consecutive activity of three distinct enzymes, Ub-activating protein. In general, proteins tagged with Lys48-linked polyubiquitin (E1), Ub-conjugating (E2) and Ub-ligating (E3) (Fig. 1). In the first step, chains are destined for proteasomal degradation (Chau et al., 1989; ubiquitin is activated by the E1 enzyme in the presence of ATP, forming Hershko & Ciechanover, 1998), whereas modifications involving a thioester bond between the carboxy-terminal glycine residue of ubiqui- Lys63-linked chains are more typically associated with non- tin and the active site cysteine of the E1 enzyme. Once activated, ubiquitin proteasomal roles such as DNA repair, DNA replication and signal is transferred from E1 to a cysteine residue of one of the 30–40 E2 ubiq- transduction (Haglund & Dikic, 2005). Other linkage types are gen- uitin carrier proteins. Substrate specificity is conferred by E3 ligases, erally less well characterized, although reports have shown that which bind target substrates and co-ordinate the covalent attachment polyubiquitin chains linked through Lys6, Lys11, Lys27, Lys29, or of ubiquitin. Two distinct families of E3 ligases exist, the HECT domain Lys33 can target proteins for proteasome-mediated degradation family that receives ubiquitin from the E2 ligase forming an ubiquitin- (Xu et al., 2009). Even Lys63 chains, which are more traditionally E3 intermediate, and the RING finger family of E3 ligases that form a mo- implicated in signaling, can target the attached protein to the pro- lecular bridge between the E2 ligase and target proteins. There are N500 teasome for degradation (Saeki et al., 2009). E3 ligases in cells, making them the main specificity factor in the UPS The process of ubiquitination is highly dynamic and can be reversed (Hershko et al., 1983; Voges et al., 1999; Pickart & Eddins, 2004). by the action of specialized enzymes known as deubiquitinases (DUBs). There are three different classes of ubiquitination: i) mono- DUBs oppose the action of the E3 ligases by cleaving the isopeptide bond ubiquitination where a single ubiquitin is attached, ii) multi- between lysine residues on target proteins and the C-terminal glycine of ubiquitination or poly-monoubiquitination where several single ubiquitin. Analysis of the human genome shows the presence of ~80

E2 SH SH O U + ATP AMP+PP HO 1

E1 E1 E2 S U O

S SH U O E3

E2 SH S E2 U O E3 E3

substrate N substrate substrate N NH2 U U O U U U O

Fig. 1. Ubiquitination of proteins. Proteins are targeted for degradation by the addition of ubiquitin chains to lysine residues by a process that involves three enzymes. Ubiquitin is activated by a ubiquitin-activating enzyme (known as E1) and transferred to a cysteine residue of a ubiquitin carrier protein (known as E2). E3 ligases bind target substrates and co-ordinate the covalent attachment of ubiquitin. The existence of a large number (N500) of E3 ligases makes them the main specificity factor in the UPS. Target proteins may be monoubiquitinated or, as in this example, polyubiquitinated. A target protein must be tagged with at least four ubiquitin monomers (forming a polyubiquitin chain) to be recognized by the proteasome. 34 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 functional DUBs (Komander et al., 2009), accounting for a major fraction of the ~460 functional proteases (Fortelny et al., 2014). Based on active Rpn15 site homology, DUBs can be divided into six classes: ubiquitin-specific Rpn7 proteases (USPs), ubiquitin carboxy-terminal hydrolases (UCHs), Rpn3 Rpn5 ovarian-tumor proteases (OTUs), Machado–Joseph disease protein do- Rpn6 main proteases, JAMM/MPN domain-associated metallopeptidases Rpn12 Rpn8 (JAMMs) and monocyte chemotactic protein-induced protein (MCPIP) Rpn11 19S RP (Fraile et al., 2012; Fortelny et al., 2014). Of these DUBs, the USP class Rpn10 is the most numerous, due to a rapid diversification during evolution, Rpn9 fi possibly in concert with the diversi cation of E3 ligases (Semple et al., Rpn1 2003). This process has presumably led to an increased capacity for Rpn2 Rpn13 specific ubiquitination and deubiquitination events, similar to that observed for protein phosphorylation, where both kinases and phospha- Rpt1 Rpt2 Rpt6 Rpt4 Rpt5 Rpt3 tases are important for regulatory circuits. A more detailed overview α1 α2 α3 α4 α5 α6 α7 of different DUBs is given below. β1 β2 β3 β4 β5 β6 β7 20S CP 2. The proteasome β7 β6 β5 β4 β3 β2 β1 α α α α α α α The 26S proteasome is a large ATP-dependent protease complex 7 6 5 4 3 2 1 found in the cytosol and the nucleus of eukaryotic cells (Tanaka et al., 1983; Bochtler et al., 1999; Goldberg, 2003). The 26S proteasome con- Rpt3 Rpt5 Rpt4 Rpt6 Rpt2 Rpt1 sists of ~50 different subunits with a combined molecular weight of Rpn1 ~2.5 MDa (Fig. 2). The 26S proteasome is arranged into two sub- Rpn2 Rpn13 complexes: a catalytic 20S core particle (CP) capped by one or two Rpn9 Rpn10 19S regulatory particle(s) (19S RP). 19S RP Rpn8 Rpn11 Rpn12 2.1. The 20S core particle Rpn6

The eukaryotic 20S CP is a ~730 kDa cylinder-shaped multimeric Rpn5 Rpn3 complex composed of two heptameric inner β-rings capped by two Rpn7 heptameric α-subunits (Groll et al., 1997; Unno et al., 2002). The Rpn15 outer α-rings provide attachment sites for the 19S RP as well as forming a 13 Å molecular gate to control substrate access to the catalytic cham- β β ber. The proteolytic activity of the 20S CP is mediated by the 1, 2and Fig. 2. Structure of the proteasome in higher eukaryotes. The various components of the 20S β5 subunits, which contain catalytically active threonine residues at core particle (CP) and 19S regulatory particles (RP) are shown. The 20S proteasome con- their N termini. The catalytic β-subunits are classiﬁed based on sub- tains 14 different subunits overall (α1–α7andβ1–β7) which show molecular masses be- strate preference with the β1-subunits associated with caspase-like ac- tween 20 and 30 kDa, totalling a molecular weight of ~700 kDa. The α-rings are responsible for the regulation of substrate entrance and for recognition and binding of the substrate tivity, β2-subunits with trypsin-like activity, and β5-subunits with whereas the catalytic centers are located in the β-rings. The β1 subunit shows a chymotrypsin-like activity. Substrate proteins are degraded into peptidyl–glutamyl–peptide hydrolyzing activity (“caspase-like” activity); the β2subunit oligopeptides ranging in length from 3 to 15 amino-acid residues, cleavesafterbasicaminoacids(“trypsin-like” activity); the β5 subunit cleaves after neutral which are subsequently hydrolyzed by cytosolic peptidases into free amino acids (“chymotrypsin-like” activity). The 19S regulatory particle consists of two amino acids (Puhler et al., 1992; Zwickl et al., 1992; Lowe et al., 1995; main structures: a ring-shaped base and a lid that recognizes and binds polyubiquitinated proteins. The base ring contains at least 10 different subunits (Rpt1–Rpt6, Rpn1, Rpn2, Groll et al., 1997). Alternatively, proteasome-processed oligopeptides Rpn10 and Rpn13). The lid contains 9 subunits (Rpn3, Rpn5–Rpn9, Rpn11, Rpn12 and may be taken up by the TAP1 complex and loaded onto histocompatibil- Rpn15). The Rpt-subunits display ATPase activity (Rpt: regulatory particle ATPase), Rpn- ity complex (MHC) class I molecules for presentation to the immune subunits do not (Rpn: regulatory particle non-ATPase). Rpn11 (POH1) is a metalloprotease system (Saeki & Tanaka, 2012). with DUB activity.

2.2. The 19S regulatory particle constitutive ubiquitin receptors, Rad23, Dsk2 and Ddi1, also associate with the proteasome and modulate the binding of ubiquitinated cargo. The 19S RP is a ~930 kDa complex consisting of at least 19 different The extrinsic ubiquitin receptors contain ubiquitin-like (UBL) domains subunits that can be further divided into lid and base sub-complexes that bind to the 19S RP subunits Rpn1, Rpn10 and Rpn13 and (Glickman et al., 1998; Lander et al., 2012)(Fig. 2). The base of the 19S ubiquitin-associated (UBA) domain that bind polyubiquitinated sub- RP is composed of ten subunits, six of which are related AAA+ ATPases strates (Hartmann-Petersen & Gordon, 2004). In addition to binding (Rpt1–Rpt6) that form a hetero-hexameric ring (Tomko et al., 2010). ubiquitin, Rpn13 serves also as a receptor for the DUBs Uch37/UCHL5, The ATPase ring plays an important role in the opening of the gated thus providing a link between chain recognition and disassembly channel to the 20S CP as well as utilizing the hydrolysis of ATP to drive (Husnjak et al., 2008). the unwinding and translocation of ubiquitinated proteins through the In order to facilitate the degradation of proteasome-targeted sub- narrow pore into the 20S CP for degradation (Smith et al., 2007; Martin strates, specialized proteasome-associated DUBs function to remove et al., 2008; Rabl et al., 2008; Zhang et al., 2009; Maillard et al., 2011; bulky ubiquitin moieties that may otherwise impede entry to the 20S Erales et al., 2012). The other four base subunits are the scaffolding pro- CP (Verma et al., 2002; Yao & Cohen, 2002). This process also leads to teins Rpn1 and Rpn2 and the ubiquitin receptors S5a/Rpn10 and Rpn13. recycling of free ubiquitin for further use by the UPS (Fig. 3). Such These receptors display preferences for the type of ubiquitin complexes DUB activity also provides a care taker function by clearing of captured. Rpn10 contains two C-terminal ubiquitin-interacting motifs substrate-free polyubiquitin chains that may otherwise become stuck (UIMs) that cooperate to bind polyubiquitin chains (Deveraux et al., to the proteasome. 1995; Elsasser et al., 2004; Finley, 2009), whereas, Rpn13 binds to Three DUBs are associated with the proteasome: Rpn11/POH1, Lys48-linked di-ubiquitin with high afﬁnity. In addition, three non- Ubp6/USP14 and Uch37/UCHL5 (yeast/human nomenclature). Rpn11/ P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 35

