Deubiquitinase Inhibitor Research Articles

Abstract 2988: Proteasomal deubiquitinases represent an attractive therapeutic target in head and neck squamous cell carcinomas (HNSCC)

Abstract Despite recent advances in treatment options for Human Papillomavirus (HPV)- head and neck squamous cell carcinoma (HNSCC), the overall survival (OS) rate for HNSCC is low, demonstrating the need for novel therapies for these cancers. Targeting the ubiquitin-proteasome system (UPS) has emerged as a potential target for the development of novel anti-cancer therapies. Bortezomib, a first generation proteasome inhibitor clinically approved for the treatment of Multiple Myeloma, has previously been demonstrated to induce tumor regression in a subset of HNSCC patients through the inhibition of canonical NFκB activity; however, the failure of Bortezomib to inhibit other pro-survival pathways such as MAPK and STAT3 signalling resulted in heterologous responses. A recently developed alternative to proteasome inhibitors is the small molecule b-AP15, which inhibits the proteasomal deubiquitinases USP14 and UCHL5, thus allowing more specificity and less toxicity than proteasome inhibitors. Here, we show b-AP15 inhibits proliferation and colony formation in multiple HPV- and HPV+ HNSCC cell lines, with minimal effects in normal oral keratinocytes. Furthermore, b-AP15 induces significant caspase-dependent apoptosis. Using siRNA targetting USP14 and UCHL5, only USP14 depletion significantly reduced cell proliferation and colony formation. Mechanistically, b-AP15 treatment reduces TNFα-induced NFκB activity and the expression of pro-survival proteins such as cIAP2 and TRAF2, promoting the formation of the TNFR complex II and sensitizing cells to TNFα-induced cell death. Finally, TCGA data demonstrates that USP14 is highly expressed in HNSCC when compared with normal tissue and is significantly correlated with worse overall survival. Together, we have identified that inhibition of proteasomal deubiquitinases inhibits the proliferation and survival of HNSCC cells and enhanced TNFα-induced cell death via the inhibition of NFκB activity. Our data suggest that combination therapies with b-AP15 could potentially offer a clinical benefit in HNSCC patients by promoting TNFα-induced cytotoxicity. Further studies will focus on the mechanism by which USP14 regulates NFκB signalling in HNSCC cells and the effect of b-AP15 activity in combination with radiation treatment in in vivo mouse xenograft models. Citation Format: Ethan L. Morgan, Tiffany Toni, Xinping Yang, Hui Cheng, Ramya Viswanathan, Zhong Chen, Carter Van Waes. Proteasomal deubiquitinases represent an attractive therapeutic target in head and neck squamous cell carcinomas (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2988.

Hepatitis B Virus X Protein Is Stabilized by the Deubiquitinating Enzyme VCPIP1 in a Ubiquitin-Independent Manner by Recruiting the 26S Proteasome Subunit PSMC3.

ABSTRACTHepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide, and the viral X protein (HBx) is an etiological factor in HCC development. HBx is a high-turnover protein, but knowledge of the role of deubiquitinating enzymes (DUBs) in maintaining HBx homeostasis is very limited. We used a 74-DUB library-based yeast two-hybrid assay and determined that a novel DUB, valosin-containing protein-interacting protein 1 (VCPIP1), interacted with HBx. VCPIP1 and its C-terminal amino acids 863 to 1221 upregulated the HBx protein expression, with or without HBV infection. Mechanistically, VCPIP1 stabilized HBx protein through a ubiquitin-independent pathway, which was validated by the HBx ubiquitination site mutant plasmid. Coimmunoprecipitation assays demonstrated the potency of VCPIP1 in recruiting 26S proteasome regulatory subunit 6A (PSMC3) and forming a ternary complex with HBx through mutual interaction. In vitro, purified His-tagged PSMC3 protein rescued HBx degradation induced by the 20S proteasome, and in vivo VCPIP1 synergized the mechanism. Functionally, HBx specifically binding to VCPIP1 significantly enhanced the transcriptional transactivation of HBx by activating NF-κB, AP-1, and SP-1 and inhibited hepatoma cell clonogenicity in Huh7 and HepG2 cells. Moreover, we further demonstrated that overexpression of VCPIP1 significantly affected the HBV covalently closed circular DNA (cccDNA) transcription in HBV-infected HepG2-NTCP cells. Altogether, our results indicate a novel mechanism by which VCPIP1 recruits PSMC3 to bind with HBx, stabilizing it in a ubiquitin-independent manner, which might be critical for developing DUB inhibitors in the future.IMPORTANCE HBx is a multifunctional viral oncoprotein that plays an essential role in the viral life cycle and hepatocarcinogenesis. HBx degradation occurs through the ubiquitin-proteasome system (UPS). However, whether novel compartments of the DUBs in the UPS also act in regulating HBx stability is not fully understood. Here, for the first time, we defined VCPIP1 as a novel DUB for preventing HBx degradation by the 20S proteasome in a ubiquitin-independent manner. PSMC3, encoding the 26S proteasome regulatory subunit, directly stabilized HBx through physical binding instead of a common approach in protein degradation, serving as the key downstream effector of VCPIP1 on HBx. Therefore, the ternary binding pattern between VCPIP1, HBx, and PSMC3 is initiated for the first time, which eventually promotes HBx stability and its functions. Our findings provide novel insights into host-virus cross talk by targeting DUBs in the UPS.

PINK1/Parkin Pathway Activation for Mitochondrial Quality Control - Which Is the Best Molecular Target for Therapy?

There has been long-term interest in drugging the PINK1-Parkin pathway with therapeutics as a treatment for Parkinson’s disease (PD). Despite significant structural data on Parkin as well as the PINK1 kinase and the multiple conformational changes it undergoes, activation of these targets is non-trivial. This review highlights small molecule screening results that suggests that activation of Parkin biochemically does not necessarily translate to activation of Parkin within cells. There are also issues with activation of PINK1 with kinetin analogs, which do not appear to rescue rodent models of PD. The counter-measure of activating the mitophagy pathway with deubiquitinase (DUB) inhibitors such as USP30 inhibitors is progressing in the clinic for kidney disease and the proof of biology for this target will be tested in these trials. An alternative mechanism of activating Parkin in response to oxidative stress via Parkin phosphorylation by the AMPK-ULK1 pathway may be a simpler way to lower the energy barrier Parkin activation.

On the Study of Deubiquitinases: Using the Right Tools for the Job.

Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design.

First‐in‐class Deubiquitylase Inhibitors Reveal New Enzyme Conformations

Cells maintain protein homeostasis by adding a small protein, ubiquitin, to regulate a variety of cellular processes, dictating protein activity, localisation or degradation. The addition of ubiquitin, known as ubiquitylation, is a reversible process making it a versatile post‐translational modification aptly suited for cell signalling. Removal of ubiquitin is catalysed by deubiquitylating enzymes, commonly referred to as DUBs. BRCC36 isopeptidase complex (BRISC) is a multi‐protein DUB complex which hydrolyses lysine‐63‐linked ubiquitin chains on Type I interferon receptors (IFNAR1/2), thus regulating interferon‐dependent signalling. Therefore, BRISC‐mediated inflammatory signalling amplification is a promising target for autoimmune disease drug development. We performed a high‐throughput screen to identify small molecules which inhibit BRISC enzymatic activity. Employing an integrative structural biology approach (cryo‐electron microscopy, native mass spectrometry, hydrogen‐deuterium exchange mass spectrometry), complemented with biochemical assays, we have uncovered new enzyme conformations, revealing a remarkable mode of action for BRISC inhibitors. Exploring these key mechanisms will expand current knowledge of inflammatory signalling pathways and establish the use of DUB inhibitors as therapeutics to combat autoimmune disease and hyperactive cytokine signalling.

