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Background CSN5, a member of Cop9 signalosome, is essential for protein neddylation. It has been supposed to serve as an oncogene in some cancers. However, the role of CSN5 has not been investigated in cervical cancer yet. Methods Data from TCGA cohorts and GEO dataset was analyzed to examine the expression profile of CSN5 and clinical relevance in cervical cancers. The role of CSN5 on cervical cancer cell proliferation was investigated in cervical cancer cell lines, Siha and Hela, through CSN5 knockdown via CRISPR–CAS9. Western blot was used to detect the effect of CSN5 knockdown and overexpression. The biological behaviors were analyzed by CCK8, clone formation assay, 3-D spheroid generation assay and cell cycle assay. Besides, the role CSN5 knockdown in vivo was evaluated by xenograft tumor model. MLN4924 was given in Siha and Hela with CSN5 overexpression. Results We found that downregulation of CSN5 in Siha and Hela cells inhibited cell proliferation in vitro and in vivo, and the inhibitory effects were largely rescued by CSN5 overexpression. Moreover, deletion of CSN5 caused cell cycle arrest rather than inducing apoptosis. Importantly, CSN5 overexpression confers resistance to the anti-cancer effects of MLN4924 (pevonedistat) in cervical cancer cells. Conclusions Our findings demonstrated that CSN5 functions as an oncogene in cervical cancers and may serve as a potential indicator for predicting the effects of MLN4924 treatment in the future.

Introduction Protein neddylation, one of the most important posttranslational modifications that tagging neuronal precursor cell‐expressed developmentally downregulated protein 8 onto substrate proteins, plays fundamental roles in the process of many cellular functions. A number of studies have demonstrated the critical roles of neddylation modification in multiple pathophysiological processes, but its regulatory role in the immune system has only been finitely unveiled. Methods In this review, the latest advances in the field of neddylation modification in regulating the immune responses are succinctly discussed. Results Neddylation modification acts as a crucial modulator of innate immune cells (neutrophils, macrophages, and dendritic cells) and lymphocytes. Dysregulation of neddylation alters characteristics and functions of those cells due to abnormal degradation of key signaling molecules involved in immunoregulation. Furthermore, the ectopic immune responses caused by the abnormal neddylation play pivotal roles in a variety of immune‐related diseases, such as infection, inflammation, and cancer. Conclusions The pivotal roles of neddylation pathway in immunoregulation are attracted more and more attention, which may provide new insights into the pathogenesis of a variety of immune‐related diseases and help to indicate new therapeutic targets and potential treatment strategies.

Background The neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved. Methods The expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 were estimated by bioinformatics analysis and western blot in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown. Findings The mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo . The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G 2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells. Interpretation These findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. Fund Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). The funders had no role in study design, data collection, data analysis, interpretation, writing of the report.

Protein neddylation is catalyzed by a three-enzyme cascade, namely an E1 NEDD8-activating enzyme (NAE), one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases. The physiological substrates of neddylation are the family members of cullin, the scaffold component of cullin RING ligases (CRLs). Currently, a potent E1 inhibitor, MLN4924, also known as pevonedistat, is in several clinical trials for anti-cancer therapy. Here we report the discovery, through virtual screening and structural modifications, of a small molecule compound HA-1141 that directly binds to NAE in both in vitro and in vivo assays and effectively inhibits neddylation of cullins 1–5. Surprisingly, unlike MLN4924, HA-1141 also triggers non-canonical endoplasmic reticulum (ER) stress and PKR-mediated terminal integrated stress response (ISR) to activate ATF4 at an early stage, and to inhibit protein synthesis and mTORC1 activity at a later stage, eventually leading to autophagy induction. Biologically, HA-1141 suppresses growth and survival of cultured lung cancer cells and tumor growth in in vivo xenograft lung cancer models at a well-tolerated dose. Taken together, our study has identified a small molecule compound with the dual activities of blocking neddylation and triggering ER stress, leading to growth suppression of cancer cells., Graphical abstract We identified HA-1141 through virtual screen and SAR-based modification. HA-1141 has dual activities in blocking cullin neddylation and in triggering severe ER stress to a) induce autophagy and b) suppress growth of cancer cells both in vitro and in vivo.Image 1

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin–proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.

Qing Yu,1– 3 Yi Sun3 1Department of Head and Neck Surgery, The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Science, Hangzhou, Zhejiang, People’s Republic of China; 2Key Laboratory of Head & Neck Cancer Translational Research of Zhejiang Province, Hangzhou, Zhejiang, People’s Republic of China; 3Cancer Institute of the Second Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of ChinaCorrespondence: Yi SunCancer Institute of the Second Affiliated Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, 268 Kaixuan Road, Hagnzhou, Zheijiang, People’s Republic of ChinaTel +86 571 86971812Fax +86 571 88981576Email yisun@zju.edu.cnAbstract: Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin neddylation, a process catalysed by three neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.Keywords: anti-cancer drug, cullin-RING E3 ligases, natural product, high-throughput screen, neddylation, small-molecule inhibitors

Neddylation, a posttranslational protein modification, refers to the specific conjugation of NEDD8 to substrates, which is of great significance to various biological processes. Besides members of the cullin protein family, other key proteins can act as a substrate for neddylation modification, which remarkably influences neurodevelopment and neurodegenerative diseases. Normal levels of protein neddylation contribute to nerve growth, synapse strength, neurotransmission, and synaptic plasticity, whereas overactivation of protein neddylation pathways lead to apoptosis, autophagy of neurons, and tumorigenesis. Furthermore, impaired neddylation causes neurodegenerative diseases. These facts suggest that neddylation may be a target for treatment of these diseases. This review focuses on the current understanding of neddylation function in neurodevelopment as well as neurodegenerative diseases. Meanwhile, the recent view that different level of neddylation pathway may contribute to the opposing disease progression, such as neoplasms and Alzheimer's disease, is discussed. The review also discusses neddylation inhibitors, which are currently being tested in clinical trials. However, potential drawbacks of these drugs are noted, which may benefit the development of new pharmaceutical strategies in the treatment of nervous system diseases.