POH1 is a metalloprotease which belongs to the JAMM domain family observations could be explained by USP14 being required for ubiquitin and is an integral part of the 19S RP lid. Uch37/UCHL5 and Ubp6/ turnover in particular cellular compartments. USP14 are cysteine proteases and members of the ubiquitin C- Ubp6/USP14 is also involved in the regulation of gate opening of the terminal hydrolase (UCH) and ubiquitin specific protease (USP) fami- 20S core particle. Binding of polyubiquitin conjugates to the 26S protea- lies, respectively. Both UCHL5 and USP14 are physically associated some increases peptide hydrolysis by increasing 20S gate opening. with the base complex of the 19S RP, and their DUB activity is stimulat- Polyubiquitin conjugates interact with Ubp6/USP14 and in this way ed upon proteasome incorporation. stimulate gate opening, enabling the substrate to be degraded (Peth Rpn11/POH1 is essential for viability in yeast and metazoan cells et al., 2009). Efficient proteolysis is a multistep process and Ubp6/ (Rinaldi et al., 1998; Gallery et al., 2007). In addition to its function as USP14 is clearly critical in integrating these multiple reactions. While a DUB, Rpn11/POH1 is essential for 26S proteasome structure and activ- helping to remove the ubiquitin chain, it also enhances gate opening ity (Lundgren et al., 2003; Gallery et al., 2007). Rpn11/POH1 contains a by the ATPase ring in order to ensure efficient destruction of the sub- JAMM/MPN+ motif sequence containing two histidine residues and an strate. It has been found that most of the cellular Ubp6/USP14 is not as- aspartic residue coordinating a zinc ion, which is important for proteo- sociated with the proteasome, indicating that it may be involved in lytic activity (Maytal-Kivity et al., 2002; Ambroggio et al., 2004). The ac- other cellular processes (Koulich et al., 2008). tivity of Rpn11/POH1 is thought to be delayed until the proteasome is The Uch37/UCHL5 deubiquitinase is well conserved from fungi to committed to degrade the substrate (Verma et al., 2002; Yao & Cohen, humans (Yao et al., 2006). An orthologue of human Uch37 has not 2002; Lee et al., 2011). Rpn11/POH1 cleaves the proximal end of the been found in S. cerevisiae. The orthologue in Saccaromyces pombe, polyubiquitin chain from the substrate, resulting in the release of a Uch2, is nonessential for viability (Li et al., 2000). Uch37/UCHL5 appears free ubiquitin chain. To allow cleavage without disengaging from the re- to be reversibly associates with the proteasome (Hamazaki et al., 2006; ceptor, an ubiquitin chain must be long enough to span the distance be- Jorgensen et al., 2006; Qiu et al., 2006; Yao et al., 2006). In contrast to tween the receptor and the DUB. At least four ubiquitin moieties are Rpn11/POH1, Uch37/UCHL5 is not important for the activity or the necessary to span the distance between receptors Rpn10 or Rpn13 structure of the 26S proteasome. The isopeptidase enzyme activity of and Rpn11/POH1 (Verma et al., 2002; Yao & Cohen, 2002; Lander Uch37/UCHL5 is enhanced after binding of the protein to the 26S pro- et al., 2012). teasome (Koulich et al., 2008) via the Rpn13/Admr1 receptor in the The DUB USP14 is important for ubiquitin recycling. This DUB is not a 19S RP base complex. The UCH-domain contains an active-site cross- constitutive proteasome subunit and reversibly associates with the over loop which must be displaced to allow substrate entry. This auto- Rpn1 subunit of the 19S RP base. Association of USP14 with the protea- inhibitory function is reversed by binding of Uch37/UCHL5 to Rpn13/ some results in enhanced DUB activity (~1000 fold) when compared to Admr1 of the proteasome (Hamazaki et al., 2006; Qiu et al., 2006; Yao unbound enzyme (Borodovsky et al., 2001). The USP14 protein contains et al., 2006). Similar to Ubp6/USP14, Uch37/UCHL5 removes ubiquitin an N-terminal 9-kDa UBL domain and a 45-kDa catalytic domain. from the distal tip of polyubiquitin chains. While Ubp6/USP14 is able Cys114, His435, and Asp451 form a catalytic triad in the active site of to release di- and tri-ubiquitin from chains, Uch37/UCHL5 releases free USP14, and the catalytic mechanism of USP14 appears to parallel only monoubiquitin (Lam et al., 1997; Hanna et al., 2006). Uch37/ that of the papain family of cysteine proteases (Hu et al., 2005). USP14 UCHL5 cleaves both Lys48- and Lys63-linked polyubiquitin chains is structurally related to USP7 (HAUSP). An important difference is (Jacobson et al., 2009). It is believed that Uch37/UCHL5 suppresses pro- that the catalytic site is misaligned in free USP7 and is properly formed tein degradation by shortening the chains of inappropriately or poorly after binding of ubiquitin (Hu et al., 2005). The ubiquitin binding pocket modified substrates (Lam et al., 1997; Koulich et al., 2008). In contrast, of Ubp6/USP14 is blocked by two loops which must be removed in order a recent study has suggested that Uch37/UCHL5 promotes the degrada- to catalyze deubiquitination (Hu et al., 2005). It has been proposed that tion of specific proteasome substrates, nitric oxide synthase and IκB-α the binding of Ubp6/USP14 to the base of the 19S RP induces a confor- (Mazumdar et al., 2010). Thus, Uch37/UCHL5 appears to suppress the mational change in the two loops to make the active site for ubiquitin degradation of some substrates while promoting the degradation of accessible (Hu et al., 2005). Lys48-linked polyubiquitin chains are the others. preferable substrates for Ubp6/USP14 and are cleaved from their distal The exact difference in catalytic function between Ubp6/USP14 and tips (Hu et al., 2005; Hanna et al., 2006). Uch37/UCHL5 is not clear. Double knockdown of Ubp6/USP14 and Ubp6/USP14 was shown to inhibit proteasome activity by delaying Uch37/UCHL5 results in inhibition of cell growth, decreased protein the breakdown of proteins by the proteasome (Lee et al., 2010, 2011). degradation, and accumulation of polyubiquitinated proteins, a pheno- It is suggested that Ubp6/USP14 prevents deubiquitination of the pro- type similar to that observed after knock-down of Rpn11/POH1 teasome substrate by Rpn11/POH1. This allows the substrate to be (Lundgren et al., 2003). RNAi-mediated down-regulation of either docked at the proteasome for a longer time, thus resulting in more ex- DUB alone creates a complete opposite phenotype where cell growth tensive trimming of ubiquitin chains, which reduces substrate binding is not affected and reduced levels of polyubiquitinated proteins are ob- affinity to the proteasome and favors release back to the cytosol served, indicating that each enzyme could compensate for loss of func- (Hanna et al., 2006; Lee et al., 2010). The Saccaromyces cerevisiae tion of the other (Koulich et al., 2008). orthologue of USP14, Ubp6, is nonessential for cell viability (Guterman & Glickman, 2004), although it appears important for cell survival fol- 3. DUBs as drug targets for cancer therapeutics lowing metabolic stress. In mammalian cells, knock-down of USP14 had no detectable effect on proteasome structure or the accumulation In the following section we will discuss parts of the literature rele- of polyubiquitin (Koulich et al., 2008). The small molecule USP14 inhib- vant to the role of DUBs in cancer, as well as the therapeutic potential itor IU1 was shown to reduce chain trimming and stimulate proteasome of targeting DUBs as a treatment option for cancer (see also excellent re- degradation, indicating the ability of USP14 to inhibit the proteasome views by Nijman et al. (2005b), Hussain et al. (2009), Komander et al. through its deubiquitinating activity (Lee et al., 2010). Although non- (2009), Sacco et al. (2010), Ramakrishna et al. (2011), and Eletr and essential for cell survival, USP14 does appear to be important for normal Wilkinson (2014)). Several DUBs have been described to play essential neuronal development. USP14 mutant mice (axJ/axJ mice) develop se- roles in the regulation of numerous cellular processes, particularly those vere tremors, hindlimb paralysis and die by 6–10 weeks of age frequently altered in tumorigenesis, e.g. cell cycle control, cell signaling (Wilson et al., 2002). In these mice, the levels of monomeric ubiquitin and apoptosis. We will briefly describe these DUBs and give examples of are decreased at synapses, suggesting that decreased recycling of ubiq- their roles in the etiology of cancer. Several functional screens have been uitin at synapses cannot be fully compensated by axonal transport of performed to identify DUBs involved in complex processes such as DNA newly synthesized ubiquitin (Chen et al., 2009). These different repair, cell cycle regulation and receptor signaling. These studies have 36 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54