Evidence that BJcuL, a C-type lectin from Bothrops jararacussu venom, influences deubiquitinase activity, resulting in the accumulation of anti-apoptotic proteins in two colorectal cancer cell lines

BJcuL is a snake venom C-type lectin (SVCTL) purified from the snake's venom Bothrops jararacussu. It has been previously demonstrated that BJcuL induces the accumulation of pro-apoptotic proteins of the extrinsic pathway, such as FADD and caspase-8, in the colorectal cancer cell line HT29, suggesting that the lectin may be able to enhance TRAIL-induced apoptosis. To test this hypothesis, we exposed two colorectal cancer cell lines, HT29 and HCT116, to increasing concentrations of BJcuL (1–20 μg/mL) in the presence or absence of TRAIL. Contrary to our expectations, however, BJcuL was unable to induce apoptosis in these cells, as shown by annexin-V/7AAD, clonogenic assays, and immunoblotting. Nevertheless, BJcuL was able to induce the accumulation of FADD and caspase-8, as well as anti-apoptotic proteins such as c-FLIP and survivin and poly-ubiquitinated proteins. Incubation with the deubiquitinase inhibitor WP1130 (10 μM) resulted in decreased BJcuL-induced survivin levels. Altogether, our results evince the effects of SVCTL on the ubiquitin-proteasome system in vitro for the first time. Compounds that can influence such system are important tools in the search for new therapeutic or diagnostic targets in cancer since they can elucidate the molecular mechanisms involved in determining cell fate as well as contributing to drug-development strategies in partnership with the pharmaceutical industry.

Aloin isoforms (A and B) selectively inhibits proteolytic and deubiquitinating activity of papain like protease (PLpro) of SARS-CoV-2 in vitro

The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC50 of 13.16 and 16.08 μM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu167, which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus.

A Synthetically Accessible Small-Molecule Inhibitor of USP5-Cav3.2 Calcium Channel Interactions with Analgesic Properties.

Cav3.2 calcium channels are important mediators of nociceptive signaling in the primary afferent pain pathway, and their expression is increased in various rodent models of chronic pain. Previous work from our laboratory has shown that this is in part mediated by an aberrant expression of deubiquitinase USP5, which associates with these channels and increases their stability. Here, we report on a novel bioactive rhodanine compound (II-1), which was identified in compound library screens. II-1 inhibits biochemical interactions between USP5 and the Cav3.2 domain III-IV linker in a dose-dependent manner, without affecting the enzymatic activity of USP5. Molecular docking analysis reveals two potential binding pockets at the USP5-Cav3.2 interface that are distinct from the binding site of the deubiquitinase inhibitor WP1130 (a.k.a. degrasyn). With an understanding of the ability of some rhodanines to produce false positives in high-throughput screening, we have conducted several orthogonal assays to confirm the validity of this hit, including in vivo experiments. Intrathecal delivery of II-1 inhibited both phases of formalin-induced nocifensive behaviors in mice, as well as abolished thermal hyperalgesia induced by the delivery of complete Freund's adjuvant (CFA) to the hind paw. The latter effects were abolished in Cav3.2 null mice, thus confirming that Cav3.2 is required for the action of II-1. II-1 also mediated a robust inhibition of mechanical allodynia induced by injury to the sciatic nerve. Altogether, our data uncover a novel class of analgesics─well suited to rapid structure-activity relationship studies─that target the Cav3.2/USP5 interface.

Role of ubiquitin-specific protease 5 in the inflammatory response of chronic periodontitis.

The systemic inflammatory response caused by chronic periodontitis is a risk factor for multiple diseases. Ubiquitin-specific protease 5 (USP5) is a kind of deubiquitinase which mainly responsible for dissociating unanchored polyubiquitin. However, the functions of USP5 in chronic periodontitis have not been reported. Chronic periodontitis patients were recruited, and their periodontal samples were collected. The levels of USP5, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in gingival crevicular fluid were evaluated by ELISA. The expression of USP5, TNF-α, IL-6, and IL-1β in human periodontal ligament stem cells (PDLSCs) was estimated by qRT-PCR assay. The activation of STAT3 signaling was examined by Western blot. USP5 was upregulated in the gingival crevicular fluid and gingival tissues of chronic periodontitis patients. USP5 expression was positively correlated with the expression of proinflammatory factors. USP5 knockdown and deubiquitinase inhibitor inhibited LPS-induced inflammatory responses in PDLSCs. Suppressing USP5 inhibited STAT3 signaling in PDLSCs. Suppression deubiquitinase USP5 inhibits the inflammatory response of chronic periodontitis by suppressing STAT3 signaling.

Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization.

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

Targeting the deubiquitinase USP7 for degradation with PROTACs.

Targeting deubiquitinating enzymes (DUBs) has emerged as a promising therapeutic approach in several human cancers and other diseases. DUB inhibitors are exciting pharmacological tools but often exhibit limited cellular potency. Here we report PROTACs based on a ubiquitin-specific protease 7 (USP7) inhibitor scaffold to degrade USP7. By investigating several linker and E3 ligand types, including novel cereblon recruiters, we discovered a highly selective USP7 degrader tool compound that induced apoptosis of USP7-dependent cancer cells. This work represents one of the first DUB degraders and unlocks a new drug target class for protein degradation.

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

Roles and Mechanisms of Deubiquitinases (DUBs) in Breast Cancer Progression and Targeted Drug Discovery.

Deubiquitinase (DUB) is an essential component in the ubiquitin—proteasome system (UPS) by removing ubiquitin chains from substrates, thus modulating the expression, activity, and localization of many proteins that contribute to tumor development and progression. DUBs have emerged as promising prognostic indicators and drug targets. DUBs have shown significant roles in regulating breast cancer growth, metastasis, resistance to current therapies, and several canonical oncogenic signaling pathways. In addition, specific DUB inhibitors have been identified and are expected to benefit breast cancer patients in the future. Here, we review current knowledge about the effects and molecular mechanisms of DUBs in breast cancer, providing novel insight into treatments of breast cancer-targeting DUBs.

Deubiquitination and Activation of the NLRP3 Inflammasome by UCHL5 in HCV-Infected Cells.

ABSTRACTChronic hepatitis C virus (HCV) infection induces liver inflammation that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Inflammation is the outcome of the action of proinflammatory cytokines and chemokines, including interleukin-1 beta (IL-1β) and tumor necrosis factor alpha. Mature IL-1β production and secretion are facilitated by active inflammasome complexes, including the NACHT-LRR pyrin domain-containing protein 3 (NLRP3) inflammasome. Our study shows that the NLRP3 inflammasome is activated in HCV-infected hepatocytes and that the activation is regulated by posttranslational modifications. NLRP3 is modified by lysine-63 ubiquitin chains in hepatocytes and is deubiquitinated during HCV infection. Inhibition of deubiquitinases (DUBs) with chemical inhibitors or blocking UCHL5 DUB expression with small interfering RNA (siRNA) abrogated NLRP3 inflammasome assembly and activation. Inhibition of inflammasome deubiquitination was correlated with a reduction in IL-1β maturation, decrease in HCV protein expression, and reduction in release of HCV from the cells. Together, this study suggests that HCV-induced activation of the NLRP3 inflammasome through posttranslational modification is crucial for the HCV life cycle and pathogenesis.IMPORTANCE HCV infection induces inflammation leading to fibrosis, cirrhosis, and cancer. The current study identifies the mechanisms leading to the activation of the NLRP3 inflammasome in hepatocytes, which is an important site of viral replication. Deubiquitination of NLRP3 by UCHL5 is required for inflammasome activation. Inhibition of deubiquitination blocks NLRP3 inflammasome activation and IL-1β maturation and also decreases HCV replication, suggesting the importance of the NLRP3 inflammasome in inflammation as well as other signaling pathways.