Estrogen-related receptor beta (ERRβ) is downregulated in breast cancer cells and its overexpression in breast cancer patients is positively correlated with an improved prognosis and prolonged relapse-free survival. Here, we unravelled a molecular mechanism for ERRβ downregulation in breast cancer. We found that ERRβ is a key substrate of the SCF complex and that NEDDylation can activate the Cullin subunits of the SCF complex to target ERRβ for degradation in breast cancer. Consistently, using in vitro and in vivo models, we demonstrated that MLN4924, a specific small molecule inhibitor of NEDDylation, can restore ERRβ expression and culminate in a reduction in cell proliferation and migration of breast cancer cells. We also showed that increased ERRβ expression promotes the upregulation of its target genes, including the tumour suppressors p21Cip1/Waf1 and E-cadherin, involved in cell proliferation and migration arrest at the gene promoter level. Interestingly, this tumour suppressive role of ERRβ does not depend on the expression of ERα in breast cancer. Moreover, our data revealed that the ERRβ recruits the transcription co-activator p300 to its targeted gene promoters to upregulate their expression. Collectively, our work revealed that restoration of ERRβ expression using the NEDDylation inhibitor MLN4924 can be a novel and effective strategy for breast cancer treatment.

Neddylation, a post-translational modification that conjugates an ubiquitin-like protein NEDD8 to substrate proteins, is an important biochemical process that regulates protein function. The best-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which, as the largest family of E3 ubiquitin ligases, control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. Recently, increasing pieces of experimental evidence strongly indicate that the process of protein neddylation modification is elevated in multiple human cancers, providing sound rationale for its targeting as an attractive anticancer therapeutic strategy. Indeed, neddylation inactivation by MLN4924 (also known as pevonedistat), a small molecule inhibitor of E1 NEDD8-activating enzyme currently in phase I/II clinical trials, exerts significant anticancer effects by inducing cell cycle arrest, apoptosis, senescence and autophagy in a cell-type and context dependent manner. Here, we summarize the latest progresses in the field with a major focus on preclinical studies in validation of neddylation modification as a promising anticancer target.

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G2–M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34+ cells and LSCs in CML-bearing mice via accumulation of p27kip1 in the nucleus. Notably, p27kip1 silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML. Significance: These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. Cancer Res; 78(6); 1522–36. ©2018 AACR.

Inhibition of protein neddylation pathway has emerged an attractive anticancer strategy in preclinical studies by using Nedd8-activating enzyme (NAE) inhibitor MLN4924 (Pevonedistat). Previous studies have reported the antitumor activity of MLN4924 mediated by its efficacy on apoptosis, autophagy and senescence. However, whether MLN4924 has any effect on renal carcinoma cells (RCC) remains unexplored. Here we reported that MLN4924 specifically inhibited protein neddylation pathway, leading to statistically significantly suppress the proliferation, survival and migration of RCC cells by inducing G2 cell-cycle arrest, followed by apoptosis in a MLN4924 dose-dependent manner. Further mechanistic study revealed that MLN4924-induced apoptosis was mediated by substantial up-regulation of pro-apoptotic NOXA. These findings highlighted the anticancer effects of the neddylation inhibitors (e.g. MLN4924) for the treatment of RCC.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA with a 5-year survival rate less than 3% to 5%. Gemcitabine remains as a standard care for PDAC patients. Although protein neddylation is abnormally activated in many human cancers, whether neddylation dysregulation is involved in PDAC and whether targeting neddylation would sensitize pancreatic cancer cells to gemcitabine remain elusive. Here we report that high expression of neddylation components, NEDD8 and NAE1, are associated with poor survival of PDAC patients. Blockage of neddylation by MLN4924, a small molecule inhibitor targeting this modification, significantly sensitizes pancreatic cancer cells to gemcitabine, as evidenced by reduced growth both in monolayer culture and soft agar, reduced clonogenic survival, decreased invasion capacity, increased apoptosis, G2/M arrest, and senescence. Importantly, combinational treatment of MLN4924-gemcitabine near completely suppressed in vivo growth of pancreatic cancer cells. Mechanistically, accumulation of NOXA, a pro-apoptotic protein and ERBIN, a RAS signal inhibitor, appears to play, at least in part, a causal role in MLN4924 chemo-sensitization. Our study demonstrates that neddylation modification is a valid target for PDAC, and provides the proof-of-concept evidence for future clinical trial of MLN4924-gemcitabine combination for the treatment of pancreatic cancer patients.

Purpose: Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a proapoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage. Experimental Design: Methods of immunohistochemistry and immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by a pulldown assay. Polyubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays. Results: UBE2F is overexpressed in non–small cell lung cancer (NSCLC) and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with a low level of NOXA and poor patient survival. Conclusions: By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target. Clin Cancer Res; 23(4); 1104–16. ©2016 AACR.