poly-ubiquitin chain

U U U U U U U U U unfolding U binding U U U U U

substrate

U U U U de-ubiquitination

release

translocation and hydrolysis

Fig. 3. Proteolysis by the proteasome. Polyubiquitinated proteins are recognized and bind to the 19S regulatory particle by an ATP-dependent mechanism. Substrates must be partially unfolded before they enter into the catalytic chamber of the 20S core particle. Substrate unfolding is an energy-dependent process. The polyubiquitin chain is removed by proteasome- associated DUBs (deubiquitinases, ubiquitin isopeptidases) prior to translocation of the substrate into the 20S particle. Proteolysis occurs within the central chamber, generally resulting in ~7–9 amino acid peptides. generally found that multiple DUBs are involved in these processes, sug- early stage tumors, suggesting a role for these enzymes in disease pro- gesting considerable redundancy in DUB-regulated processes. gression. Components of the UPS regulating H2B ubiquitination status may represent new therapeutic targets for the treatment of cancer 3.1. Examples of cellular processes involving DUBs (Cole et al., 2014). Consistent with this concept, compounds that inhibit the UPS have been shown to act in synergy with clinically used DNA The process of ubiquitination has been shown to play essential roles damaging drugs. for DNA-repair and DNA-damage response pathways. These pathways As recently reviewed (Ramakrishna et al., 2011), a number of DUBs typically involve the mono-ubiquitination of key DNA-repair proteins have been shown to regulate the process of programmed cell death (ap- that have important regulatory functions in processes such as homolo- optosis), either positively or negatively. These authors listed 14 DUBs gous recombination and trans-lesion DNA synthesis (Huang & with apoptosis promoting activity, including USP7, USP9x, USP28 and D'Andrea, 2006). One of the most studied DUBs in DNA repair is USP1, CYLD. Only 3 DUBs were regarded as negative regulators of apoptosis a negative regulator of FANCD2 mono-ubiquitination (Nijman et al., (A20, USP18 and UCHL3). From a perspective of small molecule inhibi- 2005a). USP1 also deubiquitinates PCNA (proliferating cell nuclear anti- tors as cancer therapeutics, the latter category is of course of immediate gen), an important component of the trans-lesions synthesis (TLS) re- interest. pair pathway (Huang et al., 2006). USP28 is required for the The UPS may be particularly important for the interferon response. stabilization of Chk2 and 53BP1 following DNA damage (Zhang et al., The ubiquitin-like (Ubl) protein ISG15 (interferon-stimulated gene 2006) and loss of USP28 is predicted to increase the susceptibility to product of 15 kDa) shows significant homology to ubiquitin and is cova- ionizing radiation. The DUB BRCC36 is a constituent of the BRCC lently attached to target proteins in a similar manner (Haas et al., 1987; (BRCA1 and BRCA2 Containing Complex) (Dong et al., 2003). This com- Loeb & Haas, 1992). A number of DUBs have been shown to have impor- plex is required for the response to ionizing radiation and for maintain- tant roles in interferon signaling, including OTUD5 (DUBA) (Kayagaki ing a G2 DNA checkpoint. BRCC36 counteracts the ubiquitination of et al., 2007), OTUB1/OTUB2 (Li et al., 2010), USP3 (Cui et al., 2014), H2AX and H2A to terminate the double-strand break response USP17 (Chen et al., 2010) and USP25 (Zhong et al., 2013). Conversely, (Sobhian et al., 2007). viruses have been shown to encode DUB enzyme activities that counter- Ubiquitination of H2B leads to a more open chromatin structure, en- act interferon induction as a means of escaping innate immune re- hancing the accessibility for transcription factors and DNA repair pro- sponses (Wang et al., 2011; van Kasteren et al., 2012). teins. The ubiquitination of H2B is regulated by the E3 ligase action of The UPS is involved at multiple levels of the Met receptor tyrosine ki- the RNF20–RNF40 complex and the opposing activity by a number of nase pathway (Buus et al., 2009). The scattering response of epithelial DUBs, including USP7, USP22 and USP44. Many advanced cancers typi- cells is quite complex and includes loss of cell–cell adhesion and induc- cally display low levels of ubiquitinated H2B compared to normal or tion of cellular motility. Using siRNA library screening, 12 DUBs were P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 37 identified to be required for hepatocyte growth factor (HGF)-dependent 2007; Allende-Vega et al., 2010). In distinction to USP7/HAUSP, Usp2a scattering response of A549 cells, including USP3, USP30, USP33, USP47 does not appear to bind to p53. Overexpression of USP2a leads to in- and ATXN3L (Buus et al., 2009). creased cellular levels of Mdm2/MdmX and degradation of p53 A number of DUBs are involved in direct or indirect regulation of the (Fig. 4). As expected, suppression of endogenous USP2a leads to desta- stability of the p53 tumor suppressor protein: USP2, USP7, USP10, bilization of Mdm2 and accumulation of p53 protein. Usp2a has also USP22, USP42 and OTUD5. A large body of evidence points to mutant been shown to interact with and deubiquitinate Aurora-A (Shi et al., p53 in tumors having gain-of-function and decreasing the stability of 2011). The Aurora-A protein is localized to centrosomes and is essential mutant p53 may be a viable therapeutic strategy. Similar to wild-type for centrosome duplication (Meraldi et al., 2004). Knockdown of USP2a p53, mutant p53 is regulated by Mdm2 (Terzian et al., 2008). leads to reduced protein levels of Aurora-A and abnormal mitosis. Fur- ther studies have shown that Usp2a targets cyclin A1 (Kim et al., 3.2. Genetic alterations affecting DUB genes are found in human tumors 2012) and cyclin D (Shan et al., 2009), leading to increases in the expression of this protein and enhancement of cell proliferation. Fatty Mutations in genes encoding DUBs have been detected in human acid synthase is known to be overexpressed in many epithelial tumors cancers, illuminating the importance of DUBs in processes of direct im- and important for cell survival (Dhanasekaran et al., 2001) and USP2a portance for cancer cell biology. Germline mutations in the CYLD gene has been demonstrated to interact with this enzyme (Graner et al., were identified in kindreds with familial cylindromatosis and sporadic 2004). USP2a is androgen-regulated and overexpressed in prostate can- cylindromas (Bignell et al., 2000) and also in patients with Brooke– cer, and functional inactivation of the DUB has been shown to enhance Spiegler syndrome and familial trichoepithelioma (Poblete Gutierrez apoptosis of prostate cancer cells (Graner et al., 2004). Inhibition of et al., 2002). These are autosomal dominant inherited diseases associat- Usp2a may be a general strategy to activate p53 in tumor cells ed with the development of multiple skin tumors of the head and neck. (Allende-Vega & Saville, 2010), resulting in the induction of tumor apo- Mutations are thought to affect the catalytic activity of CYLD enzyme. ptosis, at least under some conditions. For example, siRNA depletion of Aberrant USP6 expression, resulting from gene translocation, has been USP2a induced moderate levels of apoptosis in prostate cancer cell lines found to be causative in most instances of aneurysmal bone cysts after 48 h (Graner et al., 2004). In contrast to these reports, it has been (Oliveira et al., 2004), locally aggressive bone lesions that occur during reported that knock-down of USP2c results in apoptosis, while targeting the first two decades of life (Rapp et al., 2012). The translocation, USP2a does not affect cell survival (Mahul-Mellier et al., 2012). USP2a is t(16;17)(q22;p13), fuses the promoter for the osteoblast cadherin 11 a candidate therapeutic target in oncology, the inhibition of which is ex- gene to the full-length USP6 gene resulting in upregulated USP6 tran- pected to lead to decreased cell proliferation (Fig. 4). scription. A20 (TNFAIP3/tumor necrosis factor alpha-induced protein USP5 is commonly referred to as isopeptidase T and is known to 3) is required to terminate NFκB signaling in response to tumor necrosis show specificity for unanchored polyubiquitin chains (Hadari et al., factor. Inactivating A20 mutations were reported to be frequently occur- 1992). Deletion of the yeast homolog of the USP5 gene (Ubp14) results ring in cases of marginal zone lymphoma (Novak et al., 2009). Further- in accumulation of free ubiquitin chains. It was proposed that more, an almost complete loss of A20 mRNA expression has been isopeptidase T facilitates proper proteasome function by preventing un- observed in cases of Non-Hodgkin's Lymphoma (Durkop et al., 2003). anchored ubiquitin chains competing for ubiquitin receptors on the 19S Various types of genetic alterations have been found in the gene proteasome (Amerik et al., 1997). Suppression of USP5/isopeptidase T encoding the BAP1 deubiquitinase in various diseases, including lung has been shown to increase both the levels and transcriptional activity and breast tumors, clear cell renal cell carcinomas and malignant pleural of p53 without altering Mdm2 stability (Dayal et al., 2009). The mecha- mesotheliomas (Buchhagen et al., 1994; Jensen et al., 1998; Jensen & nism whereby USP5 knockdown stabilizes p53 is by decreasing its Rauscher, 1999; Bott et al., 2011; Pena-Llopis et al., 2012). Mutations oc- proteasome-mediated degradation. It was proposed that p53 is stabi- curring in BAP1 were reported to lead to loss of deubiquitinating activity lized due to competition between unanchored polyubiquitin chains (Ventii et al., 2008). Finally, the USP42 gene is a fusion partner in the and ubiquitinated p53 in cells (Dayal et al., 2009). (7;21)(p22;q22) translocation in acute myeloid leukemia (AML) USP7, also referred to as HAUSP, has also been identified as a key reg- (Paulsson et al., 2006). ulator of p53 activity (Fig. 4). p53 levels are predominantly regulated by the E3 ubiquitin ligase Mdm2, leading to low intracellular concentra- 3.3. Association of DUBs with processes relevant to cancer tions during normal homeostasis. USP7 was first identified as Herpes virus-associated cellular factor (HAUSP) (Everettetal.,1997)andlater A short overview of DUB activities relevant to cancer is presented shown to deubiquitinate and stabilize p53 (Li et al., 2002). USP7 can below. We have focussed on mechanisms with potential translational also deubiquitinate and stabilize Mdm2 providing an alternative level potential in cancer. The field is growing rapidly, and due to space restric- of p53 regulation (Cummins et al., 2004; Li et al., 2004). Polycomb re- tions the overview is not complete. pressive complex 1 (PRC1) is known to monoubiquitinate histone USP1 is involved in the DNA damage response (Nijman et al., 2005a). H2A. Both USP7 and USP11 co-purify with human PRC1-type complexes USP1 regulates DNA repair and the Fanconi anemia pathway through its and regulate the ubiquitination of some components of these com- association with UAF (USP1 associated factor 1; WDR48) and through plexes (Maertens et al., 2010). Removal of USP7 or USP11 in primary its deubiquitination of two critical DNA repair proteins, FANCD2-Ub human fibroblasts results in increased expression of the INK4a tumor and PCNA-Ub. USP1 is activated by complex formation with UAF1 suppressor and proliferative arrest (“senescence”). (USP1 associated factor 1; WDR48). USP1 deubiquitinates the DNA rep- USP8/UBPY (the mammalian ortholog of budding yeast Ubp4/Doa4) lication processivity factor, PCNA, as a safeguard against error-prone was described as a DUB that accumulates upon growth stimulation of translesion synthesis of DNA (replication at sites of DNA damage). Ultra- human fibroblasts (Naviglio et al., 1998). Down-regulation of USP8/ violet (UV) irradiation inactivates USP1 through an autocleavage event, UBPY prevents fibroblasts from entering S-phase in response to serum thus enabling monoubiquitinated PCNA to accumulate and to activate stimulation (Naviglio et al., 1998). USP8 has subsequently been found translesion DNA synthesis (Huang et al., 2006). UAF1-deficient cells to be involved in EGFR receptor turnover. Following ligand stimulation, have severe defects in homologous recombination (HR)-mediated the EGFR is ubiquitinated by the E3 ligase Cbl, resulting in receptor in- double-strand break (DSB) repair and are hypersensitive to DNA dam- ternalization. The internalized receptor is subsequently deubiquitinated aging agents such as camptothecin (Murai et al., 2011). by USP8 prior to lysosomal degradation (Alwan et al., 2003; Row et al., USP2 and its different isoforms have been extensively studied by 2006; Alwan & van Leeuwen, 2007). cancer biologists. USP2a is known to associate with Mdm2 and MdmX FLIPS is a suppressor of TRAIL-induced apoptosis. FLIPS stability is and has the capacity to deubiquitinate these proteins (Stevenson et al., controlled by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 38 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54

A U U U U U U U U U U U p53 U p53 p53 p53 p53 U p53 U U p53 U Mdm2/ Mdm2/ MdmX MdmX

+ USP2a + USP2a

U U U U Mdm2/ MdmX

E3 ligase

U U U U U U U U U U U U U U U U U U U U Aurora-A Aurora-A Aurora-A Aurora-A Aurora-A USP2a USP2a

E3 ligase U U U U U U U U U U U U U U U U U U U U Cyclin A1/D1 Cyclin A1/D1 Cyclin A1/D1 Cyclin A1/D1 Cyclin A1/D1 USP2a USP2a

B U U U U

p53 HDM2

U U U U USP7 USP7 U U U U

U U U U p53 HDM2

Fig. 4. USP2a and USP7 control the stability of p53 and other important cellular regulators. (A) USP2a deubiquitinates and stabilizes Mdm2/MdmX. Suppression of USP2a leads to destabilization of Mdm2 and accumulation of p53 protein (right). Usp2a has also been shown to deubiquitinate Aurora-A (a protein essential for centrosome duplication). Downregulation of USP2a leads to decreased levels of Aurora-A protein and abnormal mitosis (right). Usp2a also deubiquitinates and stabilizes cyclin A1 and cyclin D; inhibition of the DUB leads to decreased levels of these proteins (right). (B) USP7 deubiquitinates both p53 and HDM2. Inhibition of USP7 enzyme activity leads to stabilization of p53 protein.

4) (Panner et al., 2010). The stability of the AIP4 ligase is in its turn reg- degradation of this receptor (Diamonti et al., 2002; Qiu & Goldberg, ulated by USP8. Interestingly, USP8 levels are regulated by the PTEN– 2002). Akt-mediated phosphorylation of the USP8 threonine residue Akt signaling pathway (Panner et al., 2010). Increased AKT phosphory- T907 has been found to regulate USP8 stability (Cao et al., 2007). Expo- lation therefore leads to decreased levels of FLIPS and resistance to TRAIL sure to ErbB3 ligand (neuregulin-1; NRG1) stabilizes USP8, leading to (Fig. 5). USP8 is required for stabilization of another E3 ubiquitin ligase, stabilization of Nrdp1. Nrdp1 (Wu et al., 2004a)(Fig. 5). Nrdp1 is involved in the regulation of USP9x is an X-linked ubiquitin speciﬁc protease (also known as steady-state ErbB3 levels by mediating growth factor-independent FAM; fat facet in mouse). The corresponding Drosophila protein faf (fat P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 39

PTEN wt PTEN loss

pAkt pAkt

USP8 USP8 USP8 USP8 U U U + U U U U U U U AIP4 AIP4 AIP4 AIP4

U + U U U U

U FLIPs FLIPs U FLIPs

PS U U U

FLIPs Trail sensitive Trail resistant

Fig. 5. USP8 regulates sensitivity to TRAIL. The stability of the AIP4 E3 ubiquitin ligase (atrophin-interacting protein 4) is regulated by USP8. AIP4 regulates the stability of FLIPS. The protein levels of USP8 levels are under control of the PTEN–Akt signaling pathway (USP8 T907 is phosphorylated by Akt). Since FLIPS is a suppressor of TRAIL-induced apoptosis, the activity level of the PTEN–AKT pathway is a determinant of the degree of TRAIL sensitivity (increased AKT activity leads to increased levels of FLIPS). facet) is required for cellularization in early embryos and for cell-fate preneoplasia using an approach of transposon-mediated insertional determination in the Drosophila eye (Fischer-Vize et al., 1992). USP9x mutagenesis (Perez-Mancera et al., 2012). Low USP9X protein and is an essential component of the TGF-β signaling pathway. The activity mRNA expression in human pancreatic ductal tumor was found to cor- of the Smad4 transcription factor is impeded by monoubiquitination relate with poor patient survival. Finally, USP9x has been shown to sta- of lysine 519, a process which is counteracted by USP9x (Dupont et al., bilize the pro-survival protein MCL1 (Schwickart et al., 2010). MCL1 is 2009). USP9x has also been implicated in the regulation of MAPK path- expressed at low levels in most cell types due to rapid turn-over due ways. USP9X supports ASK1-mediated signaling by preventing to the action of ubiquitin ligases, but is expressed at high levels in hema- proteasomal degradation of activated ASK1 (Nagai et al., 2009). USP9x tological malignances such as B-cell lymphomas, chronic myeloid leuke- was recently identiﬁed as a tumor suppressor gene for pancreatic ductal mia and multiple myeloma. A correlation between USP9x expression

Nucleus Cytoplasm Nucleus Cytoplasm ATM

U U P U U Usp10 Usp10 p53 Usp10 U U U U p53 p53 p53 U p53 U U p53 Mdm2 Mdm2 U U p53 Targets p53 Targets U U U p53 p53

