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[EN] BACKGROUND & AIMS: Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. METHODS: Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. RESULTS: The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. CONCLUSION: Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. LAY SUMMARY: Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors. This article is based upon work from the COST Action CA18122 European Cholangiocarcinoma Network supported by COST (European Cooperation in Science and Technology: www.cost.eu).

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 Protein neddylation plays a tumor-promoting role in esophageal cancer. Our previous study demonstrated that neddylation inhibition induced the accumulation of ATF4 to promote apoptosis in esophageal cancer cells. However, it is completely unknown whether neddylation inhibition could induce autophagy in esophageal cancer cells and affect the expression of other members of ATF/CREB subfamily, such as ATF3. Methods The expression of relevant proteins of NF-κB/Catalase/ATF3 pathway after neddylation inhibition was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies. ROS generation upon MLN4924 treatment was determined by H2-DCFDA staining. The proliferation inhibition induced by MLN4924 was evaluated by ATPLite assay and apoptosis was evaluated by Annexin V /PI double staining. Results For the first time, we reported that MLN4924, a specific inhibitor of Nedd8-activating enzyme, promoted the expression of ATF3 to induce autophagy in esophageal cancer. Mechanistically, MLN4924 inhibited the activity of CRLs and induced the accumulation of its substrate IκBα to block NF-κB activation and Catalase expression. As a result, MLN4924 activated ATF3-induced protective autophagy, thereby inhibiting MLN4924-induced apoptosis, which could be alleviated by ATF3 silencing. Conclusions In our study, we elucidates a novel mechanism of NF-κB/Catalase/ATF3 pathway in MLN4924-induced protective autophagy in esophageal cancer cells, which provides a sound rationale and molecular basis for combinational anti-ESCC therapy with knockdown ATF3 and neddylation inhibitor (e.g. MLN4924). Graphical abstract

Introduction: Neddylation is a highly dynamic and reversible post-translatiNeddylation is a highly dynamic and reversible post-translational modification. The abnormality of neddylation has previously been shown to be closely related to some human diseases. The detection of neddylation sites is essential for elucidating the regulation mechanisms of protein neddylation.onal modification which has been found to be involved in various biological processes and closely associated with many diseases. The accurate identification of neddylation sites is necessary to elucidate the underlying molecular mechanisms of neddylation. As the traditional experimental methods are time consuming and expensive, it is desired to develop computational methods to predict neddylation sites. In this study, a novel predictor named NeddPred is proposed to predict lysine neddylation sites. An effective feature extraction method, bi-profile bayes encoding, is employed to encode neddylation sites. Moreover, a fuzzy support vector machine algorithm is proposed to solve the class imbalance and noise problem in the prediction of neddylation sites. As illustrated by 10-fold cross-validation, NeddPred achieves an excellent performance with a Matthew's correlation coefficient of 0.7082 and an area under receiver operating characteristic curve of 0.9769. Independent tests show that NeddPred significantly outperforms existing neddylation sites predictor NeddyPreddy. Therefore, NeddPred can be a complement to the existing tools for the prediction of neddylation sites. A user-friendly web-server for NeddPred is established at 123.206.31.171/NeddPred/. Objective: As the detection of the lysine neddylation sites by the traditional experimental method is often expensive and time-consuming, it is imperative to design computational methods to identify neddylation sites. Methods: In this study, a bioinformatics tool named NeddPred is developed to identify underlying protein neddylation sites. A bi-profile bayes feature extraction is used to encode neddylation sites and a fuzzy support vector machine model is utilized to overcome the problem of noise and class imbalance in the prediction. Results: Matthew's correlation coefficient of NeddPred achieved 0.7082 and an area under the receiver operating characteristic curve of 0.9769. Independent tests show that NeddPred significantly outperforms existing lysine neddylation sites predictor NeddyPreddy. Conclusion: Therefore, NeddPred can be a complement to the existing tools for the prediction of neddylation sites. A user-friendly webserver for NeddPred is accessible at 123.206.31.171/NeddPred/.

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.

Ubiquitin-like protein neddylation is overactivated in various human cancers and correlates with disease progression, and targeting this pathway represents a valuable therapeutic strategy. Our previous work disclosed an antihypertensive agent, candesartan cilexetic (CDC), serves as a novel neddylation inhibitor for suppressing tumor growth by targeting Nedd8-activating enzyme (NAE). In this study, 42 benzimidazole derivatives were designed and synthesized based on lead compound CDC to improve the neddylation inhibition and anticancer efficacy. Optimal benzimidazole-derived 35 displayed superior neddylation inhibition in enzyme assay compared to CDC (IC50 = 5.51 μM vs 16.43 μM), along with promising target inhibitory activity and killing selectivity in cancer cell. The results of cellular mechanism research combined with tumor growth suppression in human lung cancer cell A549 in vivo, accompanied with docking model, revealed that 35 has the potential to be developed as a promising neddylation inhibitor for anticancer therapy.