The maintenance of muscle function is extremely important for whole body health and exercise is essential to this process. The ubiquitin-proteasome system (UPS) is required for muscle adaptation following exercise but little is known about acute posttranslational regulation and proteome remodeling during and after high-intensity exercise. Here, we used quantitative proteomics to study ubiquitin signaling dynamics in human skeletal muscle biopsies from healthy males before, during, and after a single bout of high-intensity exercise. Exercise resulted in a marked depletion of protein ubiquitylation in the vastus lateralis muscle consistent with proteasome activation. This was also associated with acute posttranslational modification of protein abundance. Integration of these data with phosphoproteomics identified co-regulated proximal modifications suggesting a cross talk between phosphorylation and ubiquitylation. We also identified additional protein modification cross talk and showed acute activation of protein NEDDylation. In vitro experiments revealed that cAMP-dependent activation of the proteasome requires NEDD8, an ubiquitin-like protein involved in protein NEDDylation, to maintain cellular protein ubiquitylation levels. Our data reveal the complexity of ubiquitin signaling and proteome remodeling in muscle during and after high-intensity exercise. We propose a model whereby exercise and the resulting cAMP signaling activate both the proteasome and ubiquitylation via NEDDylation to rapidly remove potentially damaged proteins.

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.

Protein neddylation is one type of posttranslational modifications that regulates the activity of the substrate proteins. Neddylation modification is catalyzed by NEDD8-activating enzyme (NAE, E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3) to attach NEDD8, an ubiquitin-like molecule, to a lysine residue of a substrate protein. The best known neddylation substrates are cullin family members, which are scaffold components of cullin-RING ligases (CRLs), and cullin neddylation is required for activation of CRLs. In mammalian cells, there are one E1, two E2s (UBC12/UBE2M and UBE2F), and over a dozen E3s. MLN4924, the first-in-class small-molecule inhibitor of NAE, blocks the entire neddylation modification to inactivate activity of all CRLs. MLN4924 is currently in the Phase I/II clinical trials for anticancer application.In the last few years, targeting protein-protein interactions of the neddylation complexes has been pursued as a potential strategy to selectively inhibit the activity of individual CRL. Analysis of the co-crystal structures of DCN1, a co-E3 for neddylation, and its binding partners UBC12 (a neddylation E2) suggested that it may be amenable for the design of potent, small-molecule inhibitors. In this chapter, we will review the discovery of small-molecule inhibitors that block the interactions of DCN1 with UBC12 (hereafter called DCN1 inhibitors) from a number of laboratories, including ours, leading to selective inactivation of CRL-1 and/or CRL-3. We will also discuss potential therapeutic applications of these small-molecule inhibitors.

Protein neddylation, a process of conjugating neural precursor cell expressed, developmentally downregulated 8 (NEDD8) to substrates, plays a tumor-promoting role in lung carcinogenesis. Our previous study showed MLN4924, an inhibitor of NEDD8 activating enzyme (E1), significantly inhibits the growth of multiple cancer cells. However, resistance can develop to MLN4924 by mutation. Therefore, it is important to further understand how NEDD8 acts in lung cancer. In the present study, we demonstrated NEDD8 is overactivated in lung cancers and confers a worse patient overall survival. Furthermore, we report that in lung adenocarcinoma cells, NEDD8 depletion significantly suppressed lung cancer cell growth and progression both in vitro and in vivo. Mechanistic studies revealed that NEDD8 depletion induced the accumulation of a panel of tumor-suppressive cullin-RING ubiquitin ligase substrates (e.g., p21, p27, and Wee1) via blocking their degradation, triggering cell cycle arrest at G2 phase, thus inducing apoptosis or senescence in a cell-line-dependent manner. The present study demonstrates the role of NEDD8 in regulating the malignant phenotypes of lung cancer cells and further validates NEDD8 as a potential therapeutic target in lung cancer.

Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin–RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein–protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors., Graphical abstract The past decade has seen significant progress in the field of cullin neddylation, from the preclinical target validation, to drug screening, and to the clinical trials of small-molecule inhibitors. Future effort aims to discover more selective and potent inhibitors for anticancer therapeutics by targeting E2s, E3s or various PPI interfaces in the cullin neddylation cascade.Image 1

Mammalian oocyte meiosis is a special form of cell division that provides haploid gametes for fertilization. Unlike in mitosis, post-translational modifications (PTMs) are more crucial during meiosis because of the absence of de novo mRNA transcription. As a classic PTM, protein neddylation is a biological process that mediates protein degradation by modifying cullin proteins and activating the Cullin-Ring E3 ligases. This process plays important roles in various biological processes such as autophagy and tumorigenesis. However, the function of neddylation in germ cells is unknown. In this study, we observed that the inhibition of neddylation by its specific inhibitor MLN4924 significantly arrests mouse oocyte at the stage of metaphase during meiosis. The arrested oocytes display impaired spindles with over-activation of spindle assembly checkpoint (SAC). Accordingly, we identified early mitosis inhibitor 1 (Emi1), a key inhibitor of anaphase-promoting complex/cyclosome (APC/C(Fzr1)), as a substrate of neddylation-mediated protein degradation. Thus, our study uncovered an unknown role of neddylation in female germ cells and suggests that proper neddylation is essential for oocyte maturation.