Unstressed Genotoxic stress

Fig. 6. USP10 regulates p53 localization and stability. USP10 is a mainly cytoplasmic DUB which is phosphorylated by ATM (Ataxia telangiectasia mutated) after DNA damage. Phosphor- ylated USP10 translocates to the nucleus where it deubiquitinates p53 and reverses Mdm2-induced p53 nuclear export and degradation. Increased USP10 expression in p53 mutant cancer cells promotes cell proliferation and downregulation of USP10 inhibits cancer cell growth. Adapted from Yuan et al. (2010). 40 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 and MCL1 levels was reported in human follicular lymphomas and dif- has been suggested to show a quality control-type function, prevent im- fuse large B-cell lymphomas (Schwickart et al., 2010). proper autoubiquitination of labile ligases (Hetfeld et al., 2005; Wee USP10 is a mainly cytoplasmic DUB which deubiquitinates p53 and re- et al., 2005). verses Mdm2-induced p53 nuclear export and degradation (Fig. 6)(Yuan USP16 (also known as Ubp-M) is phosphorylated at the onset of mi- et al., 2010). USP10 is stabilized after DNA damage and translocates to the tosis by cdc-2/cyclin B complexes (Cai et al., 1999). USP16 is responsible nucleus to activate p53. USP10 is phosphorylated by ATM (Ataxia telangi- for deubiquitinating H2A during mitosis. H2A deubiquitination by USP16 ectasia mutated) at Thr42 and Ser337. USP10 suppresses tumor cell is a prerequisite for subsequent phosphorylation of histone H3 on Ser10 growth in cells with wild-type p53. Increased USP10 expression in mu- and for chromosome segregation during mitosis (Joo et al., 2007). tant p53 background increases p53 levels and promotes cancer cell prolif- USP17 has been shown to have a critical role in cell migration and to eration, while downregulation of USP10 inhibits cancer cell growth. be a potential target for anti-metastatic therapy (de la Vega et al., 2011). USP10 also suppresses ubiquitination of the sirtuin family histone Depletion of USP17 blocks chemokine-induced subcellular relocalization deacetylase SIRT6, leading to protection of SIRT6 from degradation by of GTPases essential for cell motility (Cdc42, Rac and RhoA). USP17 also the proteasome (Lin et al., 2013). This indirectly leads to decreased tran- negatively regulates the activity of Ras-converting enzyme 1 (RCE1) scriptional activity of c-Myc, and inhibition of cancer cell proliferation and (Burrows et al., 2009). RCE1 cleaves RAS at its C-terminal CAAX motif tumor formation. USP10 is not exclusively found in the cytoplasm and has and expression of USP17 leads to impaired Ras membrane localization indeed been reported to deubiquitinate the histone variant H2A.Z (Draker and activation. et al., 2011). Interestingly, other histone-modifying enzymes, including USP18 is a deubiquitinase of the ISG15 protein (ISG15: interferon- histone acetyltransferases, the deacetylase SirT1 (Vaziri et al., 2001)and stimulated gene 15) (Malakhov et al., 2002). Mice that are genetically the demethylase LSD1 (Huang et al., 2007),haveallbeenshowntotarget defective in USP18 are hypersensitive to interferon. USP18 was subse- and modify p53 as well. The autophagy regulator Beclin1 has been report- quently shown to block the interaction between JAK kinase and the ed to control the stability of USP10, and also of USP13 (Liu et al., 2011a). IFN receptor (Malakhova et al., 2006). All-trans-retinoic acid treatment USP11 has been identified as an IκBα-associated deubiquitinase ca- increases USP18 expression in acute promyelocytic leukemia (APL) pable of deubiquitinating IκBα in vitro (Sun et al., 2010). IκBα cells, leading to stabilization of the PML/RARα protein. USP18 knock- ubiquitination is required for IκBα degradation and NFκB activation. down decreases PML/RARα protein levels and inhibits APL cell prolifer- Knock-down of USP11 expression enhances TNFα-induced IκBα ation (Guo et al., 2010). ubiquitination and NF-κB activation. USP11 is therefore important for USP19 has been described to be involved in a number of cellular pro- downregulation of TNFα-mediated NFκB activation by modulating cesses. USP19 is a membrane-anchored DUB localized to the endoplas- IκBα stability. USP11 is also involved in the regulation of TGFβ signaling mic reticulum and a key component of ERAD (endoplasmic-reticulum by deubiquitinating the type I TGFβ receptor (Al-Salihi et al., 2012). associated degradation) (Hassink et al., 2009). USP19 has been sug- USP11 interacts with SMAD7 and enhances TGFβ signaling. Finally, the gested to participate in a late step of the protein quality-control machin- USP11 protein has been found in complexes with the DNA damage ery by rescuing ERAD substrates that have been retro-translocated to repair-associated protein BRCA2 (Schoenfeld et al., 2004). BRCA2 does the cytosol. Interestingly, USP19 contains a co-chaperone-like domain, not, however, appear to be a physiologic substrate of USP11 and may in- which may be involved in the proposed rescue process. USP19 also in- stead function as a molecular bridge between USP11 and its substrates. teracts with HIF-1α, promoting its stability and an appropriate hypoxia USP12 deubiquitinates non-activated Notch and is required for the response (Altun et al., 2012). USP19 stabilizes the KPC1 ubiquitin ligase, lysosomal degradation of this protein (Moretti et al., 2012). USP12 involved in regulation of the p27Kip1 cyclin-dependent kinase inhibitor down-regulation leads to an increased level of Notch molecules at the (Lu et al., 2009). Depletion of USP19 by RNA interference leads to cell surface. USP12 (and USP46) also acts as a histone H2A and H2B p27Kip1 accumulation and inhibition of the proliferation of fibroblasts deubiquitinase that regulates Xenopus development (Joo et al., 2011). (Lu et al., 2009). The ability of USP19 to regulate cell proliferation and USP13 is a deubiquitinating enzyme for MITF (microphthalmia-as- p27Kip1 levels appears to be cell context-dependent, and is lost when fi- sociated transcription factor), the activity of which leads to stabilization broblasts are transformed by an oncogenic form of Ras (Lu et al., 2011). and upregulation of MITF protein levels. MITF is a basic helix–loop– USP19 also interacts with the inhibitors of apoptosis (IAPs) c-IAP1 and helix-leucine zipper transcription factor and an important regulator in c-IAP2 (Mei et al., 2011). Knockdown of USP19 decreases levels of the development and survival of melanocytes. Amplification of MITF is these c-IAPs, whereas overexpression results in increases in the levels oncogenic in 10–20% of melanomas (Garraway et al., 2005) and inhibi- of these apoptosis inhibitors. Knock-down of USP19 enhances TNFα-in- tion of MITF induces melanoma cell death regardless of whether the duced caspase activation and apoptosis in a c-IAP1- and c-IAP2- gene is amplified or not (McGill et al., 2002). Conversely, suppression dependent manner. of USP13 (by siRNA knock-down) leads to a dramatic loss of MITF pro- USP21 was found to be unique among cellular DUBs by showing tein. USP13 has been found to be essential for melanoma growth in clear association with centrosomes and microtubules in a GFP-based animal tumor models and targeting this enzyme may provide a thera- screen (Urbe et al., 2012). Binding to microtubules occurs via a novel peutic opportunity. microtubule-binding motif at the N-terminus of USP21. Depletion of USP15 regulates the TGF-β pathway and is believed to be important USP21 is required for recovery from microtubule depolymerization for the proliferation of glioblastoma cells (Eichhorn et al., 2012). USP15 and is required for nerve growth factor-induced neurite outgrowth in binds to the SMAD7–SMAD E3 ligase complex and deubiquitinates and PC12 cells (Urbe et al., 2012). USP21 catalyzes the hydrolysis of stabilizes the type I TGF-β receptor (TGFβR-I), leading to an enhanced ubiquitinated H2A (Nakagawa et al., 2008; Okuda et al., 2013). During TGF-β signal. High expression of USP15 correlates with high TGF-β ac- chromatin assembly in vitro, ubiquitinated H2A represses di- and tivity, and the USP15 gene is found amplified in glioblastoma, breast trimethylation of H3K4. USP21 relieves this repression and is believed and ovarian cancer. Glioblastoma patients with increased (N2.5) USP15 to be associated with de-repression of transcriptional initiation by copy numbers in their tumors have a shorter overall survival time inhibiting H3K4 methylation. (Eichhorn et al., 2012). Depletion of USP15 decreases the oncogenic ca- USP22 was described as one of 11 death-from-cancer signature pacity of patient-derived glioma-initiating cells due to the repression of genes that are critical in controlling cell growth and death (Glinsky, TGF-β signaling, offering a therapeutic opportunity. USP15 is also a DUB 2006). USP22 is a positive regulator of the histone deacetylase Sirt1 for another set of components of the TGF-β signaling pathway, the (Lin et al., 2012). USP22-mediated stabilization of Sirt1 leads to de- receptor-activated SMADs (R-SMADs) (Inui et al., 2011). USP15 opposes creases in p53 acetylation and suppression of p53 function. In contrast, R-SMAD monoubiquitination, preventing promoter recognition. USP15 knock-down of USP22 leads to destabilization of Sirt1, increases in p53 has been found to be associated with the COP9 signalosome where it acetylation and induction of p53-dependent apoptosis. USP22 has, P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 41 similar to USP12 and USP46, been described to deubiquitinate H2A and transcription factor (Flugel et al., 2012). The inhibition of the enzymatic H2B (Zhao et al., 2008; Joo et al., 2011). activity of USP28 may be a potential target for cancer therapy. USP25 is suppressed by miR-200c, leading to inhibition of tumor cell USP29 has attracted interest following the finding that the gene migration and invasion in vitro and inhibition of lung metastasis forma- encoding this DUB is imprinted and is transcribed mainly from the pa- tion in vivo (Li et al., 2014a). USP25 protein and mRNA level expression ternal allele (Kim et al., 2000). USP29 is involved in the control of the were elevated in non-small cell lung cancer tumors and correlated with stability of claspin, a protein that has a key role in the ATR-Chk1 branch clinical stage and lymphatic node metastasis of patients (Li et al., of the DNA damage checkpoint (Martin et al., 2014). USP29 knockdown 2014a). USP25 has been shown to interact with the SYK non-receptor results in an impaired phosphorylation of Chk1 after DNA damage. tyrosine kinase (Cholay et al., 2010) and has been implicated in ERAD Claspin is also involved in the process of DNA replication and USP29- (Blount et al., 2012) depleted cells display defects in S-phase progression (Martin et al., USP28 plays a critical role in regulation of the Chk2–p53–PUMA-sig- 2014). In addition, USP29 has been found to be transcriptionally in- naling pathway, important for DNA-damage-induced apoptosis in reduced following oxidative stress, where it contributes to the full induc- sponse to double-strand breaks (Zhang et al., 2006; Bohgaki et al., tion of a p53 response (Liu et al., 2011b). 2013). USP28 is required for stabilization of Chk2 and 53BP1 in response USP33 (also called VDU1) was originally discovered as a DUB which to DNA damage. Upon DNA damage in the G2 phase of the cell cycle, binds to the pVHL-containing E3 ligase complex targeted for ubiquitin Usp28 protects claspin from APC/CCdh1-mediated degradation. Usp28 (Ub)-mediated degradation (Li et al., 2002). The USP33 homolog VDU2 permits claspin-mediated activation of Chk1 in response to DNA is also referred to as USP20. USP33 has been found to be localized to damage. the secretory pathway, and one splice variant accumulates at the Golgi The transcription factor MYC is dysregulated in a number of neo- apparatus (Thorne et al., 2011). Substrates identified so far for USP33 plasms, including colorectal tumors (Diefenbacher et al., 2014)and and/or its homolog USP20/VDU2 include the RAS-like GTPase RALB bladder cancers (Guo et al., 2014a). Similar to other transcription factors (Simicek et al., 2013), type 2 iodothyronine deiodinase (D2) (Curcio- such as p53, c-MYC is difficult to target directly using small molecules. Morelli et al., 2003) and HIF-1α (Li et al., 2005). Deubiquitylation of Interestingly, USP28 is required for the stability of the MYC oncoprotein RALB by USP33 promotes the assembly of complexes which contain in human tumor cells (Fig. 7)(Popov et al., 2007). USP28 controls MYC Beclin-1 and which stimulate autophagosome formation (Simicek stability by counteracting the activity of the SCFFBW7 ubiquitin ligase et al., 2013). USP33 also binds to the Robo1 receptor and is required complex (Popov et al., 2007). It was recently demonstrated that USP28 for the responsiveness of breast cancer cells to the migration factor Slit antagonizes the ubiquitin-dependent degradation of not only MYC, but (Yuasa-Kawada et al., 2009). USP33 deubiquitinates the CP110 protein, also of c-JUN and NOTCH (Diefenbacher et al., 2014). Mice lacking an important regulator of centrosome duplication (Li et al., 2013). Over- USP28 showed reduced intestinal cell proliferation and fewer intestinal expression of CP110 leads to centrosome over-duplication and genomic tumors. Depletion of USP28 reduced tumor size and increased the instability (Chen et al., 2002a). CP110 levels are controlled through lifespan of tumor-bearing mice. ubiquitination by the SCF ligase complex and through deubiquitination The chromatin modulator LSD1 controls cellular pluripotency by USP33. During duplication and elongation of centrioles in S and G2/ through histone demethylation and is overexpressed in many tumor M phases, USP33 localizes to centrioles. Down-regulation of USP33 de- types. USP28 has been shown to stabilize LSD1 via deubiquitination stabilizes CP110 and thereby inhibits centrosome amplification and mi- (Wu et al., 2013). Knock-down of USP28 using RNA interference results totic defects (Li et al., 2013). in destabilization of LSD1 and leads to the suppression of cancer stem USP34 is encoded by a gene region on 2p15-16.1 which is amplified cell-like characteristics and inhibition of tumorigenicity in vivo (Wu during progression of follicular lymphoma (FL) to diffuse large B-cell lym- et al., 2013). Another function of USP28 is its ability to antagonize the phoma (DLBCL) (Kwiecinska et al., 2014). Haploinsufficiency for USP34 ubiquitin ligase Fbw7, resulting in stabilization of the HIF-1α appears to affect the regulation of developmental processes (Fannemel et al., 2014). USP34 has been reported to act downstream of the β- catenin destruction complex in the Wnt pathway (Lui et al., 2011). Knock-down of USP34 by RNA interference leads to degradation of axin, β U a scaffolding protein, and to inhibition of -catenin-mediated transcrip- U U USP28 tion. Finally, USP34 has a role in proper maintenance of genome stability U due to its role in stabilizing the E3 ligase RNF168, important for assembly of K63-linked chains at DNA double strand breaks (Sy et al., 2013). c-Myc Max c-Myc Max USP42 is a fusion partner in the (7;21)(p22;q22) translocation in acute myeloid leukemia (AML) (Paulsson et al., 2006). The genomic Fbw7 breakpoint is in intron 7 of RUNX1 and intron 1 of USP42. The RUNX1 transcription factor is a key regulator of hematopoiesis and is involved in numerous translocations in AML (Osato, 2004). USP42 was subsequently described as an additional deubiquitinating enzyme for p53, similarly to USP2a, USP7 and USP10. USP42 was reported not to affect the basal levels of p53, but to be important during the early phase of a U U stress response, helping to rapidly induce p53 levels (Hock et al., 2011). U USP28 U USP44 is an important regulator of the mitotic spindle checkpoint. USP44 prevents the premature activation of the anaphase-promoting N-Myc Max N-Myc Max complex (APC), an ubiquitin ligase that promotes sister chromatid sep- aration. This is achieved by stabilizing the APC-inhibitory Mad2–Cdc20 complex (Stegmeier et al., 2007a). Cells that overexpress Usp44 have Fbw7 been found to be prone to chromosome segregation errors and aneuploidization (Zhang et al., 2011; Zhang et al., 2012). USP44 has also been shown to be a H2B deubiquitinase (Fuchs et al., 2012). USP47 is responsible for deubiquitination the key base excision re- Fig. 7. USP28 regulates c-Myc stability. USP28 binds to c-MYC and N-MYC through an in- β teraction with the F-box protein FBW7α. USP28 stabilizes MYC in the nucleus. Stabiliza- pair (BER) DNA polymerase (Pol )(Parsons et al., 2011). Knockdown tion of MYC by USP28 is essential for the proliferation of tumor cells. of USP47 results in decreased levels of Pol β and a deficiency in BER. 42 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54

