Proteasome-associated deubiquitinases and cancer

At its simplest level, the UPS consists of a relay of enzymes that tag proteins for destruction with the small-molecule ubiquitin (Ub) and the proteasome, a highly specific molecular shredder that degrades Ub-tagged substrates (Fig. 1). The ubiquitination of proteins occurs via three sequential steps performed by ubiquitin-activating (E1), ubiquitin conjugation (E2), and ubiquitin ligase (E3) enzymes. The E1 enzyme initially forms a high-energy thio-ester bond with Ub in an ATP dependent manner, resulting in the activation of the Ub molecule. The active Ub forms a complex with the active cysteine in the E2 enzyme resulting in the formation of an intermediate E2-Ub complex. A compatible E3 enzyme interacts with the E2-Ub complex and target substrate, thus serving as a highly specific molecular scaffold that facilitates the conjugation of the Ub residue from the E2 to consensus lysine residues in the target protein. The number of E1, E2, and E3 enzymes is highly variably with 2 E1, over 40 E2, and several hundred varieties of E3 enzymes described [3]. A high degree of variability and target specificity exists within the E3s, with enzymes classified into three families based on sequence homology, namely the RING, HECT, or RING-between-RING ubiquitin ligases. The E3 ligases play a central role in substrate recognition and specificity in Ub conjugation. To finalize the ubiquitination process, the Ub chain attached to target proteins may be elongated by a specific E3 subtype, commonly referred to as an E4 ligase that catalyzes the formation of extended poly-Ub chains. The nature of Ub attachment whether as mono-Ub or as poly-Ub chains, composed of multiple Ub moieties, can have profound effects on a proteins activity and/or stability. Since Ub contains seven internal lysine residues, multiple conformations of linkages within the polyubiquitin chains are possible. The linkage between the Ub moieties located in the chain functions as a highly specific ubiquitin code that determines the fate of the conjugated protein. Ubiquitin linkages occurring at Lysine 11, Lysine 29, and Lysine 48 generally serves as a destruction signal for proteasomal degradation, whereas other linkage types usually regulate non-proteolytic activities that alter activity, location, or interactions of the substrate protein [4].

Ubiquitination of proteins is not a one-way process and can be reversed by a class of isopeptidases known as deubiquitinating enzymes (DUBs), which catalyze the breaking of the isopeptide bond between the C-terminal glycine of Ub and the ε-amino group of lysine residues in target proteins. In total, around 80 DUBs have thus far been identified and classified into six groups based on sequence homology of the catalytic domain: ubiquitin-specific proteases (USP), ubiquitin carboxy-terminal hydrolases (UCH), ovarian-tumor proteases [5], Machado-Joseph disease protein domain proteases (MJD), JAMM/MPN domain-associated metallopeptidases (JAMM), and monocyte chemotactic protein-induced protein (MCPIP) [6‐8]. While the function of many DUBs remains to be determined, deubiquitination of mono-Ub or poly-Ub chains can induce altered protein localization, trafficking, or enhanced stability [9]. While the majority of DUBs are presumed to exist as free enzymes, several have been identified through their association of large enzyme complexes. Considering the total number of DUBs identified and the degree of variability within the different family members, they are considered to be highly druggable with various DUB inhibitors currently in various stages of pre-clinical development.

Proteins conjugated with poly-Ub chains containing Lys-11, Lys-29, and Lys-48 linkages are generally transported to the 26S proteasome, where they are degraded by the proteolytic activities of the 20S core particle (20S CP). Entry of ubiquitinated proteins to the 20S CP is controlled by the 19S regulatory particle (19S RP), which functions as a highly specific gatekeeper allowing only those proteins displaying the correct Ub tags to enter. Poly-Ub tagged substrates are recognized by highly specific Ub receptors, Rpn10 and Rpn13 localized within the 19S RP. Once a poly-Ub protein has been captured by the Ub receptors, the AAA-ATPase subunits Rpt1–6 located at the base of the 19S RP facilitate unfolding of captured protein, gate opening of the 20S CP, and translocation into the catalytic chamber where proteolysis occurs. In addition to the 19S RP, entry into the 20S CP can also be modulated by other regulatory particles such as the 11S regulator (also referred to as PA28) that replaces the 19S RP on the 20S CP and forms the immunoproteasome. While not as extensively characterized as the conventional 26S proteasome, studies have shown that immunoproteasome is involved in diverse functions, such as antigen processing and modulating the response to oxidative stress. However, the role of the immunoproteasome is outside the scope of this review. One particular aspect of the 19S RP is how polyubiquitin chains are removed from target proteins following capture by the proteasome. The presence of large, bulky poly-Ub chains poses a potential problem for proteasomal degradation, since polyubiquitinated proteins can have molecular weights much greater than non-ubiquitinated species. In order to facilitate unfolding and translocation through the narrow gate into to the 20S CP, the poly-Ub chain must be removed from the target protein. This process of proteasome deubiquitination is mediated by the action of 3 DUBs, POH1/Rpn11, UCHL5/UCH37, and USP14 [10‐14].