Sorafenib remains the standard care for patients with hepatocellular carcinoma (HCC) even though it has low antitumor efficacy. Protein neddylation is abnormally activated in many types of human cancer. However, whether dysregulation of neddylation is involved in HCC progression and whether targeting neddylation sensitizes HCC cells to sorafenib need to be ascertained. In the present study, it was demonstrated that high expression of neddylation components, neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and NEDD8‑activating enzyme 1 (NAE1), were associated with poor survival of patients with HCC. Inhibition of neddylation by MLN4924, a small‑molecule inhibitor of NAE1, significantly inhibited HCC growth, reduced clonogenic survival, increased apoptosis, and decreased migration capacity. Sorafenib alone exhibited minimal anticancer efficacy. However, a combination of sorafenib with MLN4924 at a low concentration significantly enhanced the inhibition of cell proliferation and migration as well as the induction of apoptosis induced by sorafenib. In vivo HCC xenograft mouse models also showed that MLN4924 increased the antitumor efficacy of sorafenib. Mechanistically, MLN4924 enhanced the antitumor activity of sorafenib in HCC cells via upregulation of cullin‑RING E3 ubiquitin ligase (CRL)/Skp1‑Cullin1‑F box (SCF) E3 ubiquitin ligase substrates p21, p27, Deptor and IκBɑ. Taken together, these findings suggest that combination therapy of MLN4924 with sorafenib appears to present an additive effect with a maximal in the treatment of HCC.

Background Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. Methods An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. Results FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1–452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn’t attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. Conclusion These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer. Electronic supplementary material The online version of this article (10.1186/s12964-019-0338-2) contains supplementary material, which is available to authorized users.

Protein posttranslational modifications such as neddylation play crucial roles in regulating protein function. Only a few neddylated substrates have been validated to date, and the role of neddylation remains poorly understood. Here, using Trypanosoma brucei as the model organism, we investigated the function and substrates of TbNedd8. TbNedd8 is distributed throughout the cytosol but enriched in the nucleus and the flagellum. Depletion of TbNedd8 by RNAi abolished global protein ubiquitination, caused DNA re-replication in postmitotic cells, impaired spindle assembly, and compromised the flagellum attachment zone filament, leading to flagellum detachment. Through affinity purification and mass spectrometry, we identified 70 TbNedd8-conjugated and -associated proteins, including known Nedd8-conjugated and -associated proteins, putative TbNedd8 conjugation system enzymes, proteins of diverse biological functions, and proteins of unknown function. Finally, we validated six Cullins as bona fide TbNedd8 substrates and identified the TbNedd8 conjugation site in three Cullins. This work lays the foundation for understanding the roles of protein neddylation in this early divergent parasitic protozoan.

Protein neddylation is essential for the viability of most organisms and is widely involved in the regulation of immunity, DNA damage and repair, cell signaling and cell cycle. Unlike RING-type neddylation ligases, HECT-type neddylation ligase remains less defined. Here, we show that Itch is a novel HECT-type neddylation E3 ligase and we identify JunB as a substrate of Nedd8 modification by Itch. JunB neddylation attenuates its transcriptional activity. In addition, JunB neddylation mediated by Itch promotes its ubiquitination-dependent degradation. Therefore, these findings define a new HECT-type neddylation ligase and its neddylation substrate.

Purpose: Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor–mediated extrinsic apoptosis during treatment remained to be determined. Experimental Design: The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo. The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies. Results: Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)–mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival. Conclusions: Our findings highlight a critical role of ATF4–CHOP–DR5 axis-mediated extrinsic apoptosis in neddylation-targeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation. Clin Cancer Res; 22(16); 4145–57. ©2016 AACR.

Backgrounds: The neddylation pathway is overactivated in human cancers. Inhibition of neddylation pathway has emerged as an attractive anticancer strategy. The mechanisms underlying neddylation overactivation in cancer remain elusive. MLN4924/Pevonedistat, a first-in-class NEDD8-activating enzyme (NAE, E1) inhibitor, exerts significant anti-tumor effects, but its mutagenic resistance remains unresolved. Methods: The expression of NEDD8-conjugating enzyme UBC12/UBE2M (E2) and NEDD8 was estimated by bioinformatics analysis and western blotting in human lung cancer cell lines. The malignant phenotypes of lung cancer cells were evaluated both in vitro and in vivo upon UBC12 knockdown. Cell-cycle arrest was evaluated by quantitative proteomic analysis and propidium iodide stain and fluorescence - activated cell sorting (FACS). The growth of MLN4924 - resistant H1299 cells was also evaluated upon UBC12 knockdown. Results: The mRNA level of UBC12 in lung cancer tissues was much higher than that in normal lung tissues, increased with disease deterioration, and positively correlated with NEDD8 expression. Moreover, the overexpression of UBC12 significantly enhanced protein neddylation modification whereas the downregulation of UBC12 reduced neddylation modification of target proteins. Functionally, neddylation inactivation by UBC12 knockdown suppressed the malignant phenotypes of lung cancer cells both in vitro and in vivo. The quantitative proteomic analysis and cell cycle profiling showed that UBC12 knockdown disturbed cell cycle progression by triggering G2 phase cell-cycle arrest. Further mechanistical studies revealed that UBC12 knockdown inhibited Cullin neddylation, led to the inactivation of CRL E3 ligases and induced the accumulation of tumor-suppressive CRL substrates (p21, p27 and Wee1) to induce cell cycle arrest and suppress the malignant phenotypes of lung cancer cells. Finally, UBC12 knockdown effectively inhibited the growth of MLN4924-resistant lung cancer cells. Conclusions: These findings highlight a crucial role of UBC12 in fine-tuned regulation of neddylation activation status and validate UBC12 as an attractive alternative anticancer target against neddylation pathway. Funding Statement: This work was supported by the Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81401893, 81625018, 81820108022, 81772470, 81572340 and 81602072), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The authors state: "Not applicable."