Deubiquitinase Inhibitor WP1130 Blocks FLT3-ITD to Induce Apoptosis in Leukemic Cells

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is the most frequent kinase mutation seen in acute myeloid leukemia (AML). The ITD mutation promotes ligand-independent constitutive activation of FLT3 kinase, resulting in altered downstream signal pathway activation to enhance cell proliferation and prevent cell death. AML with FLT3-ITD is associated with poor prognosis, which neither high-dose chemotherapy nor allogeneic hematopoietic stem cell transplantation can overcome. Several tyrosine kinase inhibitors targeting FLT3 have been developed and tested in clinical trials, but their efficacies are limited so far partly due to resistance to kinase inhibition. Therefore, development of novel treatment strategies with distinct mechanisms to block FLT3 is warranted.Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin from target proteins, resulting in changes in protein stability and signaling. WP1130 is an inhibitor for DUBs, which has been reported to inhibit BCR/ABL not through blocking the kinase activity but through promoting ubiquitination and translocation of BCR/ABL protein into perinuclear aggresomes. WP1130 also downregulates JAK2 tyrosine kinase in the same manner. However, the effect of WP1130 against FLT3-ITD has not been reported.Here we demonstrate that WP1130 exerts anti-leukemic activity against leukemic cell lines and primary AML cells harboring FLT3-ITD. WP1130 efficiently downregulated activated form of FLT3-ITD through translocation of this mutant into aggresomes and activated the mitochondria-mediated intrinsic apoptotic pathway at least partly through accumulation of intracellular reactive oxygen species (ROS).First, we examined the anti-proliferative effect of WP1130 and found that proliferation of the FLT3-ITD-harboring AML cell line MV4-11 was suppressed in a dose dependent manner with IC50 of 1.3 μM. It was notable that MV4-11 was more susceptible to WP1130 compared with K562 (IC50 = 3.3 μM) and HEL (IC50 = 3.4 μM), leukemic cell lines harboring BCR/ABL and JAK2-V617F aberrant kinases, respectively.Next, we assessed the effect of WP1130 against FLT3-ITD in MV4-11 cells. WP1130 rapidly downregulated FLT3-ITD to suppress downstream signaling pathways. The aggresomal translocation of FLT3-ITD was confirmed by confocal microscopy and western blot analysis of detergent-insoluble fraction of cellular proteins. Importantly, the downregulation was prominently observed for the activated form of FLT3-ITD autophosphorylated on Y591. Consistent with this, inhibition of FLT3 autophosphorylation by AC220 (quizartinib) resulted in attenuation of WP1130-induced FLT3-ITD downregulation.Importantly, WP1130 induced apoptosis in MV4-11 cells much more efficiently than in other leukemic cell lines that we examined, including K562 and HEL. WP1130 activated the mitochondria-mediated intrinsic apoptotic pathway as demonstrated by Bax conformation change and loss of mitochondrial membrane potential (MMP). Forced overexpression of Bcl-xL, a member of pro-survival Bcl-2 family proteins that preserve MMP, blocked WP1130-induced apoptosis. Moreover, the apoptosis was synergistically enhanced by inhibition of Bcl-xL using ABT-737 or navitoclax, further supporting the involvement of Bcl-xL in WP1130-induced apoptosis. Interestingly, the apoptosis induction was also enhanced by 17-AAG, an HSP90 inhibitor that we previously demonstrated to disrupt the chaperone association of HSP90 with FLT3-ITD resulting in enhanced ubiquitination of FLT3-ITD. FLT3-ITD down regulation and apoptosis induction by WP1130 were also confirmed using other cell lines expressing FLT3-ITD and primary cells derived from FLT3-ITD positive AML patients.Finally, since FLT3-ITD has been reported to increase intracellular ROS, we examined if ROS was involved in the anti-leukemic effect of WP1130. Treatment with WP1130 increased ROS levels in MV4-11 cells. Antioxidants N-acetyl-cysteine (NAC) and tert-butylhydroquinone (tBHQ) both attenuated WP1130-induced apoptosis, suggesting that intracellular ROS accumulation may play a role in induction of apoptosis.Further studies are in progress to elucidate the exact molecular mechanisms involved in WP1130-mediated effects and to develop novel strategies to enhance its anti-leukemic effects in synergistic manners. DisclosuresNo relevant conflicts of interest to declare.

Identification and validation of selective deubiquitinase inhibitors

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes.

VLX1570 induces apoptosis through the generation of ROS and induction of ER stress on leukemia cell lines.

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL‐60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in “heat shock transcription factor 1 (HSF1) activation”, “HSF1 dependent transactivation”, and “Regulation of HSF1 mediated heat shock response”. VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase‐1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP‐1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti‐leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.