Protein NEDDylation emerges as an important post-translational modification and an attractive target for therapeutic intervention. Modification of NEDD8 onto substrates is finely balanced by the co-ordinated activity of conjugating and deconjugating enzymes. The NEDP1/DEN1/SENP8 protease is a NEDD8 specific processing and deconjugating enzyme that regulates the NEDDylation mainly of non-cullin substrates. Here, we report the development and characterisation of nanobodies as first-in-class inhibitors for NEDP1. The nanobodies display high-affinity (low nM) against NEDP1 and specifically inhibit NEDP1 processing activity in vitro and NEDP1 deconjugating activity in tissue-culture cells and in cell extracts. We also isolated nanobodies that bind to NEDP1 with high-affinity but do not affect NEDP1 activity. The developed nanobodies provide new tools to study the function of NEDP1 and to prevent deNEDDylation in cell extracts used in biochemical assays.

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.

Glioblastoma is a highly aggressive and lethal brain tumor, with limited treatment options. Abnormal activation of the neddylation pathway is observed in glioblastoma, and the NEDD8-activating enzyme (NAE) inhibitor, MLN4924, was previously shown to be effective in glioblastoma cell line models. However, its effect has not been tested in patient-derived glioblastoma stem cells. We first analyzed public data to determine whether NEDD8 pathway proteins are important in glioblastoma development and patient survival. NAE1 and UBA3 levels increased in glioblastoma patients, high NEDD8 levels were associated with poor clinical outcomes. Immunohistochemistry results also supported this result. The effects of MLN4924 were evaluated in 4 glioblastoma cell lines and 15 patient-derived glioblastoma stem cells using high content analysis. Glioblastoma cell lines and patient-derived stem cells were highly susceptible to MLN4924, while normal human astrocytes were resistant. In addition, there were various responses in 15 patient-derived glioblastoma stem cells upon MLN4924 treatment. Genomic analyses indicated that MLN4924 sensitive cells exhibited enrichment of Extracellular Signal Regulated Kinase (ERK) and Protein kinase B (AKT, also known as PKB) signaling. We verified that MLN4924 inhibits ERK and AKT phosphorylation in MLN4924 sensitive cells. Our findings suggest that patient-derived glioblastoma stem cells in the context of ERK and AKT activation are sensitive and highly regulated by neddylation inhibition.

Protein neddylation is a posttranslational modification of conjugating the neuronal precursor cell-expressed developmentally down-regulated protein 8 (Nedd8) to substrates. Our previous work revealed that neddylation pathway is overactivated in various human lung cancers and correlates with the disease progression, whereas pharmacologically targeting this pathway has emerged as an attractive therapeutic strategy. As a follow-up research, 1331 approved drugs were investigated the inhibitory activities of cullin1 neddylation for screening the hit compounds via an improved enzyme-based assay. An antihypertensive agent, candesartan cilexetic (CDC), was identified as a novel neddylation inhibitor that ATP-competitively suppressing Nedd8-activating enzyme (NAE, E1) in mechanism, which inhibited the cullins neddylation superior than two representative non-covalent NAE inhibitors, M22 and mitoxantrone. Following with the findings such as apoptotic induction and tumor growth suppression in human lung cancer A549 in vitro and in vivo, CDC represents a potential anticancer lead compound with promising neddylation inhibitory activity.

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.

Purpose: To determine the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of the investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) and to investigate pevonedistat pharmacokinetics and pharmacodynamics in patients with advanced nonhematologic malignancies. Experimental Design: Pevonedistat was administered via 60-minute intravenous infusion on days 1 to 5 (schedule A, n = 12), or days 1, 3, and 5 (schedules B, n = 17, and C, n = 19) of 21-day cycles. Schedule B included oral dexamethasone 8 mg before each pevonedistat dose. Dose escalation proceeded using a Bayesian continual reassessment method. Tumor response was assessed by RECIST 1.0. Results: Schedule A MTD was 50 mg/m2; based on the severity of observed hepatotoxicity, this schedule was discontinued. Schedules B and C MTDs were 50 and 67 mg/m2, respectively. DLTs on both these schedules included hyperbilirubinemia and elevated aspartate aminotransferase. There were no grade ≥3 treatment-related serious adverse events reported on schedules B or C. Twenty-three (74%) evaluable patients on schedules B and C had stable disease. Intermittent dexamethasone use did not significantly influence pevonedistat pharmacokinetics. NAE inhibition by pevonedistat was demonstrated in multiple tumor types via IHC detection of pevonedistat-NEDD8 adduct and accumulation of Cullin-RING ligase substrates CDT1 and NRF2 in tumor biopsies. Conclusions: Pevonedistat was generally well tolerated on a day 1, 3, 5 schedule every 3 weeks with an MTD between 50 mg/m2 and 67 mg/m2. DLTs were predominantly hepatic enzyme elevations. Pharmacodynamic studies demonstrated that pevonedistat inhibited NAE in tumors. Clinical trials are ongoing. Clin Cancer Res; 22(4); 847–57. ©2015 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.

