Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization.

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice.

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Integrated analysis reveals the regulatory mechanism of the neddylation inhibitor MLN4924 on the metabolic dysregulation in rabbit granulosa cells.

BACKGROUND: Neddylation, an important post-translational modification (PTM) of proteins, plays a crucial role in follicular development. MLN4924 is a small-molecule inhibitor of the neddylation-activating enzyme (NAE) that regulates various biological processes. However, the regulatory mechanisms of neddylation in rabbit ovarian cells have not been emphasized. Here, the transcriptome and metabolome profiles in granulosa cells (GCs) treated with MLN4924 were utilized to identify differentially expressed genes, followed by pathway analysis to precisely define the altered metabolisms. RESULTS: The results showed that 563 upregulated and 910 downregulated differentially expressed genes (DEGs) were mainly enriched in pathways related to cancer, cell cycle, PI3K-AKT, progesterone-mediated oocyte maturation, and PPAR signaling pathway. Furthermore, we characterized that MLN4924 inhibits PPAR-mediated lipid metabolism, and disrupts the cell cycle by promoting the apoptosis and proliferation of GCs. Importantly, we found the reduction of several metabolites in the MLN4924 treated GCs, including glycerophosphocholine, arachidic acid, and palmitic acid, which was consistent with the deregulation of PPAR signaling pathways. Furthermore, the increased metabolites included 6-Deoxy-6-sulfo-D-glucono-1,5-lactone and N-Acetyl-D-glucosaminyldiphosphodolichol. Combined with transcriptome data analyses, we identified genes that strongly correlate with metabolic dysregulation, particularly those related to glucose and lipid metabolism. Therefore, neddylation inhibition may disrupt the energy metabolism of GCs. CONCLUSIONS: These results provide a foundation for in-depth research into the role and molecular mechanism of neddylation in ovary development.

Neddylation of Enterovirus 71 VP2 Protein Reduces Its Stability and Restricts Viral Replication.

Posttranslational modifications (PTMs) of viral proteins play critical roles in virus infection. The role of neddylation in enterovirus 71 (EV71) replication remains poorly defined. Here, we showed that the structural protein VP2 of EV71 can be modified by neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in an E3 ligase X-linked inhibitor of apoptosis protein (XIAP)-dependent manner. Mutagenesis and biochemical analyses mapped the neddylation site at lysine 69 (K69) of VP2 and demonstrated that neddylation reduced the stability of VP2. In agreement with the essential role of VP2 in viral replication, studies with EV71 reporter viruses with wild-type VP2 (enhanced green fluorescent protein [EGFP]-EV71) and a K69R mutant VP2 (EGFP-EV71-VP2 K69R) showed that abolishment of VP2 neddylation increased EV71 replication. In support of this finding, overexpression of NEDD8 significantly inhibited the replication of wild-type EV71 and EGFP-EV71, but not EGFP-EV71-VP2 K69R, whereas pharmacologic inhibition of neddylation with the NEDD8-activating enzyme inhibitor MLN4924 promoted the replication of EV71 in biologically relevant cell types. Our results thus support the notion that EV71 replication can be negatively regulated by host cellular and pathobiological cues through neddylation of VP2 protein. IMPORTANCE Neddylation is a ubiquitin-like posttranslational modification by conjugation of neural precursor cell-expressed developmentally downregulated protein 8 (NEDD8) to specific proteins for regulation of their metabolism and biological activities. In this study, we demonstrated for the first time that EV71 VP2 protein is neddylated at K69 residue to promote viral protein degradation and consequentially suppress multiplication of the virus. Our findings advance knowledge related to the roles of VP2 in EV71 virulence and the neddylation pathway in the host restriction of EV71 infection.

Effects of neddylation on viral infection: an overview.