The 20S core subunit is a cylindrical structure comprised of two outer heptameric α subunits stacked on two central heptameric β subunits [15, 16]. The α ring forms a physical barrier that protects against promiscuous entry to the proteolytic chamber formed by the β subunits. The catalytic activity of the 20S CP is exerted by β1, β2, and β5 subunits responsible for caspase-like, trypsin-like, and chymotrypsin-like activities, respectively [17]. The cumulative effect of these different activities results in the degradation of protein substrates into short oligopeptides generally between 3 and 15 amino acids in length. Oligopeptides are further hydrolyzed to single amino acid residues by oligopeptidases located in the cytosol, thus facilitating the recycling of amino acids for protein synthesis.

The manner by which proteasome inhibition induces apoptosis in cancer cells is not fully understood. Since the UPS is involved in such a broad range of processes, the manner in which apoptosis is induced is at least in part context dependent. Four potential mechanisms by which proteasome inhibition induces apoptosis and cell death are discussed below.

Bortezomib (PS-341, NSC 681239, Velcade®; Millennium Pharmaceuticals, Inc.) was the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed MM [44] and MCL [45]. Bortezomib was initially used in the clinic as single agent for the treatment of MM patients who relapsed after standard dexamethasone therapy, where it resulted in an improved overall survival rate. Subsequent studies have shown that bortezomib treatment for newly diagnosed MM significantly increased patient survival rates [46].

Bortezomib is a synthetic dipeptide boronic acid, which slowly and reversibly inhibits the chymotrypsin-like and to a lesser extent the trypsin-like and caspase-like activities of the 20S CP. Inhibition is achieved through an initial nucleophilic attack on the active N-terminus threonine residues located on the β5, β2, and β1 subunits, leading to loss of proteolytic activity [47]. It has been reported that bortezomib inhibits the activation of NF-κB pathway in squamous cell carcinoma [48], MM [49]and in MCL [50]. However, other studies have reported that canonical NF-κB activation is induced by bortezomib in MM cells suggesting that its cytotoxic effects cannot be entirely attributed to inhibition of NF-κb [28]. For this, the anti-cancer and pro-apoptotic activities of bortezomib may cell type-dependent and driven by specific signaling pathways. It has been reported that bortezomib induces both transcriptional and post-translational increases in cyclin-dependent kinases, p21 and p27 in hepatocellular carcinoma cells, and NSCLC [51, 52]. Bortezomib-mediated proteasome inhibition is p53-dependent in renal and NSCLC cell lines [52, 53]. Bortezomib activates the stress kinase JNK and the c-Jun/AP-1 signal pathway, thus inducing the expression of the cyclin-CDK inhibitor p21, leading to growth arrest and apoptosis of NSCLC cells [52]. Finally, the induction of pro-apoptotic protein NOXA by bortezomib is directly dependent on the oncogene MYC in melanoma cell lines [54].