Similar to USP30 and USP33, USP47 is involved in the Met receptor tyro- an ovarian tumor (OTU) domain (Borodovsky et al., 2002; Balakirev sine kinase-activated scattering response of epithelial cells (Buus et al., et al., 2003). Ovarian tumor domain DUBs show specificity for different 2009). USP47 also interacts with the E3 ubiquitin ligase SCF(beta- Ub chain linkages. OTUB1 and A20 are specific to K48-linked chains, Trcp) (Skp1/Cul1/F-box protein beta-transducin repeat-containing pro- Cezanne shows specificity to K11-linked chains and TRABID cleaves tein). Depletion of USP47 increases Cdc25A levels in cells and decreases K29- and K33-linked chains (Eletr & Wilkinson, 2014). cell survival, suggesting possibilities of therapeutic intervention OTUB1 inhibits UBC13 (also known as UBE2N) and other E2 en- (Peschiaroli et al., 2010). zymes. UBC13 heterodimerizes with UEV1A to synthesize K63-linked USP50 is believed to be involved in repressing entry into mitosis fol- polyubiquitin chains at double strand breaks in a process also requiring lowing activation of the DNA damage checkpoint (Aressy et al., 2010). the ubiquitin ligase (E3) RNF168 (Stewart et al., 2009). OTUB1 directly USP50 accumulates in the nucleus in response to treatment with DNA suppresses MDM2-mediated p53 ubiquitination, and overexpression damaging agents. HSP90 is a major interacting partner for USP50 and of OTUB1 leads to stabilization and activation of p53 (Sun et al., 2012). depletion of USP50 results a loss in accumulation of the HSP90 client Monoubiquitination of OTUB1 increases the interaction with UbcH5, Wee1. resulting in a suppression of UbcH5 activity (Li et al., 2014b). OTUB1 CYLD germline mutations occur in familial cylindromatosis and deubiquitinates estrogen receptor α (ERα) and negatively regulates some other rare autosomal dominant inherited disorders associated ERα mediated transcription (Stanisic et al., 2009). OTUB1, as well as with the development of multiple skin tumors of the head and neck OTUB2, negatively regulates virus-induced type I IFN induction and an- (Bignell et al., 2000; Poblete Gutierrez et al., 2002). Hyperactive Wnt tiviral responses by deubiquitinating TRAF3 and TRAF6 (Li et al., 2010). signaling has been demonstrated in CYLD mutant human cylindroma OTUD1 was reported to be highly expressed in thyroid carcinomas tumors (Tauriello et al., 2010). The underlying mechanism of activation and an OTUD1 antibody was able to distinguish carcinomas from benign has been shown to be via enhanced K63-linked ubiquitination of Dvl lesions (Carneiro et al., 2014). (Dishevelled), the cytoplasmic effector of Frizzled, in the absence of OTUD4 is a deubiquitinase which appears to be involved in XPC functional CYLD (Tauriello et al., 2010). An important function of CYLD recycling. OTUD4 has been demonstrated to interact with the is regulation of NFκB activation (Trompouki et al., 2003). CYLD inhibits xeroderma pigmentosum complementation group C (XPC) protein NFκB-mediated activation by different TNF receptor family members (Lubin et al., 2014). due to deubiquitination and inactivation of TNFR-associated factor 2 OTUD5 (DUBA) is activated by phosphorylation (Huang et al., 2012) (TRAF2) and TRAF6. It was later shown that CYLD removes K63-linked and suppresses the type I interferon response by cleaving the polyubiquitin chains from the Bcl-3 protein, resulting in retention of polyubiquitin chain from tumor necrosis factor receptor-associated fac- Bcl-3 in the cytoplasm (Massoumi et al., 2006). Bcl-3 associates with tor 3 (TRAF3) (Kayagaki et al., 2007). In addition, OTUD5 is also a the NFκB p50 or p52 subunits and enhances cell proliferation through deubiquitinating enzyme for p53 which is required for the stabilization activation of the cyclin D1 promoter. In CYLD-deficient keratinocytes, and the activation of a p53 response (Luo et al., 2013) Bcl-3 accumulates in the nucleus, leading to activation of NFkB target A20 (TNFα-induced protein 3 (TNFAIP3)) is an important negative genes. CYLD also has another cell-cycle regulatory function, being im- regulator of the transcription factor NFκB. A20 inhibits the NFκBpath- portant for proper regulation of mitotic entry (Stegmeier et al., 2007b). way by removing the K63-linked polyubiquitin chains on RIP1 (receptor UCHL1 was identified as being overexpressed in lung adenocarci- interacting protein 1) (Wertz et al., 2004) and TRAF6 (TNF receptor- nomas using a proteomics approach (Chen et al., 2002b). UCHL1 over- associated factor 6) (Ma & Malynn, 2012)(Fig. 8). The N-terminus of expression in tumors was associated with patients having a smoking A20 contains an OTU domain whereas the C-terminal portion contains history. Interestingly, UCHL1 was reported to be consistently up- seven zinc finger structures. A20 is considered as an ubiquitin-editing regulated in airway epithelial samples obtained from smokers, com- enzyme with both DUB and E3 ligase activity (Ma & Malynn, 2012; pared with nonsmokers (Carolan et al., 2006). Overexpression of Shembade & Harhaj, 2012). During TNF-mediated stimulation, A20 ex- UCHL1 was speculated to represent an early event in the transformation pression levels increase, leading to deubiquitination of K63-linked process of normal lung epithelium (Carolan et al., 2006). In contrast to chains on the RIP1 protein, but after some hours A20 conjugates K48- these findings, the UCHL1 gene was found to be silenced in giant cell tu- linked polyubiquitin chains on the same RIP1 substrate, triggering its mors of bone (Fellenberg et al., 2010). Silencing was associated with degradation by the proteasome. A20 acts as a tumor suppressor in lym- methylation of the CpG island covering the UCHL1 promoter. The phoid malignancies due to its NFκB inhibiting role (reviewed in Harhaj UCHL1 gene has also been found to be silenced by methylation in and Dixit (2012)). As discussed in a previous section, the A20 gene is colon cancer cell lines (Fukutomi et al., 2007). mutated/deleted in various types of lymphoma (Honma et al., 2009; BAP1 (BRCA1 associated protein-1) is a nuclear-localized DUB of the Novak et al., 2009; Schmitz et al., 2009; Dong et al., 2011; Zhang et al., UCH family originally identified as an interacting partner of the BRCA1 2012). In contrast, forced overexpression of A20 in breast cancer cells tumor suppressor protein (Jensen et al., 1998). BAP1 has not been dem- leads to tamoxifen resistance (Vendrell et al., 2007). onstrated to deubiquitinate BRCA1, but fulfills various criteria of having Cezanne is an OTU deubiquitinase with sequence homology to A20 a tumor suppressor function. Deletions and missense mutations in the (Evans et al., 2003). Cezanne is induced by TNF-α in cultured cells and BAP1 gene are found in lung and breast tumors and lung cancer cell silencing of Cezanne leads to elevated NFκB activity in response to this lines (Buchhagen et al., 1994; Jensen et al., 1998; Jensen & Rauscher, cytokine (Enesa et al., 2008). It was suggested that miR-218 regulates 1999). The BAP1 protein is also inactivated in a fraction of clear cell the ability of TGF-β to induce myofibroblast differentiation via a path- renal cell carcinomas (RCC) (Pena-Llopis et al., 2012)andBAP1 way involving Cezanne (Guo et al., 2014b). inactivating mutations have been demonstrated in ~25% of malignant Ataxin-3 was identified as the protein product of the Machado– pleural mesotheliomas (Bott et al., 2011). Cancer-associated BAP1 mu- Joseph disease gene locus (Kawaguchi et al., 1994). Machado–Joseph tants are deficient in deubiquitinating activity (Ventii et al., 2008). disease is a dominantly inherited form of spinocerebellar ataxia. BAP1 can suppress tumorigenicity of lung cancer cells in athymic nude Ataxin-3 contains a poly-glutamine stretch, the expansion of which mice (Ventii et al., 2008). BAP1 loss sensitizes RCC cells in vitro to is associated with the disease. Additional proteins (ataxin 3-like genotoxic stress (Pena-Llopis et al., 2012). BAP1 interacts with the tran- and Josephin domain containing 1) showing pronounced homology scriptional cofactor HCF-1 (host cell factor 1). By regulating HCF-1 pro- to ataxin-3 were later identified and named Josephins (Albrecht et al., tein levels, BAP1 may be involved in cell cycle control by associating 2003). Ataxin-3 is a DUB which has been implicated in ERAD and the with genes involved in G1–Stransition(Misaghi et al., 2009). regulation of autophagy. Ataxin-3 is a poly-glutamine containing OTUB enzymes (OTU domain-containing ubiquitin aldehyde- deubiquitinating enzyme (DUB) that interacts with the p97 ATPase binding proteins) constitute a subfamily of 14 DUBs characterized by and with various shuttling factors such as HHR23A and -B (Boeddrich P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 43