Background: Protein neddylation is a posttranslational modification of conjugating the neuronal precursor cell-expressed developmentally down-regulated protein 8 (Nedd8) to substrates. The best characterized substrates of neddylation are cullin family members, the essential components of cullin-RING E3 ubiquitin ligases (CRLs). Previous studies have shown that the neddylation pathway is overactivated in various types of human cancers and correlates with the disease progression, whereas pharmacologically targeting this pathway has emerged as an attractive therapeutic strategy. Methods: To screen the potential neddylation inhibitors for anticancer therapy, the inhibitory effects on neddylation modification of 1331 approved drugs were investigated by an improved enzyme-based assay. Neddylation inhibiting mechanism of hit compound was illuminated by exploring the levels of Nedd8 modification on cullins and the subsequent changes of CRLs substrates in enzyme- and cellular-based assays, along with the molecular docking of hit compound with the target neddylation enzyme. A series of phenotypic experiments, including assays on in vitro cell proliferation, cell cycle arrest and in vivo tumor growth, were applied to investigate the anticancer activities and mechanisms of hit compound. Findings: An antihypertensive agent candesartan cilexetic (CDC) was identified as a new neddylation inhibitor from 1331 approved drugs based on the strategy of drug repurposing. CDC inhibited neddylation pathway by ATP-competitively suppressing Nedd8-activating enzyme (NAE, E1), which was superior to two representative non-covalent NAE inhibitors, M22 and mitoxantrone. Following with the findings such as apoptotic induction and tumor growth suppression, CDC represents a potential antitumor lead compound with promising neddylation inhibitory activity. Interpretation: In this study, we identified CDC as a hit compound that blocks the neddylation modification of cullins. Our findings highlighted the potential of developing CDC as a novel neddylation inhibitor. Funding Statement: This work was supported by the Chinese Minister of Science and Technology grant (2016YFA0501800), National Natural Science Foundation of China (Grant Nos. 81625018, 81820108022), Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-10-E00056), Program of Shanghai Academic/Technology Research Leader (18XD1403800), National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development (2017ZX09304001). Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: All experimental protocols were approved by the Animal Experimental Ethics Committee of Longhua Hospital of Shanghai University of Traditional Chinese Medicine.

Protein neddylation, a newly characterized posttranslational modification that adds the ubiquitin-like molecule NEDD8 to substrates, modulates important biological processes, whereas dysfunction of neddylation may cause several serious diseases, such as cancer. Inhibition of neddylation pathway has emerged as a promising anticancer strategy, as evidenced by development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924. Due to its potent anti-cancer efficacy and well-tolerated toxicity, MLN4924 has been evaluated in multiple Phase I clinical trials for solid tumors and hematologic malignancies. Recently, accumulating evidences indicate that neddylation pathway also plays a pivotal role in the regulation of multiple processes of tumor microenvironment (TME), such as tumor angiogenesis and the function of immune cells. In this review, we briefly summarize the latest progresses in this field and highlight neddylation pathway as an attractive therapeutic target against human cancer.

Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma.

Acquired resistance to first-line chemotherapeutics, including paclitaxel (PTX), is a primary factor contributing to chemotherapy failure in non-small cell lung cancer (NSCLC) patients. Previous studies have identified that targeting NEDD8-activating enzyme (NAE) with MLN4924 effectively overcomes platinum resistance in preclinical models of ovarian cancer. However, the underlying mechanisms are yet to be fully elucidated. The present study demonstrates that the inhibition of the neddylation pathway with MLN4924 an NAE inhibitor inhibited protein neddylation, inactivated cullin-RING E3 ligase and exhibited a potent antiproliferative effect on PTX-resistant A549 and H460 cells (A549/PTX and H460/PTX). The application of MLN4924 promotes apoptosis and DNA damage in A549/PTX and H460/PTX cells. Additionally, MLN4924 abrogated the 3-dimensional growth potential of these cells and inhibited the formation of the A549/PTX and H460/PTX spheroids. Notably, combining MLN4924 with PTX did not exhibit synergy in PTX-resistant NSCLC cells. Taken together, the results of the current study suggest that MLN4924 may be utilized as an effective strategy for the treatment of PTX-resistant NSCLC.

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm.

NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin-RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here, we re-evaluate studies of non-cullin targets of NEDD8 in light of the current understanding of the neddylation pathway, and suggest criteria for identifying genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights.

Significance: Protein neddylation is catalyzed by an E1 NEDD8-activating enzyme (NAE), an E2 NEDD8-conjugating enzyme, and an E3 NEDD8 ligase. Known physiological substrates of neddylation are cullin family members. Cullin neddylation leads to activation of cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases responsible for ubiquitylation and degradation of many key signaling/regulatory proteins. Thus, through modulating CRLs, neddylation regulates many biological processes, including cell cycle progression, signal transduction, and tumorigenesis. Given that NEDD8 is overexpressed and CRLs are abnormally activated in many human cancers, targeting protein neddylation, in general, and cullin neddylation, in particular, appears to be an attractive anticancer approach. Recent Advances: MLN4924, a small molecule inhibitor of NAE, was discovered that inactivates CRLs and causes accumulation of CRL substrates to suppress tumor cell growth both in vitro and in vivo. Promising preclinical results advanced MLN4924 to several clinical trials for anticancer therapy. Critical Issues: In preclinical settings, MLN4924 effectively suppresses tumor cell growth by inducing apoptosis, senescence, and autophagy, and causes sensitization to chemoradiation therapies in a cellular context-dependent manner. Signal molecules that determine the cell fate upon MLN4924 treatment, however, remain elusive. Cancer cells develop MLN4924 resistance by selecting target mutations. Future Directions: In the clinical side, several Phase 1b trials are under way to determine the safety and efficacy of MLN4924, acting alone or in combination with conventional chemotherapy, against human solid tumors. In the preclinical side, the efforts are being made to develop additional neddylation inhibitors by targeting NEDD8 E2s and E3s. Antioxid. Redox Signal. 21, 2383–2400.