Despite promising results in MM patients, several limitations of bortezomib treatment have emerged. The lack of response observed in some patients as a first-line therapy and relapses and/or resistance after initial favorable responses in MM patients have been described as the main limitations of the drug [55]. Moreover, bortezomib treatment has been correlated to the onset of adverse effects such as thrombocytopenia, fatigue, GI symptoms, and asthenia. Bortezomib-induced peripheral neuropathy (BIPN) was identified as the main significant dose-limiting toxicity potentially leading to permanent nerve damage to extremities probably due to an off-target effect of the drug [56] . The incidence of BIPN was reduced by changing the dose frequency from twice weekly to once weekly and choosing subcutaneous administration over intravenous injection [57‐61]. In addition, bortezomib has shown successful results when used in combination with other type of treatments. Combination treatments of bortezomib with doxorubicin, dexamethasone, panobinostat, and daratumumab showed improved clinical outcomes in patients with relapsed and refractory MM, suggesting its ability to sensitize malignant cells to conventional chemotherapy [61‐63]. However, less promising results were obtained in patients with solid tumor compare to those with hematologic malignancies. The combinational treatment of bortezomib with docetaxel in prostate tumor did not reveal any significant anti-cancer effect [64].

Several mechanisms of resistance have been reported and investigated in pre-clinical and clinical settings. Alterations at the proteasome level include mutations and overexpression of the catalytic subunits. For instance, alteration of the gene PSMB5 encoding for the proteasome subunit β5 was found in tumors resistant to bortezomib in vitro and in pre-clinical studies [65‐67]. However, no such mutations were found in MM patients who developed resistance to bortezomib [68]. Resistance to bortezomib downstream of the proteasome includes enhanced activation of the aggresome-autophagy pathway, alterations in apoptotic signaling, increased expression of anti-oxidants, and dampening of the ER stress response. The role of the aggresome-autophagy pathway has been investigated as a resistance mechanism exploited by cells to overcome proteotoxicity induced by proteasome inhibition. It has been shown that pancreatic cancer cells treated with bortezomib form aggresomes, which are large aggregates of Ub-conjugated proteins that are subsequently cleared by autophagy. Studies revealed that inhibition of aggresome formation with histone deacetylase inhibitors such as panobinostat increases bortezomib anti-cancer activity, suggesting that aggresome formation may be a potential resistance mechanism [69, 70]. Anti-apoptotic Bcl-2 mediators have also found to be overexpressed in bortezomib-resistant cells, also implicating a reduction in apoptotic capacity as a potential resistance mechanism [71, 72]. Consistent with this, it has been shown that combination treatments with inhibitors of Bcl-2 and bortezomib showed a synergistic effect [71, 73]. Overexpression of the chaperone BIP/Grp78 protein involved in the ER stress response to proteotoxicity has also been associated with a reduced sensitivity to bortezomib, which is not surprising considering the role of ER stress in triggering apoptosis. Pretreatment of bortezomib-resistant cells with inhibitors of ER-mediated protein folding restored cell sensitivity to bortezomib, implying that enhanced chaperone activity could also attribute to bortezomib resistance [74].

Carfilzomib (PR-171; Kyprolis; Onyx Pharmaceutical) is a tetrapeptide epoxyketone, selective and irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Carfilzomib was approved by the FDA in 2012 for the treatment of relapsed MM patients who had previously received at least two therapies, including bortezomib and immunomodulatory drugs, and displayed disease progression within 60 days after the first cycle of therapy [75]. It is used as single agent in third-line treatments and in combination with lenalidomide and dexamethasone as second-line therapy [76].

Carfilzomib covalently irreversibly inhibits the β5 subunit, responsible for the chymotrypsin-like activity of the 20S proteasome, resulting in a similar stress response to that observed following bortezomib [77, 78].

Carfilzomib displayed higher cytotoxicity than bortezomib in several cell lines derived from hematologic tumors as well as solid cancer [76]. The explanation is found in the higher selectivity of the epoxyketone for the N-terminal threonine active site of the proteasome compared to the boronic acids of bortezomib and β-lactone of salinosporamide A [78, 79]. Carfilzomib is considered the most specific and potent proteasome inhibitor with good cytotoxic activity in bortezomib-resistant MM cell lines and in samples from patients with bortezomib-resistant MM [80].

To date, most of the clinical trials with carfilzomib have been performed in patients with relapsed MM. However, many other studies are ongoing and/or recruiting patients with other hematological diseases, such as Hodgkin lymphoma or solid tumors like ovarian and kidney cancer. The treatment design consists of the use of carfilzomib as a single agent or in combination with conventional treatments, such as dexamethasone, melphalan, panobinostat, and irinotecan. The new phase III CLARION study is recruiting patients with newly diagnosed MM to treat with carfilzomib or bortezomib in combination with conventional drugs to evaluate if carfilzomib can replace bortezomib as front line treatment for MM [81].