TNF

DDDD DD DD TRADD

TRAF2 RIP1 U U A20 U U U P U TAK1 P

IKKγ

P IKKβ IKKα

USP15

USP11 USP11 P USP11 P P P Rel A κ α Rel A Iκ Bα Rel A Iκ Bα I B P U P p50 p50 p50 U U U U U

Fig. 8. Role of A20, USP11 and USP15 in the regulation of TNFα-mediated IκBα ubiquitination and NF-κB activation. A20 (TNFα-induced protein 3 (TNFAIP3)) inhibits the NFκB pathway by removing K63-linked polyubiquitin chains on RIP1 (receptor interacting protein 1). USP11 is associated with IκBα and inhibits TNFα-induced IκBα ubiquitination and degradation. USP15 also inhibits IκBα ubiquitination and degradation. USP11 and USP15 have been shown to be important for downregulation of NFκB activation. The model is simplified: other DUBs are known to deubiquitinate RIP1 and all DUBs shown have additional functions. Adapted from Sun et al. (2010). et al., 2006). Ataxin-3 associates with parkin, HDAC6 (histone BRCC36 was observed in breast cancer cell lines and tumors (Dong deacetylase 6) and other aggresome components (Wang et al., 2012). et al., 2003). Knock-down of BRCC36 in breast cancer cells leads to Ataxin-3 trims K63-linked chains from misfolded ubiquitinated pro- sensitization to ionizing radiation (Chen et al., 2006). This effect teins and enhances the rate of aggresome formation (Burnett & was found to be associated with a loss of BRCA1 activation. Pittman, 2005). Josephin DUBs have roles in cell signaling: all three members are implicated in regulation of PTEN (phosphatase and tensin homolog) expression (Sacco et al., 2014). Interestingly, DUB-mediated 4. Small molecule DUB inhibitors regulation occurs at the level of transcription by an unidentified mechanism. Importantly, knock-down of ataxin-3 using siRNA leads to PTEN Similar to protein kinase inhibitors, a span of small molecule DUB in- induction and downregulation of PI3K signaling. hibitors has been described ranging from broad pan-enzyme inhibitors to BRCC36 is a member of the JAMM (JAB1/MPN/MOV34 specific inhibitors of single DUB enzymes. Also in analogy with kinase in- metalloenzyme) class of DUBs. BBRC36 was isolated as a constituent hibitors, unspecific inhibitors are likely to elicit more profound biological of an enzyme complex also containing BRCA1, BRCA2, and RAD51 effects but also to show stronger unspecific toxicity. Inhibition of multiple (Dong et al., 2003). BRCA1 is recruited to DNA double-strand breaks DUBs is predicted to induce cellular changes such as (a) increased accu- by the ubiquitin-binding protein RAP80 (Sobhian et al., 2007). The mulation of polyubiquitinated proteins/unanchored polyubiquitin chains role of BRCC36 in this complex may be to terminate signaling follow- (if the proteasome/USP5 is inhibited); (b) declines in the pool of mono- ing completion of DNA repair (Shao et al., 2009). Cancer-associated meric ubiquitin, (c) slower rates of polyubiquitin disassembly, (d) an BRCA1 mutations decrease the association between BRCC36 and overall decrease in individual DUB activities, and (e) affect cellular BRCA1 (Dong et al., 2003). Interestingly, aberrant expression of levels/activities of DUB-regulated oncoproteins (discussed by Kapuria 44 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 et al. (2010)). The subject has recently been reviewed elsewhere LDN-57444 is an isatin O-acyl oxime reported to selectively inhibit (Edelmann et al., 2011; Buckley & Crews, 2014). UCHL1 (Liu et al., 2003). LDN-57444 is a reversible, competitive, and ac- A number of individual DUBs have been reported to be involved in the tive site-directed inhibitor. The inhibitor increased proliferation of the control of programmed cell death (apoptosis) (Ramakrishna et al., 2011). H1299 lung tumor cell line but had no effect on a lung tumor line that An example is USP7 inhibition that results in decreased levels of Mdm2, does not express UCHL1 (Liu et al., 2003). LDN-57444 was found to in- leading to stabilization of p53. Inhibition of multiple DUBs may be ex- crease the levels of polyubiquitinated proteins and to induce apoptosis as- pected to amplify these affects (if they regulate essential substrates) sociated with ER stress in SK-N-SH neuroblastoma cells (Tan et al., 2008). but may also be predicted to increase the quantity of polyubiquitinated Pimozide (an anti-psychotic drug) and GW7647 (a PPAR-α agonist) proteins/unanchored polyubiquitin chains. The inhibition of some were identified as inhibitors of USP1/UAF1 in a high-throughput screen DUBs, particularly those associated with the proteasome, can lead to im- (Chen et al., 2011). These drugs were found to act synergistically with paired proteasome function and accumulation of misfolded proteins in cisplatin in inhibiting proliferation of cisplatin-resistant non-small cell cells. Such accumulation will lead to proteotoxicity and ultimately cell lung cancer cells (Chen et al., 2011). death. HBX 19,818 is a specific inhibitor of USP7 that covalently modifies the active Cys223 residue of this enzyme (Reverdy et al., 2012). LS1 is an UCHL3 inhibitor which was identified in a FRET-based screen 4.1. Synthetic small molecule DUB inhibitors where fluorescently labeled ubiquitin was attached to substrates containing quenchers (Ohayon et al., 2012). PR-619 is a general DUB en- fi USP7 (HAUSP) has attracted signi cant interest from drug devel- zyme inhibitor, but does not inhibit other classes of cysteine proteases fi opers due to its role in controlling p53 stability. P022077 was identi ed (Altun et al., 2011). LDN91946 was identified in an in vitro screen for in- fi as a speci c USP7 inhibitor by Progenra (Tian et al., 2011). P5091 is an hibitors of Ub-AMC activity (Mermerian et al., 2007). The compound inhibitor of USP7 and USP47 (Chauhan et al., 2012; Weinstock et al., was reported to be a non-competitive inhibitor of UCHL1, but did not in- 2012) and was found to induce apoptosis of multiple myeloma cells re- hibit other cysteine proteases such as UCHL3, USP5 and caspase-3. sistant to the 20S proteasome inhibitor bortezomib. Specificity was val- idated using USP7 knockout cells. P5091 was reported to be well tolerated by animals and to inhibit tumor growth (Chauhan et al., 4.2. DUB inhibition by cyclopentone prostaglandins 2012). A more potent analog of P5091 has subsequently been described (cpd 14) (Weinstock et al., 2012)(Table 1). Fitzpatrick and coworkers (Mullally et al., 2001) originally reported USP14 is one of the three distinct DUBs that are associated with that cyclopentone prostaglandins of the PGJ2 class induce accumulation proteasomes (RPN11, UCHL5 and USP14). USP14 disassembles of polyubiquitinated proteins in cells and inhibit DUB activity. Prosta- proteasome-bound polyubiquitin chains from their distal tips. USP14 glandin PGJ2 is a metabolite of PGD2 that is sequentially metabolized activity leads to suppression of substrate degradation by disengaging to Δ12-PGJ2 or to 15Δ-PGJ2 (Fitzpatrick & Wynalda, 1983). PGJ2 prosta- the substrates from the proteasomes (Lee et al., 2011). A small molecule glandins contain α,β-unsaturated ketones that function as Michael ac- inhibitor of USP14 (IU1; Fig. 9) has been described which indeed en- ceptors (Fig. 9). Cyclopentenone PGs react with thiol molecules at the hances proteasome function in cells (Lee et al., 2010). This compound β-position within the cyclopentenone ring (Suzuki et al., 1997). The fi binds speci cally to the activated, proteasome bound, form of USP14. mechanism(s) of UPS inhibition by PGs may be complex. One study re- Increasing proteasomal-mediated degradation by IU1 or similar inhibi- ported that a major intracellular target of protein carbonylation was the fi tors may be of therapeutic bene t in diseases such as Alzheimer's dis- S6 ATPase of the 26S proteasome. Protein carbonylation and oxidative ease (Lee et al., 2010). The situation appears to be quite complex, stress induced by PGs decreased the activities of the S6 ATPase and however, since USP14 appears to be essential for the maintenance of that of the proteasome (Ishii et al., 2005). Other investigators have re- synaptic ubiquitin levels and the development of neuromuscular junc- ported that cyclopentone PGs inhibit cellular DUB activities: UCHL3 tions (Wilson et al., 2002; Chen et al., 2009). It is, however, quite possi- was found to be inhibited by Δ12-PGJ2 (Li et al., 2004) and UCHL1 by ble that loss of USP14 protein does not elicit the same effect on cells as 15Δ-PGJ2 (Liu et al., 2011c). 15Δ-PGJ2 affects UCHL1 overall structure, inhibition of enzyme activity. resulting in inhibition of enzyme activity (Koharudin et al., 2010). 15Δ-PGJ2 has also been reported to stabilize p53, but p53 function was functionally inactivated by binding of the PG to the Cys-277 residue Table 1 (Kim et al., 2010). Cysteine modification sites of 15Δ-PGJ2 have been Synthetic small molecule DUB inhibitors. identified in UCHL1 (Koharudin et al., 2010) and in Ras (Renedo et al., Compound Reported activity/ Ref 2007). Cyclopentenone PGs are known to induce apoptosis by mecha- target nisms that do not require the p53 pathway (Mullally et al., 2001). Oxi- P022077 USP7 (Tian et al., 2011) dative stress induced by 15Δ-PGJ2 was reported to be associated with P5091 USP7 (Chauhan et al., 2012) the formation of adducts between PG and thioredoxin 1 (Shibata et al., Cpd 14 USP7 and USP47 (Weinstock et al., 2012) 2003). Two cysteine residues in thioredoxin 1, Cys-35 and Cys-69, P22077 USP7/USP47 (Altun et al., 2011) fi Δ HBX 41,108 USP7 (Colland et al., 2009) were found to be modi ed by 15 -PGJ2. Interestingly, UCHL1 was re- HBX-19,818 USP7 (Reverdy et al., 2012) ported to be among the proteins that were oxidized in cells exposed HBX-28,258 USP7 (Reverdy et al., 2012) to 15Δ-PGJ2 (Ishii & Uchida, 2004)(Table 2). HBX 90,397 USP8 (Edelmann et al., 2011) The DUB inhibitory activity of cyclopentone PGs is very likely due to Ethyloxyimino-9H-indeno USP8 (Colombo et al., 2010; the presence of Michael acceptors in these compounds and the fact that [1,2-b]pyrazine-2,3- Byun et al., 2013) dicarbonitrile the vast majority of DUBs are cysteine proteases. Among the amino acid IU1 USP14 (Lee et al., 2010) residues in proteins, cysteine is the most nucleophilic and reactive. Isatin O-acyl UCHL1 (Liu et al., 2003) Whereas the pKa of free cysteine is 8.4, the pKa of a cysteine residue in oxime derivatives a protein is affected by the environment and varies between 4 and 9. LDN91946 UCHL1 (Mermerian et al., 2007) LS1 UCHL3 (Ohayon et al., 2012) Basic residues close to cysteine sulfhydryls in the protein structure de- NSC112200, TRABID/ZRANB1 (Shi et al., 2012) crease the pKa of cysteine and result in ionization to cysteine thiolate NSC267309 (RS−) at neutral pH. The nucleophilic nature of cysteine thiolate allows PR-619 Broad spectrum DUB (Edelmann et al., 2011) it to react with Michael acceptors such as those present in maleimides, inhibition vinyl sulfones, acrylamides, and α,β-unsaturated ketones and aldehydes. P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 45

IU1 15-Deoxy- α prostaglandin J2

O N COOH N F

O b-AP15 RA-9 O

NO N NO Cl N Cl 2 2 O Cl CO - arginine - lysineCl

O F6 G5 O

N NO NO 2 S 2 OO

WP1130 Eeyarestatin-1 Cl O O HN O N Br N N N N NO2 O H C HO N

N O HN

Curcumin OO

O O

HO OH

Gambogic acid OH AC17 O O O O O O O O O

O O

OH OH O

Fig. 9. Chemical structures of some of the compounds discussed in the text.