Purpose: Ovarian cancer has the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure and novel therapeutic strategies are urgently needed. MLN4924 is a NEDDylation inhibitor currently under investigation in multiple phase I studies. We investigated its anticancer activity in cisplatin-sensitive and -resistant ovarian cancer models. Experimental Design: Cellular sensitivity to MLN4924/cisplatin was determined by measuring viability, clonogenic survival, and apoptosis. The effects of drug treatment on global protein expression, DNA damage, and reactive oxygen species generation were determined. RNA interference established natural born killer/bcl-2–interacting killer (NBK/BIK) as a regulator of therapeutic sensitivity. The in vivo effects of MLN4924/cisplatin on tumor burden and key pharmacodynamics were assessed in cisplatin-sensitive and -resistant xenograft models. Results: MLN4924 possessed significant activity against both cisplatin-sensitive and -resistant ovarian cancer cells and provoked the stabilization of key NEDD8 substrates and regulators of cellular redox status. Notably, MLN4924 significantly augmented the activity of cisplatin against cisplatin-resistant cells, suggesting that aberrant NEDDylation may contribute to drug resistance. MLN4924 and cisplatin cooperated to induce DNA damage, oxidative stress, and increased expression of the BH3-only protein NBK/BIK. Targeted NBK/BIK knockdown diminished the proapoptotic effects of the MLN4924/cisplatin combination. Administration of MLN4924 to mice bearing ovarian tumor xenografts significantly increased the efficacy of cisplatin against both cisplatin-sensitive and -resistant tumors. Conclusions: Our collective data provide a rationale for the clinical investigation of NEDD8-activating enzyme (NAE) inhibition as a novel strategy to augment cisplatin efficacy in patients with ovarian cancer and other malignancies. Clin Cancer Res; 19(13); 3577–90. ©2013 AACR.

The ubiquitin-like molecule NEDD8 modifies cullin-RING ubiquitin E3 ligases. NEDD8 has been shown to have a few additional substrates, but the extent to which this modification targets non-cullins and the functional significance of such modifications remain unclear. Here, we demonstrate that the cell-cycle-regulating transcription factor E2F-1 is a substrate for NEDD8 post-translational modification. NEDDylation results in decreased E2F-1 stability, lower transcriptional activity and slower cell growth. The lysine residues in E2F-1 targeted for NEDDylation can also be methylated, pointing to a possible interplay between these modifications. These results identify a new mode of E2F-1 regulation and highlight the emerging role of NEDD8 in regulating transcription factor stability and function.

E3 ubiquitin ligases are pivotal effectors of the ubiquitin-proteasome system as they determine the substrate specificity and transfer ubiquitin to the substrate. HECT-type ubiquitin ligase Smad ubiquitination regulatory factor 2 (Smurf2) has been demonstrated functions as a tumor suppressor. However, the mechanisms underlying regulation of Smurf2 is still unclear. Here we show that ubiquitin-like protein Nedd8 targets the HECT-type ubiquitin ligase Smurf2 for neddylation, and promotes Smurf2 degradation. Neddylation of Smurf1 activates its ubiquitin ligase activity and Smurf2 exerts Nedd8 ligase activity. This study provided new clues of Smurf2 activation regulation.

The small ubiquitin-like molecule NEDD8 has been identified as an essential regulator of the activity of the cullin-RING E3 ubiquitin ligases (CRLs), which control the turnover of multiple proteins with fundamental roles in cancer biology. The aberrant function of the NEDD8 cascade within the context of malignancy makes it an attractive target for the development of novel anticancer agents. MLN4924 is a first-in-class inhibitor of the proximal regulator of the NEDD8 system (NEDD8-activating enzyme, NAE) that has entered Phase-I trials for cancer therapy and has established that significant therapeutic benefit can be achieved by antagonizing NEDD8-mediated protein degradation.This review provides a detailed overview of the NEDD8 system and discusses the mechanisms of action of MLN4924, a novel small molecule NAE inhibitor. Key findings from preclinical investigations of MLN4924 in a broad range of cancer models and preliminary findings from ongoing Phase-I clinical trials with MLN4924 are also discussed.Targeting protein NEDDylation represents an exciting new anticancer strategy with demonstrable therapeutic benefit. Ongoing and future studies focused on dissecting the functional status/regulation of the NEDD8 system in individual tumor types will facilitate the design of novel approaches that yield optimal therapeutic benefit.