Carfilzomib is associated with several side effects mainly correlated not only to the cardiovascular system, such as hypertension, but also to the urinary tract. Moreover, carfilzomib can lead to the onset of fever, anemia, diarrhea, fatigue, and nausea [82, 83]. The main adverse effects of carfilzomib differ from bortezomib suggesting a potential difference in downstream effects of the two drugs [84].

Oprozomib (ONX0912; PR-047), a truncated derivate of carfilzomib, is a tripeptide epoxyketone that functions as an irreversible and selective inhibitor of the chymotrypsin-like activity 20S CP. The need to find better proteasome inhibitor with better dose flexibility and convenience for patients led to the design and synthesis of oprozomib as a new generation of proteasome inhibitor with better oral bioavailability compared to the intravenously administrated carfilzomib [85].

Oprozomib has shown similar anti-tumor activity, potency, and selectivity as carfilzomib on MM cell derived from relapsed patients after treatment with conventional anti-MM drugs and therefore can be used to treat patience with resistance to bortezomib, dexamethasone, or lenalidomide. In vitro studies showed that oprozomib has synergistic/additive anti-MM activity when combined with bortezomib, lenalidomide, and dexamethasone and inhibits the migration of MM cells and angiogenesis. Moreover, oprozomib inhibits the tumor growth in human MM xenograft models reducing tumor progression and increasing the survival [86]. The inhibitory activity of oprozomib in pre-clinical models of solid tumors had also been investigated. Results showed that oprozomib induced apoptotic pathway through Bik upregulation and activation of caspase-8, caspase-9, caspase-3, and PARP cleavage in head and neck squamous cell carcinoma (HNSCC) cells, leading to cell death. This effect is antagonized by upregulation of Mcl-1 and anti-apoptotic Bcl2 family member that inhibits cytochrome C release form the mitochondria. In response to proteasome inhibition, HNSCC cells upregulate autophagy and ATF4 resulting in increased cell survival. Oprozomib has shown promising inhibition of tumor growth in HNSCC xenograft mice models providing the bases for a further use of this drug in the clinic [87]. Oprozomib also inhibits NF-κB and activates the JNK pathways [88]. Oprozomib, similar to carfilzomib, not only targets myeloma growth directly but also decreases myeloma-associated bone disease by inhibition of osteoclast differentiation and reabsorption while enhancing osteoblast formation and function [89]. Oprozomib is in phase I/II clinical trial for treatment of newly diagnosed, relapsed, and refractory MM as a single agent or in combination with other anti-MM drugs such as dexamethasone. A phase I study in solid tumors has also been performed; however, gastrointestinal toxicities and minimal anti-tumor activity in patients with advanced solid tumors were reported [90].

Ixazomib citrate (MLN9708, Ninlaro®, Takeda Pharmaceutical, Cambridge, MA, USA) and its biologically active form ixazomib (MLN2238) are the first orally administrated proteasome inhibitors tested in the clinic for the treatment of relapsed and refractory MM [91, 92].

Ixazomib, like bortezomib, is a dipeptide boronate that reversibly and selectively targets the β5 proteasome subunit inhibiting the chymotrypsin-like proteasome activity with IC₅₀ value of 3.4 nmol/L. Moreover, at higher doses, it also inhibits the β1 caspase-like and β2 trypsin-like proteolytic subunits with an IC₅₀ of 31 and 3500 nmol/L, respectively [93].

Ixazomib differs from bortezomib in its physiochemical properties leading to improved pharmacokinetics and pharmacodynamics. In fact, although both drugs have a similar potency and selectivity, ixazomib has a shorter proteasome dissociation half-life (18 min vs. 110 min). This correlated to an improved blood and tissue distribution, which make administration of higher doses possible. Moreover, increased expression of biomarkers of proteasome inhibition, such as ER stress markers in xenograft tumor tissue, suggests improved pharmacodynamic properties. Improved pharmacokinetic and pharmacodynamic profiles have been correlated to the increased anti-cancer activity of ixazomib compared with bortezomib. In fact, ixazomib showed a better anti-cancer activity in pre-clinical studies performed on solid tumor and hematologic xenograft mice models [93, 94].

Ixazomib also showed synergistic anti-MM activity when used in combination with dexamethasone or lenalidomide [93, 95].