Deubiquitinating enzyme inhibitor alleviates cyclin A1-mediated proteasome inhibitor tolerance in mixed-lineage leukemia.

Drug resistance is a significant obstacle to effective cancer treatment. Drug resistance develops from initially reversible drug‐tolerant cancer cells, which offer therapeutic opportunities to impede cancer relapse. The mechanisms of resistance to proteasome inhibitor (PI) therapy have been investigated intensively, however the ways by which drug‐tolerant cancer cells orchestrate their adaptive responses to drug challenges remain largely unknown. Here, we demonstrated that cyclin A1 suppression elicited the development of transient PI tolerance in mixed‐lineage leukemia (MLL) cells. This adaptive process involved reversible downregulation of cyclin A1, which promoted PI resistance through cell‐cycle arrest. PI‐tolerant MLL cells acquired cyclin A1 dependency, regulated directly by MLL protein. Loss of cyclin A1 function resulted in the emergence of drug tolerance, which was associated with patient relapse and reduced survival. Combination treatment with PI and deubiquitinating enzyme (DUB) inhibitors overcame this drug resistance by restoring cyclin A1 expression through chromatin crosstalk between histone H2B monoubiquitination and MLL‐mediated histone H3 lysine 4 methylation. These results reveal the importance of cyclin A1‐engaged cell‐cycle regulation in PI resistance in MLL cells, and suggest that cell‐cycle re‐entry by DUB inhibitors may represent a promising epigenetic therapeutic strategy to prevent acquired drug resistance.

Deubiquitinating enzymes as possible drug targets for schistosomiasis

Deubiquitinating enzymes (DUBs) are conserved in Schistosoma mansoni and may be linked to the 26S proteasome. Previous results from our group showed that b-AP15, an inhibitor of the 26S proteasome DUBs UCHL5 and USP14 induced structural and gene expression changes in mature S. mansoni pairs. This work suggests the use of the nonselective DUB inhibitor PR-619 to verify whether these enzymes are potential target proteins for new drug development. Our approach is based on previous studies with DUB inhibitors in mammalian cells that have shown that these enzymes are associated with apoptosis, autophagy and the transforming growth factor beta (TGF-β) signaling pathway. PR-619 inhibited oviposition in parasite pairs in vitro, leading to mitochondrial changes, autophagic body formation, and changes in expression of SmSmad2 and SmUSP9x, which are genes linked to the TGF-β pathway that are responsible for parasite oviposition and SmUCHL5 and SmRpn11 DUB maintenance. Taken together, these results indicate that DUBs may be used as targets for the development of new drugs against schistosomiasis.

Downregulation of SOX2 by inhibition of Usp9X induces apoptosis in melanoma.

Melanoma tumors driven by BRAF mutations often do not respond to BRAF/MEK/ERK pathway inhibitors currently used in treatment. One documented mechanism of resistance is upregulation of SOX2, a transcription factor that is essential for tumor growth and expansion, particularly in melanoma tumors with BRAF mutations. Targeting transcription factors pharmacologically has been elusive for drug developers, limiting treatment options. Here we show that ubiquitin-specific peptidase 9, X-linked (Usp9x), a deubiquitinase (DUB) enzyme controls SOX2 levels in melanoma. Usp9x knockdown in melanoma increased SOX2 ubiquitination, leading to its depletion, and enhanced apoptotic effects of BRAF inhibitor and MEK inhibitors. Primary metastatic melanoma samples demonstrated moderately elevated Usp9x and SOX2 protein expression compared to tumors without metastatic potential. Usp9x knockdown, as well as inhibition with DUB inhibitor, G9, blocked SOX2 expression, suppressed in vitro colony growth, and induced apoptosis of BRAF-mutant melanoma cells. Combined treatment with Usp9x and mutant BRAF inhibitors fully suppressed melanoma growth in vivo. Our data demonstrate a novel mechanism for targeting the transcription factor SOX2, leveraging Usp9x inhibition. Thus, development of DUB inhibitors may add to the limited repertoire of current melanoma treatments.