Electrophiles differ with regard to their degree of reactivity to nucle- cysteine thiolates in proteins (Aldini et al., 2006). Proteomic studies ophiles based on the “hard/soft” acid–base principle (Lopachin et al., have shown that electrophiles react with “hyper-reactive” cysteines in 2012). α,β-Unsaturated ketones are considered as relatively soft elec- proteins (Weerapana et al., 2010; Codreanu et al., 2014). Acrolein (an trophiles and believed to be selective in their reaction to a subset of α,β-unsaturated aldehyde), glyoxal (a dialdehyde) and various keto-

Table 2 DUB inhibitors containing Michael acceptors.

Compound Reported activity Ref

12Δ-PGJ2 UCHL3 (Li et al., 2004; Lee et al., 2010) 15-Deoxy-Δ12,14-prostaglandin J2 UCHL1 (Liu et al., 2011) G5 Inhibition of cellular Ub-AMC cleavage (Aleo et al., 2006) F6 (NSC 632839) Inhibition of cellular Ub-AMC cleavage (Aleo et al., 2006; Nicholson et al., 2008) USP2, USP7 SENP2 deSUMOylase WP1130 USP5/USP9x/USP14/UCHL1/UCHL5 (Kapuria et al., 2010) b-AP15 USP14/UCHL5 (D'Arcy et al., 2011) AC17 USP14/UCHL5 (Zhou et al., 2013) AM146, RA-9 and RA-14 USP2a/USP2b/USP5/USP8 (Issaenko and Amerik, 2012) Eeyarestatin 1 Ataxin-3 (Wang et al., 2008) 46 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 aldehydes are more reactive and cytotoxic compared to α,β-unsaturat- activity in animal models, both against solid tumors (D'Arcy et al., ed ketones (Dalle-Donne et al., 2006). Acrolein reacts with guanine 2011) and against multiple myeloma (Tian et al., 2013). The cytotoxic ac- bases in DNA, and with cysteine, lysine, histidine, and arginine residues. tivity of the drug is strongly associated with functional proteasome inhi- Punaglandins, chlorinated cyclopentenone PGs, are more potent in- bition. Thus, monitoring accumulation of a proteasome-degraded hibitors of cellular DUB activity than the J-series PGs (Verbitski et al., reporter (UbG76V-YFP) in cells exposed to low concentrations of b-AP15 2004). The increased inhibitor activity was speculated to be due in- showed a very strong correlation between blocking of proteasome activ- creased reactivity towards the nucleophilic addition of the DUB catalytic ity and cell death (Brnjic et al., 2013; Wang et al., 2014). Importantly, ex- cysteine thiol group (Verbitski et al., 2004). amination of the cellular response to b-AP15 using gene expression profiling and CMAP analysis (Lamb et al., 2006) showed that this com- 4.3. Chalcone DUB inhibitors pound induced a very similar change in gene expression as a number of proteasome inhibitors and to 15Δ-PGJ2 (D'Arcy et al., 2011). A chalcone is an aromatic ketone and an enone that forms the central AM146, RA-9, RA-14 and RAMB1 are all chalcones (RA-9 shown in core for a variety of important biological compounds. A number of Fig. 9)(Anchoori et al., 2011; Issaenko & Amerik, 2012). These com- chalcone compounds (e.g. G5, b-AP15, RA-9) have been described to in- pounds were described to act as partially selective DUB inhibitors and hibit cellular deubiquitinase activity (Fig. 9). These compounds are un- to induce rapid accumulation of polyubiquitinated proteins and to de- related to PGs, but contain cross-conjugated α,β-unsaturated ketones plete the cellular pools of free ubiquitin. AM146, RA-9, and RA-14 and accessible β-carbons, and generally inhibit DUB activity. These com- were found to inhibit UCHL1, UCHL3, USP2 and USP8, but did not sup- pounds have been reported to be either relatively specific (b-AP15) or press the activity of Ataxin-3, A20, BAP1, Otubain 1 or USP7 (Issaenko broad spectrum (G5) DUB inhibitors. The reasons for these apparent & Amerik, 2012). These compounds elicit apoptosis of breast, ovarian discrepancies are unknown. and cervical cancer cells but not of primary human cells. Compounds G5 and F6 were identified in a cell based screen for Bcl- 2-independent apoptosis (Aleo et al., 2006). Both compounds are 4.4. Other DUB inhibitors containing Michael acceptors chalcones that contain cross-conjugated α,β-unsaturated ketones. These compounds were reported to inhibit total DUB activity (Aleo WP1130 (degrasyn, Fig. 9) is a small-molecule compound that in- et al., 2006) and F6 was reported to inhibit USP2 and USP7 and also hibits several DUBs of both USP and UCH subclasses, including USP9x, the SENP2 deSUMOylase (Nicholson et al., 2008). G5 induces apoptosis USP5, USP14 and UCHL5 (Kapuria et al., 2010). In cells, the inhibition at concentrations of ~1 μM, and necrotic cell death at higher concentra- of DUB activity resulted in the accumulation of polyubiquitinated conju- tions (10 μM) (Fontanini et al., 2009). gates. WP1130 induces the accumulation of free polyubiquitin chains, b-AP15 (Fig. 9)wasidentified in cell based screens for compounds in- presumably due to inhibition of USP5 (isopeptidase-T). Downregulation ducing apoptosis independently of p53 and cathepsin-D (Erdal et al., of the anti-apoptotic protein MCL1 was observed in cells exposed to 2005; Berndtsson et al., 2009) and was found to induce the accumulation WP1130. This effect is expected from the inhibition of USP9x, and is of polyubiquitin in cells (Berndtsson et al., 2009). Subsequent work not observed in cells exposed to b-AP15 (X.W., unpublished observa- showed that this compound blocks degradation of a proteasome- tion). Pham and colleagues showed that WP1130 in combination with degraded reporter protein, suggesting that DUB inhibition was linked bortezomib had antitumor activity in a mantle cell lymphoma animal to a decrease in proteasome function (D'Arcy et al., 2011). b-AP15 in- model (Pham et al., 2010). In distinction to b-AP15, WP1130 was re- hibits the activity of proteasome associated cysteine DUBs (USP14 and ported not to induce oxidative stress (Kapuria et al., 2010). The inhibi- UCHL5) at concentrations of ~5 μM(D'Arcy et al., 2011; Wang et al., un- tion of USP14 and UCHL5 by WP1130 is expected to induce a published data) (Fig. 10). Dual inhibition of USP14 and UCHL5 using RNA functional block of proteasome function in cells, but this has not been interference is known to lead to strong accumulation of proteasomal sub- tested. strates and loss of cell viability (Koulich et al., 2008; Tian et al., 2013; Eeyarestatin-1 (Eer1) (Fig. 9) was identified in a screen for inhibitors Wang et al., 2014). Surprisingly, b-AP15 did not inhibit total cellular of the ERAD pathway (Fiebiger et al., 2004). Subsequent studies have DUB activity at concentrations of 20–50 μM(D'Arcy et al., 2011). Consid- shown that Eer1 inhibits p97/VCP-associated DUB activity, and that ering the structural similarity between b-AP15 and other chalcones, ataxin-3 activity is inhibited by this compound (Wang et al., 2008). these differences in specificity are surprising and are as of yet unex- Using surface plasmon resonance and pull-down assays, Eer1 has been plained (see further below). b-AP15 shows considerable anti-neoplastic shown to directly bind to the p97 ATPase (261). Eer1 appears to have