A study on microRNA profiling of MM cells treated with ixazomib showed upregulation of the small long-coding RNA miR33b, which seems to be constitutively under-expressed in MM patients. Overexpression of miR33b led to tumor growth inhibition and increased survival in human MM xenograft mice model, elucidating its tumor suppressor role during apoptosis induced by ixazomib treatment [96].

Ixazomib was approved in 2015 in the USA by FDA and in 2016 in EU by EMA. Ongoing clinical trials are investigating its activity, as single agent or in combination with other standard anti-MM drugs, like melphalan, prednisone, lenalinomide, and dexamethasone, in hematological and solid tumors. Results have shown an evident anti-MM activity and good tolerability and safety [97]. Like bortezomib, ixazomib’s most common side effects are thrombocytopenia, gastrointestinal symptoms, neutropenia, and fatigue but with a lower incidence of neuropathy, probably due to its higher selectivity [37].

b-AP15 (3E,5E-bis[(4-nitrophenyl)methylene]-1-(1-oxo-2-propen-1-yl)-4-piperidinone) (Fig. 3A) is a small-molecule inhibitor of proteasomal DUBs USP14 and UCHL5 [98]. Its predicted method of action is inhibition of these proteasomal DUBs, blocking the deubiquitination process. The b-AP15 molecule, and its optimized lead VLX1570, contains two α,β-unsaturated carbonyl groups (Table 2, indicated by blue arrow) which, together with electron drawing capacity from side groups (Table 2, indicated by red arrow), can facilitate Michael addition to the thiols of cysteine residues of the DUB active sites.

In pre-clinical models, b-AP15 shows to be potent in causing accumulation of ubiquitinated proteins without affecting the activity of the 20S [23, 99], consistent with the idea that proteasomal DUB inhibition selectively causes a block in the deubiquitination and subsequent degradation of proteins by the proteasome. b-AP15 response is characterized by strong induction of HMOX-1 indicating a crucial role for oxidative stress in apoptosis induced by b-AP15 [23]. As of yet, it is unclear whether this induction of HMOX-1 is caused as a direct consequence of accumulation of ubiquitinated proteins, or whether off-target activity is the cause. While some off-target activity of b-AP15 has been determined, in the form of inhibition of thioredoxin reductase (TrxR), TrxR inhibition by itself accounted for neither the ubiquitinated protein accumulation nor the induction of apoptosis [23]. Of particular interest in the context of overcoming bortezomib resistance is the finding that b-AP15-induced apoptosis in bortezomib-resistant MM cells. Cells resistant to bortezomib show marked induction of anti-apoptotic regulator BCL2 and reduction of pro-apoptotic proteins Bax and Bak [23, 98, 100]. While sufficient to perturb apoptosis induced by bortezomib, bortezomib-resistant cells maintained their sensitivity to b-AP15. This indicates that b-AP15 induces apoptosis by a distinct pathway from bortezomib suggesting a potential treatment for patients with acquired bortezomib resistance.

Resistance to b-AP15 itself in clinical setting is likely inevitable, but pre-clinical assessment of acquired resistance to b-AP15 shows promising results. After 9 months of continues low-grade exposure to b-AP15, cells only became ~ twofold more resistant [101]. Mutations to the active site of USP14 or UCHL5 that would silence inhibition by b-AP15 are unlikely to be functional and to the best of our knowledge have not been observed. Upregulation of detoxification enzyme glutathione is another likely method by which resistance could be induced, but screening of cell lines with known levels of glutathione showed no correlation between these levels and the ability of b-AP15 to induce apoptosis [102].

WP1130 (degrasyn, (2E)-3-(6-bromo-2-pyridinyl)-2-cyano-N-[1S-phenylbutyl]-2-propenamide) (Fig. 3A) is a partially selective DUB inhibitor initially identified as a stabilizer of Bcr/Abl in chronic myelogenous leukemia [103] and a negative regulator of MYC in melanoma [104]. WP1130 inhibited tumor growth in vivo at doses that were well tolerated. Further studies in MM identified the compound as an inhibitor of USP9x [5], a DUB necessary for stabilizing the anti-apoptotic BCL2 family protein MCL1 [105]. In response to WP1130 treatment, USP24 was upregulated and acted as a compensatory mechanism for USP9x inhibition by WP1130. Consequently, stability of MCL1 was eventually reestablished after WP1130 treatment. Within the same study, however, the novel WP1130 analogue EOAI3402143 was able to inhibit both USP9x and USP24 suggesting improved clinical potential [5].