Posttranslational neddylation of cullins in the Cullin-Ring E3 ligase (CRL) complexes is needed for proteolytic degradation of CRL substrates, whose accumulation induces cell-cycle arrest, apoptosis, and senescence. The Nedd8-activating enzyme (NAE) is critical for neddylation of CRL complexes and their growth-promoting function. Recently, the anticancer small molecule MLN4924 currently in phase I trials was determined to be an inhibitor of NAE that blocks cullin neddylation and inactivates CRL, triggering an accumulation of CRL substrates that trigger cell-cycle arrest, apoptosis, and senescence in cancer cells. Here, we report that MLN4924 also triggers autophagy in response to CRL inactivation and that this effect is important for the ability of MLN4924 to suppress the outgrowth of liver cancer cells in vitro and in vivo. MLN4924-induced autophagy was attributed partially to inhibition of mTOR activity, due to accumulation of the mTOR inhibitory protein Deptor, as well as to induction of reactive oxygen species stress. Inhibiting autophagy enhanced MLN4924-induced apoptosis, suggesting that autophagy is a survival signal triggered in response to CRL inactivation. In a xenograft model of human liver cancer, MLN4924 was well-tolerated and displayed a significant antitumor effect characterized by CRL inactivation and induction of autophagy and apoptosis in liver cancer cells. Together, our findings support the clinical investigation of MLN4924 for liver cancer treatment and provide a preclinical proof-of-concept for combination therapy with an autophagy inhibitor to enhance therapeutic efficacy. Cancer Res; 72(13); 3360–71. ©2012 AACR.

Radiotherapy is used in locally advanced pancreatic cancers in which it can improve survival in combination with gemcitabine. However, prognosis is still poor in this setting in which more effective therapies remain needed. MLN4924 is an investigational small molecule currently in phase I clinical trials. MLN4924 inhibits NAE (NEDD8 Activating Enzyme), a pivotal regulator of the E3 ubiquitin ligase SCF (SKP1, Cullins, and F-box protein), that has been implicated recently in DNA damage and repair. In this study, we provide evidence that MLN4924 can be used as an effective radiosensitizer in pancreatic cancer. Specifically, MLN4924 (20–100 nmol/L) effectively inhibited cullin neddylation and sensitized pancreatic cancer cells to ionizing radiation in vitro with a sensitivity enhancement ratio of approximately 1.5. Mechanistically, MLN4924 treatment stimulated an accumulation of several SCF substrates, including CDT1, WEE1, and NOXA, in parallel with an enhancement of radiation-induced DNA damage, aneuploidy, G2/M phase cell-cycle arrest, and apoptosis. RNAi-mediated knockdown of CDT1 and WEE1 partially abrogated MLN4924-induced aneuploidy, G2/M arrest, and radiosensitization, indicating a causal effect. Furthermore, MLN4924 was an effective radiosensitizer in a mouse xenograft model of human pancreatic cancer. Our findings offer proof-of-concept for use of MLN4924 as a novel class of radiosensitizer for the treatment of pancreatic cancer. Cancer Res; 72(1); 282–93. ©2011 AACR.

Cullin-RING ubiquitin ligase (CRL), with its founding member of SKP1-Cullins-F-box proteins (SCF) E3 ubiquitin ligase, is the largest family of E3 ligases, which requires cullin neddylation for its activation. Recently, an inhibitor of NEDD8 activating enzyme (NAE), MLN4924, was reported to block cullin neddylation and inactivate CRL/SCF E3, resulting in apoptosis induction and tumor suppression both in vitro and in vivo. We report here that apoptosis is not the sole mechanism by which MLN4924 suppresses tumor cell growth because apoptosis is moderately induced by the drug in some cancer cell lines and drug-induced growth suppression is only partially blocked by a pan-caspase inhibitor, z-VAD. MLN4924 treatment induces the characteristics of senescence phenotypes as evidenced by enlarged and flattened cellular morphology and positive staining of senescence-associated β-Gal. MLN4924-induced senescence is associated with cellular response to DNA damage, triggered by accumulation of DNA-licensing proteins CDT1 and ORC1, as a result of inactivation of CRL/SCF E3s. The senescence occurs in the manner independent of pRB/p16 and p53, but dependent on p21, a known substrate of CRL/SCF E3s and a mediator of senescence, which accumulates on CRL/SCF inactivation by MLN4924. Furthermore, MLN4924-induced senescence is irreversible and coupled with persistent accumulation of p21 and sustained activation of DNA damage response. Our study reveals a novel mechanism of MLN4924 action and showed that MLN4924 could be further developed as an effective anticancer agent by inducing apoptosis and irreversible senescence.

There are 17 known ubiquitin-like proteins (UBLs) from nine phylogenetically distinct classes (NEDD8, SUMO, ISG15, FUB1, FAT10, Atg8, Atg12, Urm1, and UFM1) that have been identified to conjugate to substrates in a manner analogous to ubiquitin. NEDD8 is one of the most studied UBLs and shares the highest amino acid similarity to ubiquitin. Here, we review the current knowledge of the NEDD8 conjugation cascade derived from functional studies in genetic model organisms, structural insights from crystallographic studies, biochemical studies identifying a growing list of NEDD8 substrates with oncogenic implications, and attempts to pharmacologically target the NEDD8 pathway in cancer.

The NEDD8 (neural precursor cell-expressed developmentally downregulated 8) conjugation pathway regulates the post-translational modification of oncogenic proteins. This pathway has important potential for cancer therapeutics. Several proteins vital in cancer biology are regulated by protein neddylation. These observations led to the development of a small molecule inhibitor that disrupts protein neddylation and leads to cancer cell death and important activity in early phase clinical trials.This review provides an extensive coverage of cellular protein homeostasis with particular emphasis on the NEDD8 conjugation pathway. Insights into a new investigational drug that specifically disrupts the NEDD8 pathway are discussed. The clinical data for this agent are also updated.Neddylation controls key cellular pathways found to be dysregulated in many cancers. Protein neddylation is a relatively under-explored pathway for pharmacologic inhibition in cancer. Selective disruption of this pathway has demonstrated clinical activity in patients with myeloid neoplasms and is worth exploring further in combination with other anti-leukemia agents.