Neddylation is a post-translational modification that plays an important role not only in cancer development but also in regulating viral infection and replication. Upregulation of neddylation occurs in viral infections, and inhibition of neddylation can suppress viral replication. Neddylation is thought to enhance viral protein stability and replication. Neddylation has been reported to enhance the stability of the regulatory hepatitis B virus (HBV) X protein, modulate viral replication, and enhance hepatocarcinogenesis. Inhibition of neddylation using the NEDD8-activating enzyme E1 inhibitor MLN4924 inhibits viral replication, including that of HBV. Understanding of the role of neddylation in viral infections is critical for developing new therapeutic targets and potential treatment strategies. In this review, we discuss recent progress in the understanding of the effects of neddylation during viral infection, particularly in HBV infection, and strategies for curing viral infection by targeting the neddylation pathway.

Neddylation of EphB1 Regulates Its Activity and Associates with Liver Fibrosis.

Liver fibrosis is a pathological process characterized by the excessive synthesis and accumulation of extracellular matrix proteins (ECMs) contributed mainly by the activated hepatic stellate cells (HSCs). Currently, no direct and effective anti-fibrotic agents have been approved for clinical use worldwide. Although the dysregulation of Eph receptor tyrosine kinase EphB2 has been reported to associate with the development of liver fibrosis, the involvement of other Eph family members in liver fibrosis remains underexplored. In this study, we found that the expression of EphB1 is significantly increased accompanying remarkable neddylation in activated HSCs. Mechanistically, this neddylation enhanced the kinase activity of EphB1 by the prevention of its degradation, thereby promoting the proliferation, migration, and activation of HSCs. Our findings revealed the involvement of EphB1 in the development of liver fibrosis through its neddylation, which provides new insights into the Eph receptor signaling and a potential target for the treatment of liver fibrosis.

Neddylation inactivation affects cell cycle and apoptosis in sheep follicular granulosa cells.

Protein neddylation inactivation is a novel topic in cancer research. However, there are few studies on the mechanism of neddylation underlying the development of sheep follicular granulosa cells (GCs). In this study, the development of follicular GCs in sheep was inactivated by MLN4924, a neddylation-specific inhibitor, which significantly attenuated the proliferation and cell index of sheep follicular GCs. Further, the inactivation of neddylation by MLN4924 caused the accumulation of the cullin ring ligase (CRLs) substrates Wee1 and c-Myc, which could upregulate NOXA protein expression. Meanwhile, the B-cell lymphoma/leukemia 2 (BCL2) family members Bcl-2 and MCL-1 were downregulated, subsequently inducing apoptosis in follicular GCs of sheep. Increasing Wee1 levels caused G2/M-phase arrest. The effects of neddylation inactivation on Akt, the JAK2/STAT3 signaling pathway, and Forkhead box class O(FOXO) family members were evaluated. Neddylation inactivation by MLN4924 increased the levels of phospho-Akt, JAK2, phospho-STAT3, and FOXO1 (p < 0.05) and decreased the levels of phospho-FOXO3a and STAT3 (p < 0.05). In addition, MLN4924 could alter the mitochondrial morphology of GCs, increase cellular glucose utilization and lactate production, increase reactive oxygen species (ROS) generation, and promote sheep follicular GCs glycolysis, thus causing changes in mitochondrial functions. Together, these findings point to an unrecognized role of neddylation in regulating follicular GCs proliferation in sheep.

Blocking neddylation elicits antiviral effect against hepatitis B virus replication.

BACKGROUND: Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we show that blocking of the neddylation elicits antiviral effect against HBV replication, indicating that NEDD8 supports viral production. METHODS AND RESULTS: To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication. Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection. CONCLUSION: The manipulation of the neddylation pathway can thus provide new tools interfering with HBV persistence as well as novel therapeutic strategies against chronic hepatitis B.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

Neddylation inhibitor MLN4924 has anti-HBV activity via modulating the ERK-HNF1α-C/EBPα-HNF4α axis.