Surgery and chemotherapy are the gold-standard treatments for ovarian cancer. The major cause of treatment failure in patients with ovarian cancer is tumoral heterogeneity and drug resistance. Paclitaxel (PTX) is one of the most commonly used first-line drugs for ovarian cancer chemotherapy. Unfortunately, the mechanisms of PTX chemoresistance remain unclear. Here, we examined the effects of post-translational neddylation on the sensitivity of ovarian cancer cells (OCCs) to PTX-induced apoptosis. Disruption of protein neddylation with the first-in-class inhibitor MLN4924 dramatically neutralized PTX-mediated antiproliferative, antimigration, and apoptotic effects in human OCCs. Moreover, MLN4924 treatment interrupted PTX-induced microtubule polymerization. Importantly, two neddylation conjugating E2 enzymes, UBE2M and UBE2F, were found to play essential roles in PTX-induced cytotoxicity and tubulin polymerization in OCCs. In summary, our findings demonstrated that disruption of protein neddylation by MLN4924 conferred resistance to PTX and provided insights into the potential mechanisms of PTX chemoresistance in ovarian cancer.

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.

Background Protein neddylation is a post-translational modification by a covalent conjugation with the neural precursor cell expressed, developmentally downregulated 8 (NEDD8). Although this process has been reported to participate in diverse cellular signaling, little is known about its role in cancer cell migration. Given a recent proteomics report showing that NEDD8 is downregulated in prostate cancer tissues versus normal prostate tissues, we tested the possibility that neddylation plays a role in cancer evolution, and then tried to identify target proteins of the neddylation. Methods The neddylation process was inhibited by transfecting cancer cells with NEDD8-targeting siRNAs or by treating the cells with a NAE1 inhibitor MLN4924. Cell migration was evaluated by an in vitro wound-healing assay and a Transwell migration assay. His/NEDD8-conjugated proteins were pulled down with nickel-affinity beads under a denaturing condition, and identified by Western blotting. All data were processed using the Microsoft Excel program and analyzed statistically by two-sided, unpaired Student’s t-test. Results Caveolin-1, which plays a critical role in cell migration, was identified to be conjugated with NEDD8. When the neddylation was inhibited, the phosphorylation of caveolin-1 at Tyr14 was augmented in PC3 and U373MG cells, thereby leading to increased cell migration. Such consequences by neddylation inhibition were abolished in the presence of a Src family kinase inhibitor PP2. Conclusions NEDD8 seems to inhibit the Src-mediated phosphorylation of caveolin-1 by modifying the structure of caveolin-1 protein, which blocks the migration of cancer cells. Although the neddylation process is currently regarded as an emerging target for cancer therapy, our results suggest the possibility that the inhibition of neddylation could facilitate cancer invasion or metastasis at least in some types of cancers. Electronic supplementary material The online version of this article (doi: 10.1186/s12885-017-3942-9) contains supplementary material, which is available to authorized users.

Inhibiting the protein neddylation pathway using the NEDD8-activating enzyme inhibitor MLN4924 represents an attractive anticancer strategy having been demonstrated to exhibit promising anticancer efficacy and with tolerable levels of toxicity; however, the mechanism by which MLN4924 inhibits cell proliferation in human esophageal squamous cell carcinoma (ESCC) cells requires further investigation. The present study revealed that MLN4924 treatment led to G2 cell cycle arrest and enhanced the protein stability of Wee1-like protein kinase and cyclin dependent protein kinase inhibitor 1A and B and p27. Furthermore, MLN4924 induced DNA damage and sensitized esophageal cancer cells to cisplatin by enhancing apoptosis. These findings extend the understanding of the function and mechanism of MLN4924 in ESCC and provide further evidence for the future development of neddylation inhibitors in clinical trials of esophageal cancer therapy, either alone or in combination.

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.