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.

Several studies have shown that ribosomal proteins (RPs) are important mediators of p53 activation in response to nucleolar disruption; however, the pathways that control this signalling function of RPs are currently unknown. We have recently shown that RPs are targets for the ubiquitin-like molecule NEDD8, and that NEDDylation protects RPs from destabilization. Here, we identify NEDD8 as a crucial regulator of L11 RP signalling to p53. A decrease in L11 NEDDylation during nucleolar stress causes relocalization of L11 from the nucleolus to the nucleoplasm. This not only provides the signal for p53 activation, but also makes L11 susceptible to degradation. Mouse double minute 2 (MDM2) -mediated NEDDylation protects L11 from degradation and this is required for p53 stabilization during nucleolar stress. By controlling the correct localization and stability of L11, NEDD8 acts as a crucial, new regulator of nucleolar signalling to p53.

E3 ligases confer specificity to ubiquitination by recognizing target substrates and mediating transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to substrate. The activity of most E3s is specified by a RING domain, which binds to an E2∼ubiquitin thioester and activates discharge of its ubiquitin cargo. E2-E3 complexes can either monoubiquitinate a substrate lysine or synthesize polyubiquitin chains assembled via different lysine residues of ubiquitin. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent proteolysis to modulation of protein function, structure, assembly, and/or localization. Not surprisingly, RING E3-mediated ubiquitination can be regulated in a number of ways. RING-based E3s are specified by over 600 human genes, surpassing the 518 protein kinase genes. Accordingly, RING E3s have been linked to the control of many cellular processes and to multiple human diseases. Despite their critical importance, our knowledge of the physiological partners, biological functions, substrates, and mechanism of action for most RING E3s remains at a rudimentary stage.

Conjugation of ubiquitin-like protein Nedd8 to cullin (i.e. neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here we show that neddylation stimulates recruitment of ubiquitin-conjugating enzyme (E2) esterified with ubiquitin (E2~Ub), helps bridge the ~50 Å gap between E2 and substrate bound to SCF to enable their reaction, and facilitates formation of amide bonds in E2's active site. Together, these effects potently stimulate transfer of ubiquitin to substrate. We propose that the initiator ubiquitin spans the gap, and the impact of neddylation on transfer of subsequent ubiquitins by the E2 Cdc34 arises from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires ≥4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.

The ubiquitin-like protein Nedd8/Rub1 covalently modifies and activates cullin ubiquitin ligases. However, the repertoire of Nedd8-modified proteins and the regulation of protein neddylation status are not clear. The cysteine protease DEN1/NEDP1 specifically processes the Nedd8 precursor and has been suggested to deconjugate Nedd8 from cullin proteins. By characterizing the Drosophila DEN1 protein and DEN1 null (DEN1null) mutants, we provide in vitro and in vivo evidence that DEN1, in addition to processing Nedd8, deneddylates many cellular proteins. Although purified DEN1 protein efficiently deneddylates the Nedd8-conjugated cullin proteins Cul1 and Cul3, neddylated Cul1 and Cul3 protein levels are not enhanced in DEN1null. Strikingly, many cellular proteins are highly neddylated in DEN1 mutants and are deneddylated by purified DEN1 protein. DEN1 deneddylation activity is distinct from that of the cullin-deneddylating CSN. Genetic analyses indicate that a balance between neddylation and deneddylation maintained by DEN1 is crucial for animal viability.

Neddylation is the post-translational protein modification that is most closely related to ubiquitination. However, ubiquitination is known to regulate a myriad of processes in eukaryotic cells, whereas only a limited number of neddylation substrates have been described to date. Here, we review the principles of protein neddylation and highlight the mechanisms that ensure the specificity of neddylation over ubiquitination. As numerous neddylation substrates probably remain to be discovered, we propose some criteria that could be used as guidelines for the characterization of neddylated proteins.

Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate–proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC–MS/MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here can provide clues on the biological role of Nedd8 and lay the foundation for an in-depth analysis of the regulation of the Nedd8 pathway.

Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located approximately 18 A from the site of neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin neddylation.

Background: Neddylation is a reversible post-translational modification that plays a vital role in maintaining cellular machinery. It is shown to affect localization, binding partners and structure of target proteins. Disruption of protein neddylation was observed in various diseases such as Alzheimer's and cancer. Therefore, understanding the neddylation mechanism and determining neddylation targets possibly bears a huge importance in further understanding the cellular processes. This study is the first attempt to predict neddylated sites from protein sequences by using several sequence and sequence-based structural features. Results: We have developed a neddylation site prediction method using a support vector machine based on various sequence properties, position-specific scoring matrices, and disorder. Using 21 amino acid long lysinecentred windows, our model was able to predict neddylation sites successfully, with an average 5-fold stratified cross validation performance of 0.91, 0.91, 0.75, 0.44, 0.95 for accuracy, specificity, sensitivity, Matthew's correlation coefficient and area under curve, respectively. Independent test set results validated the robustness of reported new method. Additionally, we observed that neddylation sites are commonly flexible and there is a significant positively charged amino acid presence in neddylation sites. Conclusions: In this study, a neddylation site prediction method was developed for the first time in literature. Common characteristics of neddylation sites and their discriminative properties were explored for further in silico studies on neddylation. Lastly, up-to-date neddylation dataset was provided for researchers working on post-translational modifications in the accompanying supplementary material of this article.