Hepatitis B virus (HBV) infection is a major public health problem. The high levels of HBV DNA and HBsAg are positively associated with the development of secondary liver diseases, including hepatocellular carcinoma (HCC). Current treatment with nucleos(t)ide analogues mainly reduces viral DNA, but has minimal, if any, inhibitory effect on the viral antigen. Although IFN reduces both HBV DNA and HBsAg, the serious associated side effects limit its use in clinic. Thus, there is an urgent demanding for novel anti-HBV therapy. In our study, viral parameters were determined in the supernatant of HepG2.2.15 cells, HBV-expressing Huh7 and HepG2 cells which transfected with HBV plasmids and in the serum of HBV mouse models with hydrodynamic injection of pAAV-HBV1.2 plasmid. RT-qPCR and Southern blot were performed to detect 35kb mRNA and cccDNA. RT-qPCR, Luciferase assay and Western blot were used to determine anti-HBV effects of MLN4924 and the underlying mechanisms. We found that treatment with MLN4924, the first-in-class neddylation inhibitor currently in several phase II clinical trials for anti-cancer application, effectively suppressed production of HBV DNA, HBsAg, 3.5kb HBV RNA as well as cccDNA. Mechanistically, MLN4924 blocks cullin neddylation and activates ERK to suppress the expression of several transcription factors required for HBV replication, including HNF1α, C/EBPα and HNF4α, leading to an effective blockage in the production of cccDNA and HBV antigen. Our study revealed that neddylation inhibitor MLN4924 has impressive anti-HBV activity by inhibiting HBV replication, thus providing sound rationale for future MLN4924 clinical trial as a novel anti-HBV therapy.

Nedd8-activating enzyme inhibitor MLN4924 (Pevonedistat), inhibits miR-1303 to suppress human breast cancer cell proliferation via targeting p27Kip1.

MLN4924/Pevonedistat, a Nedd8-activating enzyme (NAE, E1) inhibitor, has shown notable anti-cancer effect in pre-clinical trials, but it still faces tolerance resistance risk. Combination target therapy indicates a much better clinical effect than single target, and miRNAs are beneficial for easy detection in bodily fluids and tissues. Up to now, MLN4924 and miRNA-targeting combination approaching to treat breast cancer patients remains largely unknown. Here, microRNA-seq analysis showed that the expression of miR-1303 was significantly decreased after MLN4924 treatment in breast cancer cells. Moreover, miR-1303 was abnormally high in breast cancer tissues, and breast cancer patients with high miR-1303 showed poor prognosis. Functionally, excessive miR-1303 promoted the malignant phenotypes of breast cancer cells. Excessive miR-1303 accelerated cell cycle progression by promoting G2/M arrest. Furthermore, we revealed that miR-1303 targeted p27Kip1 to release G2/M arrest. Notably, excessive miR-1303 partially disturbed the anti-cancer effect of MLN4924. These findings provide potential evidences for combined anti-cancer target therapy of breast cancer patients in the future.

Overexpression of ABCG2 confers resistance to pevonedistat, an NAE inhibitor.

Pevonedistat is a potent, selective, first-in-class NEDD8 activating enzyme inhibitor. It is now under multiple clinical trials that investigate its anticancer effect against solid tumors and leukemia. ATP-binding cassette (ABC) transporters are membrane proteins that are involved in mediating multidrug resistance (MDR). In this article, we reveal that pevonedistat is a substrate of ABCG2 which decreases the therapeutic effect of pevonedistat. The cytotoxicity of pevonedistat was significantly weakened in ABCG2-overexpressing cells, and the drug resistance can be reversed by ABCG2 inhibitors. The ATPase assay suggested that pevonedistat can stimulate ABCG2 ATPase activity in a concentration-dependent manner. Pevonedistat showed little effect on the expression level or subcellular localization of ABCG2 after 72 h treatment. Furthermore, a pevonedistat resistance cell line S1-PR was established and overexpressed ABCG2. Generally, our study provides evidence that ABCG2 can be a prominent factor leading to pevonedistat-resistance. Furthermore, ABCG2 may also be utilized as a biomarker to monitor the development of pevonedistat resistance during cancer treatment.