P5091 (1-[5-[(2,3-dichlorophenyl)thio]-4-nitro-2-thienyl]-ethanone; 1-[5-(2,3-dichlorophenyl)sulfanyl-4-nitro-2-thienyl]ethanone) (Fig. 3B) was first identified as a specific inhibitor of USP7 [106]. The compound was shown to inhibit stabilization of HDM2 by USP7, exerting its pro-apoptotic effect through the resulting stabilization of p21 and p53. Interestingly, p53 negative cells were also sensitive to P5091 suggesting p53 independent mechanisms [106]. A remarkable inhibition of angiogenic markers and synthesis of tumor vasculature in xenograft models was observed, contributing to its anti-tumor effect. P5091 has been studied extensively in recent times, and various new studies have shown the efficacy of the compound in a range of different cancer types and through different mechanisms such as destabilization of DNA repair regulator CCDC6 in lung-neuroendocrine cancer in conjunction with PARP inhibitors [107] and prostate cancer [108], destabilization of β-catenin in colorectal cancer [109], and activation of the p53/p21 signaling axis in chronic lymphocytic leukemia [110]. P5091 further showed strong synergy in combination treatment with RRx-001 (1-bromoacetyl-3,3-dinitroazetidine) in MM [111]. RRx-001 selectively induces oxidative and nitrative stress in hypoxic environments [112, 113], acting as a potent inhibitor of tumor growth, invasiveness, angiogenesis, and overcoming resistance to various MM treatment modalities including bortezomib.

USP8i (9-ethyloxyimino-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile) (Fig. 3C) is a selective inhibitor of USP8, identified in a screen for compounds to overcome resistance to EGFR inhibitors for the treatment of NSCLC [114]. EGFR commonly acquires mutations after treatment with this class of inhibitors [115], spurring the research for alternative methods of targeting this pathway. USP8i was shown to overcome resistance to EGFR inhibitor gefitinib and reduced the cell surface expression levels of EGFR and other receptor tyrosine kinases (RTK) ERBB2, ERBB3, and MET.

Betulinic acid (BA, (3β)-3-hydroxy-lup-20(29)-en-28-oic acid) is a natural compound of plant origin with pro-apoptotic effects and high specificity to cancer cells [116, 117]. This high specificity has caused widespread interest in the compound, with over 100 publications on the subject listed as published in 2016 on PubMed. Yet, neither the exact mechanism by which BA causes its apoptotic effect nor its specificity is fully understood. BA was determined to be a promiscuous DUB inhibitor in the context of prostate cancer, where it induced apoptosis by release of mitochondrial proteins and caused downregulation of angiogenic markers in vitro [118]. Interestingly, a recent study of BA suggested an entirely novel pathway in which BA induces apoptosis. In this newly suggested mechanism inhibition of de novo fatty acid synthesis is the primary mode of action of the compound [119]. The specificity of BA is explained by the unique reliance of de novo synthesis of fatty acids by cancer cells, rather than the uptake from blood as is the case for healthy cells [120]. BA is postulated to cause accumulation of mitochondrial lipid, cardiolipin, which causes structural failure of the mitochondria and consequently the activation of the mitochondria apoptotic pathway. As of yet, it is unclear whether this effect of BA is mutually exclusive with its role as a DUB inhibitor or whether two distinct mechanisms are at play.

Capzimin (quinolone-8-thiol (8TQ)) selectively inhibits the proteasomal DUB Rpn11/POH1 through direct binding to the catalytic Zn²⁺ ion in POH1’s active site. Recently identified, this compound represents a first-in-class inhibitor selective for POH1 capable of inducing polyubiquitinated protein accumulation and cell death [121, 122]. The compound has so-far been screened against the NCI 60 panel of cell lines and has shown to be effective in leukemia cell lines as well as cell lines derived from solid tumors [121]. Interestingly, capzimin was found to remain efficacious against cells with acquired resistance to bortezomib. Together with the finding that POH1 inhibition induced a distinctly different change in the ubiquitination profile from bortezomib, this compound may have potential as a treatment modality in patients previously treated with bortezomib.