U U U U U U U U U U U U U U UCHL5 USP14 UCHL5 USP14 U U

b-AP15 AC17

Fig. 10. Inhibition of proteasomal DUBs impairs proteasome function and leads to the accumulation of polyubiquitinated substrates in cells. Compounds b-AP15 and AC17 have been reported to show this mechanism of action (D'Arcy et al., 2011; Zhou et al., 2013). P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 47 complex mechanisms of action in the ER compartment since the com- 2013). As expected from this result, GA induces the accumulation of pound also inhibits anterograde translocation of proteins over the ER polyubiquitinated proteins. The presence of α,β-unsaturated ketones (Cross et al., 2009; Aletrari et al., 2011). Eer1 has anti-cancer activities suggested that GA may inhibit cellular DUB activity, and this was indeed resembling those of bortezomib (Wang et al., 2009). found to be the case (Felth et al., 2013). The compound also inhibits 20S proteasome activity (Felth et al., 2013). 4.5. Natural products with DUB inhibitory activity 4.6. Specificity of inhibitors containing α,β-unsaturated ketones A number of compounds have been described in the literature to affect the UPS without direct inhibition of proteasome activity. Some of Michael acceptor-based inhibitors will achieve maximal selectivity these compounds have been reported to be DUB inhibitors. by targeting poorly conserved, noncatalytic cysteines (Singh et al., Curcumin is a yellow pigment from the herb Curcuma longa present 2011). Inhibitors with Michael acceptors that are believed to target cat- in the Indian spice turmeric (associated with curry powder). Curcumin alytic cysteines in DUBs nevertheless appear to show considerable spec- is one of the most studied phytochemicals; in August 2014 a PubMed ificity. Some inhibitors such as b-AP15 and AC17 were reported to show search for “curcumin” and “cancer” resulted in 2579 hits (reviewed in specificity to proteasomal DUBs (D'Arcy et al., 2011; Zhou et al., 2013), Shishodia et al. (2005)). Curcumin has been linked with suppression whereas others such as WP1130 showed a larger range of activities of tumorigenesis as well as a number of other pathological conditions but nevertheless some specificity (Kapuria et al., 2010). Some of these and has been shown to be extremely safe even at very high doses. differences may reflect different binding to target DUBs due to 3-D con- Curcumin is a homodimer of feruloylmethane containing a methoxy formation of the inhibitors. However, it is surprising that G5/F6 is broad group and a hydroxyl group (Fig. 9). Importantly, curcumine contains specificity DUB inhibitors and b-AP15 is specific to proteasomal DUBs, two α,β-unsaturated ketone moieties which are important for its anti- considering the similar molecular structures (i.e. chalcone) of these cancer activity. Using a shotgun proteomic approach, it was found that drugs. b-AP15 has a low solubility in aqueous buffers, and it is conceiv- curcumin induces the accumulation of polyubiquitinated proteins at able that b-AP15 would inhibit non-proteasomal DUBs if soluble at 10– concentrations of 40 μM(D'Aguanno et al., 2012). The ability of 50 μM. However, analysis of myeloma cells exposed to 1 μMb-AP15 curcumin to modulate molecular targets relevant to cancer, such as showed inhibition of USP14 in the same protein extracts where USP7, NFκB, cyclin D1 and p21, may be speculated to be secondary to inhibi- UCHL3 or UCHL5 was not inhibited (Tian et al., 2013). Considering the tion of the UPS (Shishodia et al., 2005)(Table 3). expected cytotoxicity resulting from blocking of proteasome function, The 4-arylidene curcumin analog AC17 (Fig. 9) has been shown to we believe it to be likely that the inhibition of proteasomal DUBs inhibit the deubiquitinase activity of the 19S RP (Zhou et al., 2013) (USP14/UCHL5) is of key importance to the biological effects of (Fig. 10). AC17 inhibits NFκB activity and activates p53 in human lung chalcones and other α,β-unsaturated ketone-containing compounds. cancer cells and has anti-tumor activity in an in vivo model of human Compounds containing α,β-unsaturated ketones frequently induce lung cancer. AC17 was reported to be an irreversible inhibitor of 19S cellular oxidative stress (Raj et al., 2011; Adams et al., 2012). Such com- DUB activity, presumably USP14/UCHL5 (Zhou et al., 2013). AC17 does pounds have also been proposed to directly react with proteins, creating not inhibit total DUB activity in cell lysates (Zhou et al., 2013). “electrophilic stress” (Adams et al., 2012). Cyclopentone PGs, curcumin Betulinic acid is triterpene that has been isolated from many diverse and b-AP15 have all been found to inhibit thioredoxin reductase (Moos plants, including the birch tree, Betula. Betulinic acid is cytotoxic against et al., 2003; Fang et al., 2005; Wang et al., 2014) and 15Δ-PGJ2 binds a range of cancer cell lines, and shows significant in vivo activity in two cysteines in thioredoxin (Shibata et al., 2003). In the instance of human melanoma xenograft animal models (Pisha et al., 1995). A 20% b-AP15, various pieces of evidence suggested that thioredoxin reduc- betulinic acid ointment has been evaluated in the treatment of dysplas- tase inhibition contributes to oxidative stress by this compound but tic nevi (http://www.cancer.gov/drugdictionary?CdrID=496932). does not appear essential for cytotoxicity (Wang et al., 2014). Induction Betulinic acid was recently reported to be a broad spectrum DUB inhib- of an oxidative stress response is a general feature of proteasome inhibitor (Reiner et al., 2013). Betulinic acid has previously been used as a re- itors (including bortezomib and MG-262 (Ling et al., 2003; Wu et al., agent in cancer research based on its ability to induce loss of 2002)), and may not necessarily require specific molecular sensors transmembrane potential in isolated mitochondria and to induce apo- such as Keap1–Nrf2 (Gao et al., 2007). When compared side-by-side, ptosis (Fulda et al., 1998). b-AP15 and the thioredoxin reductase inhibitor auranofininducesimi- Gambogic acid (GA) is a cytotoxic compound isolated from gam- lar levels of oxidative stress, but only b-AP15 induces proteasome inhi- boge, a brownish resin of the tree Garcinia hanburyi in Southeast Asia. bition (Wang et al., 2014). This plant product has been used in Chinese traditional medicine for Cysteine-dependent enzymes are not necessarily inhibited by α,β- centuries (Zhang et al., 2004; Chen et al., 2008). GA has been shown unsaturated ketone-containing compounds. Neither glutathione reduc- to be an anticancer drug candidate with documented cytotoxic activity tase nor caspase-3, enzymes which both contain cysteines in their active in several types of cancer cells (Wu et al., 2004b; Reutrakul et al., sites, are inhibited by b-AP15 (Wang et al., 2014) (X.W., unpublished in- 2007; Yi et al., 2008). The Chinese Food and Drug Administration has ap- formation). PGJ2 induces a decline in proteasome activity and a shift in proved a phase II clinical trial of GA as an antitumor candidate (Qi et al., the molecular make-up of proteasomes (decreases in 26S particles and 2008). GA has an α,β-unsaturated ketone structure which is essential increases in 20S core particles) (Wang et al., 2006). b-AP15 does not in- for growth inhibition and apoptosis induction in cancer cell lines hibit proteasome activity (D'Arcy et al., 2011) and does not affect the as- (Fig. 9)(Zhang et al., 2004; Reutrakul et al., 2007). The gene signature sembly of proteasomes in cells (M. Mazurkiewicz, unpublished data). observed in cells exposed to GA is similar to that of cells exposed to 15Δ-PGJ2 has also been shown to covalently bind to a cysteine residue UPS inhibitors such as PGJ2, celastrol and withaferin A (Felth et al., in the PPARγ ligand (Shiraki et al., 2005) and to bind actin and other

Table 3 Natural products which are candidate DUB inhibitors.

Compound Reported activity

Curcumin Accumulation of polyubiquitin, contains α,β unsaturated ketones (Mullally and Fitzpatrick, 2002; D'Aguanno et al., 2012) AC17 (curcumin analog) USP14, UCH-L5 (Zhou et al., 2013) Betulinic acid Broad spectrum DUB inhibition (Reiner et al., 2013) Gambogic acid Accumulation of polyubiquitin, contains α, β unsaturated ketones (Felth et al., 2013) 48 P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 abundant cellular proteins (Aldini et al., 2007). Morphologic changes in to be under kinetic control whereas enone reactivity is under thermody- the actin filament network were observed using high concentrations namic control. Cross-conjugated dienone PGs undergo reversible (20 μM) of 15Δ-PGJ2 (Aldini et al., 2007). Exposure of cells to 15Δ- Michael addition reactions with glutathione, generating an equilibrium PGJ2 does not induce major shifts in the position of proteins found to mixture shifted to the free PG over the conjugated PG (Suzuki et al., react with the PG on two-dimensional gels (Aldini et al., 2007), suggest- 1997). In contrast, enone PGs bind glutathione in an irreversible manner ing that only a minor fraction of the protein was covalently modified. so the equilibrium favors thiol conjugate formation. CDDO (2-cyano-3, With regard to the reactivity of dienones, see further below. 12-dioxooleana-1, 9-dien-28-oic acid) is a semi-synthetic triterpenoid It is difficult to deduce the molecular mechanism of action of 15Δ- which contains two α,β-unsaturated carbonyls and is a promising che- PGJ2, b-AP15 and other compounds using proteomic data since identi- mopreventive and chemotherapeutic agent. CDDO has been demon- fied targets are not necessarily associated with induction of apoptosis. strated to react with thiol nucleophiles, but nucleophilic addition is However, using CMAP analysis of induced gene expression profiles, both selective and reversible (Couch et al., 2005). 15Δ-PGJ2 and b-AP15 show profiles which are similar (but not identi- The pattern of reactivity of compounds containing α,β-unsaturated cal) to those of 20S proteasome inhibitors (D'Arcy et al., 2011). carbonyls can be fine-tuned by manipulating their electronic properties (Amslinger, 2010). Some of the differences in the specificity by these 4.7. Pharmacological properties of compounds with regard to DUB inhibition could be envisioned to be compounds containing α,β-unsaturated ketones due to the varying strength of the electrophiles. Another important issue is the potential general reactivity of these compounds, which Curcumin is safe to humans at doses of 12 g/day but exhibits poor would seriously compromise the bioavailability of the drugs by non- bioavailability (Anand et al., 2007). Low plasma/tissue levels appear to selective addition to scavengers such as glutathione, and could also be be mainly due to poor absorption and rapid metabolism. The anti- envisioned to lead to general toxicity. However, most of the evidence tumor effects observed with orally administered curcumin in some in the literature suggests that nucleophile addition to dienones is re- studies are somewhat surprising considering the limited bioavailability versible and that a major fraction of various compounds are free to of this compound. The use of piperine, an agent that interferes with bind various intracellular targets. It may nevertheless be advantageous glucuronidation, has been reported to increase bioavailability (Shoba to attempt to develop prodrugs where the α,β-unsaturated carbonyl re- et al., 1998; Anand et al., 2007). activity is activated in intracellular compartments. An intriguing property of some of the inhibitors discussed here is their stronger inhibitory effect in cellular assays compared to biochem- 5. Conclusions ical in vitro assays. b-AP15 blocks proteasome activity in cellular assays (accumulation of polyubiquitin and proteasome-degraded reporters) in Great efforts have been made to develop inhibitors of targets in the the 0.1–0.5 μM range, but inhibits proteasomal DUB activity in vitro at a UPS other than the proteasome. So far, however, no compounds have concentration of ~10 μM(Wang et al., 2014). This compound was found been approved for clinical use. The potential to influence processes to be rapidly taken up and enriched in cells (Wang et al., 2014), such as signal transduction, proliferation and apoptosis by affecting explaining this apparent inconsistency. Enrichment appears to be ubiquitination and proteasomal degradation of key regulators is both thiol-driven (Wang et al., 2014). Eer1 shows in vitro activity at promising and exciting. DUBs are likely to be more druggable than E3 li- ~70 μM in biochemical assays whereas 8 μM Eer1 is sufficient to block gases owing to the lack of defined catalytic residues in the latter. Most translocation of proteins over the ER in cultured cells (Cross et al., DUBs are cysteine enzymes which should be easy to drug, particularly 2009). This discrepancy was suggested to result from metabolism of using compounds containing Michael acceptors. The degree of specific- Eer1 in vivo (Wang et al., 2008). Cyclopentenone PGs have been found ity which can be achieved by such compounds is uncertain. The overall to be enriched in cells by a factor of ~20-fold (Narumiya & Fukushima, importance of DUBs for central cellular processes such as proper 1986). Cellular enrichment of cyclopentenone PGs was reported to be proteasomal function may explain the cytotoxic profiles of interesting due to active transport over cell membranes (Narumiya & Fukushima, natural products such as curcumin and synthetic chalcones. Bioavail- 1986). ability is a hurdle that needs to be overcome, but if progress can be made in this area molecules containing functional dienones as well as 4.8. Reactivity of α,β-dienones present in DUB inhibitors other types of compounds may be developed into useful cancer therapeutics. Cysteine residues are ideal targets for covalent drugs with the potential of target specificity and a prolonged residence time and, hence, Conflicts of interest duration of action (Serafimova et al., 2012). The BTK inhibitor ibrutinib and the EGFR inhibitor afatinib represent examples of covalent kinase Two of the authors (P.D. and S.L.) are share holders in Vivolux AB, inhibitors that target noncatalytic cysteine thiols (Liu et al., 2013; a company which develops DUB inhibitors to be used as cancer Schwartz et al., 2014). Irreversible Michael acceptors such as acrylam- therapeutics. ides may be converted to electrophiles that react with cysteine thiols in a rapidly reversible fashion (Serafimova et al., 2012). We have References found that the b-AP15 inhibitor induces reversible inhibition of Adams, D. J., Dai, M., Pellegrino, G., Wagner, B. K., Stern, A. M., Shamji, A. F., et al. (2012). proteasome-associated DUB activity both in vitro (enzyme assays Synthesis, cellular evaluation, and mechanism of action of piperlongumine analogs. using 26S proteasomes) or in vivo (determining the levels of Proc Natl Acad Sci U S A 109,15115–15120. polyubiquitin after removal of drug) (D'Arcy et al., 2011; Wang et al., Albrecht, M., Hoffmann, D., Evert, B. O., Schmitt, I., Wullner, U., & Lengauer, T. (2003). 2014). Issaenko and Amerik (2012) reported that the chalcone AM146 Structural modeling of ataxin-3 reveals distant homology to adaptins. Proteins 50, 355–370. elicited an irreversible commitment for cell death, suggesting that the Aldini, G., Carini, M., Vistoli, G., Shibata, T., Kusano, Y., Gamberoni, L., et al. (2007). Identi- compound is an irreversible inhibitor. Whether DUB activity was irre- fication of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma versibly inhibited was not tested, however, and it is possible that com- cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement. Biochemistry 46,2707–2718. mitment to cell death was achieved even after short exposures to Aldini, G., Dalle-Donne, I., Colombo, R., Maffei Facino, R., Milzani, A., & Carini, M. (2006). AM146, as found using b-AP15 (Wang et al., 2014). Lipoxidation-derived reactive carbonyl species as potential drug targets in preventing Addition of thiols such as glutathione to dienone PGs occurs more protein carbonylation and related cellular dysfunction. ChemMedChem 1,1045–1058. Aleo, E., Henderson, C. J., Fontanini, A., Solazzo, B., & Brancolini, C. (2006). Identification of rapidly but results in more labile thiol conjugates compared to enone new compounds that trigger apoptosome-independent caspase activation and apo- PGs (Suzuki et al., 1997). Interestingly, dienone reactivity is considered ptosis. Cancer Res 66,9235–9244. P. D'Arcy et al. / Pharmacology & Therapeutics 147 (2015) 32–54 49

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