Lipin-2 degradation elicits a proinflammatory gene signature in macrophages.

Lipin-2 is a phosphatidate phosphatase with key roles in regulating lipid storage and energy homeostasis. LPIN2-genetic deficiency is associated with an autoinflammatory disorder, underscoring its critical role in innate immune signaling; however, the regulatory mechanisms underlying protein stability remain unknown. Here, we demonstrate that Lipin-2 interacts with ß-TRCP, a substrate receptor subunit of the SCFß-TRCP E3 ligase, and undergoes ubiquitination and proteasomal degradation. ß-TRCP-knockout in RAW264.7 macrophages resulted in Lipin-2 accumulation, leading to the suppression of LPS-induced MAPK activation and subsequent proinflammatory gene expression. Consistent with this, treatment with MLN4924, a Cullin-neddylation inhibitor that suppresses SCF E3 activity, increased Lipin-2 protein and concomitantly decreased Il1b expression. These findings suggested that ß-TRCP-mediated Lipin-2 degradation affects macrophage-elicited proinflammatory responses and could lead to new therapeutic approaches to treat inflammatory diseases.

The NEDD8-activating enzyme inhibition with MLN4924 sensitizes human cancer cells of different origins to apoptosis and necroptosis.

OBJECTIVES: MLN4924 is an inhibitor of NEDD8-activating enzyme (NAE) that interferes with the cullin-RING ubiquitin ligase complexes formation and the nuclear factor kappa B (NF-κB) activation. Here, we investigated the cytotoxic effect of MLN4924 and its ability to sensitize a broad range of cancer cells of different origins to tumour necrosis factor-α (TNF)-induced cell death alongside unravelling its mechanism of action. MATERIALS AND METHODS: Cell viability and caspases processing were determined after MLN4924 treatment either alone or with zVAD-fmk (pan caspase inhibitor), necrostatin-1 (nec-1, RIPK1 inhibitor) and necrosulfonamide (NSA, MLKL inhibitor). Moreover, MLN4924 ability to potentiate TNF-induced cell death was evaluated in 24 cell lines of different cancer origins. The impact of NAE inhibition with MLN4924 on TNF-induced apoptosis and necroptosis was evaluated using zVAD-fmk and nec-1, respectively. RESULTS: MLN4924 alone was able to induce cell death in different cell lines that was attributed to apoptosis induction. Also, MLN4924 sensitized different cancer cell lines to TNF-induced cell death. MLN4924/TNF-induced cell death was apoptosis and necroptosis dependent that may be attributed to MLN4924 inhibition of NF-κB pathway activation. CONCLUSIONS: Targeting NAE and NF-κB pathway with MLN4924 represents a substantial approach to enhance the sensitivity of diverse types of cancer cells. Moreover, the broad in vitro screening of MLN4924 anticancer activity provides a valuable guidance for elucidating the susceptible cancer types for the prospective clinical application of MLN4924.

Neddylation inhibition activates the protective autophagy through NF-κB-catalase-ATF3 Axis in human esophageal cancer cells.

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). Video abstract.

Neddylation-Independent Activities of MLN4924.