MLN4924 (sulfamic acid, [(1S,2S,4R)-4-[4-[[(1S)-2,3-dihydro-1H-inden-1-yl]amino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl]-2-hydroxycyclopentyl]methyl ester) is a first-in-class compound that mimics E1 in the ubiquitination process. However, MLN4924 is incompatible with the following enzymatic reactions, thereby blocking the ubiquitination process by de facto inhibiting E1 [123, 124]. In pre-clinical studies, MLN4924 showed potent anti-tumor effects at well-tolerated doses in xenograft models through stabilization of cell cycle regulatory substrates, causing deregulation of DNA synthesis in S phase and apoptosis induction [124]. This novel compound is in early phases of clinical trials for various malignancies and is showing promising initial toxicity profiles in MM and lymphoma [125], melanoma [126], and advanced solid tumors [127].

PYZD-4409 (1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furanyl)methylene]-3,5-pyrazolidinedione) and PYR-41 (ethyl 4-[(4Z)-4-[(5-nitrofuran-2-yl)methylidene]-3,5-dioxopyrazolidin-1-yl]benzoate) are pyrazone-derived compounds that inhibit E1 [128‐130] . PYR-41 shows promising effects on the NF-κB pathway and stabilization of p53 [129]. The compound may have further clinical validity through stabilization of ionotropic testosterone receptor TRPM8 in prostate cancer, implicated to sensitize this type of cancer to apoptosis [131]. PYZD-4409 shows promising anti-tumor activity in primary leukemia patient cells as well as in vivo leukemia models [128]. Accumulation of p53 is observed in these models; however, the authors postulate that accumulation of misfolded proteins by inhibition of the UPS and consequential ER stress, rather than the regulation of specific factors, is the primary driver of apoptosis.

The study of E3 ligase inhibitors has largely focused on antagonizing the binding of E3 ligases to specific substrates [18]. Inhibition of E3 ligases hinges on competing with the binding of specific protein substrates to the E3 ligase, making this treatment potentially highly specific for the intended substrate but also a challenging mechanism to design drugs for. The HDM2-p53 interaction is so far the most promising E3-substrate interaction, for which some compounds have been suggested. Cis-imidazolines, benzodiazepinediones, spiro-oxindoles, and oxindoles are examples of classes of compounds under investigation as stabilizers of p53 through E3 antagonism. To date, p53 stabilization and subsequent induction of apoptosis have been determined in various studies showing both in vitro and in vivo efficacy, giving strong clinical validity to this class of compounds [131‐137]. Importantly, cis-imidazoline Nutlin-3 treatment remained effective in the context of mutated p53 in combination treatment with TrxR inhibitor dasatinib. Nutlin-3 has further shown to inhibit angiogenic factors by p53-dependent inhibition of HIF-1α and p53-independent inhibition of HIF-1 [138].

The UPS is a complex system, and not all of its constituents have been fully assessed. What is well understood is that the UPS is crucially involved in nearly all cellular processes. Considering the importance of the UPS in cancer formation and its validity as a cancer treatment, there is great potential for progress yet to be made in this field. Inhibition of the 20S is clinically established and remains a promising field of study. There is currently a high volume of ongoing clinical trials testing established and new inhibitors of the 20S. These trials aim to find novel 20S inhibitors with increased efficacy and decreased toxicity, as well as discovery of synergistic combination therapies and application in a broader range of cancer types of established inhibitors. Pre-clinically, the unraveling of the role of the vast range of ubiquitinating and deubiquitinating proteins is certain to provide additional targets for treatment, broadening our toolkit for targeting the stabilization and degradation of specific proteins as well as overcoming resistance to 20S inhibitors. Inhibition of the DUBs associated with the proteasome is a direct alternative to the proteasome inhibition facilitated by 20S inhibitors, causing accumulation of poly-Ub tagged proteins and ER stress. Ours and various other labs are working intensively on elucidating the effect of proteasomal DUB inhibition and to facilitate its advancement into clinical practice. New small-molecules with optimized DUB inhibitory effects and pharmacodynamics are being investigated for this goal. Overall, advancement of targeting the UPS in cancer treatment is of significant clinical importance, and we expect to see an increased role for UPS-based treatment in the clinic in the near future.