MLN4924, also known as pevonedistat, is a highly selective small-molecule inhibitor of NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8)-activating enzyme (NAE) to block the entire neddylation modification cascade, leading to inactivation of cullin-RING ligases (CRLs), since activation of CRLs requires cullin neddylation. MLN4924 showed impressive anticancer activity in many preclinical studies and is currently in several Phase I/II clinical trials for anticancer therapy as a single agent or in combination with chemotherapeutic drugs.In addition to well-characterized anti-neddylation activity, recent studies showed that MLN4924 has several neddylation-independent activities. First, MLN4924 triggers EGFR dimerization to activate EGFR and its downstream RAS/MAPK and PI3K/AKT1 signals, leading to enhanced tumor sphere formation, accelerated EGF-mediated wound healing, and inhibited ciliogenesis. Second, MLN4924 induces PKM2 tetramerization to promote glycolysis, thus affecting energy metabolism. Third, MLN4924 inhibits the interaction between ACT1 (NF-κB activator 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) and attenuates IL-17A-mediated activation of NF-κB to reduce pulmonary inflammation. Fourth, MLN4924 inhibits IRF3 binding to the IFN-ß promoter to inhibit IFN-ß production. And finally, MLN4924 activates the JNK signaling pathway to reduce c-FLIP levels, thus enhancing TRAIL-induced apoptosis. This chapter will summarize these neddylation-independent activities of MLN4924 and discuss the underlying mechanisms and potential therapeutic applications.

Inhibition of atherogenesis by the COP9 signalosome subunit 5 in vivo.

Constitutive photomorphogenesis 9 (COP9) signalosome 5 (CSN5), an isopeptidase that removes neural precursor cell-expressed, developmentally down-regulated 8 (NEDD8) moieties from cullins (thus termed "deNEDDylase") and a subunit of the cullin-RING E3 ligase-regulating COP9 signalosome complex, attenuates proinflammatory NF-κB signaling. We previously showed that CSN5 is up-regulated in human atherosclerotic arteries. Here, we investigated the role of CSN5 in atherogenesis in vivo by using mice with myeloid-specific Csn5 deletion. Genetic deletion of Csn5 in Apoe-/- mice markedly exacerbated atherosclerotic lesion formation. This was broadly observed in aortic root, arch, and total aorta of male mice, whereas the effect was less pronounced and site-specific in females. Mechanistically, Csn5 KO potentiated NF-κB signaling and proinflammatory cytokine expression in macrophages, whereas HIF-1α levels were reduced. Inversely, inhibition of NEDDylation by MLN4924 blocked proinflammatory gene expression and NF-κB activation while enhancing HIF-1α levels and the expression of M2 marker Arginase 1 in inflammatory-elicited macrophages. MLN4924 further attenuated the expression of chemokines and adhesion molecules in endothelial cells and reduced NF-κB activation and monocyte arrest on activated endothelium in vitro. In vivo, MLN4924 reduced LPS-induced inflammation, favored an antiinflammatory macrophage phenotype, and decreased the progression of early atherosclerotic lesions in mice. On the contrary, MLN4924 treatment increased neutrophil and monocyte counts in blood and had no net effect on the progression of more advanced lesions. Our data show that CSN5 is atheroprotective. We conclude that MLN4924 may be useful in preventing early atherogenesis, whereas selectively promoting CSN5-mediated deNEDDylation may be beneficial in all stages of atherosclerosis.

MLN4924 protects against interleukin-17A-induced pulmonary inflammation by disrupting ACT1-mediated signaling.

An excessive inflammatory response in terminal airways, alveoli, and the lung interstitium eventually leads to pulmonary hypertension and chronic obstructive pulmonary disease. Proinflammatory cytokine interleukin-17A (IL-17A) has been implicated in the pathogenesis of pulmonary inflammatory diseases. MLN4924, an inhibitor of NEDD8-activating enzyme (NAE), is associated with the treatment of various types of cancers, but its role in the IL-17A-mediated inflammatory response has not been identified. Here, we report that MLN4924 can markedly reduce the expression of proinflammatory cytokines and chemokines such as IL-1ß, IL-6, and CXCL-1 and neutrophilia in a mouse model of IL-17A adenovirus-induced pulmonary inflammation. MLN4924 significantly inhibited IL-17A-induced stabilization of mRNA of proinflammatory cytokines and chemokines in vitro. Mechanistically, MLN4924 significantly blocked the activation of MAPK and NF-κB pathways and interfered with the interaction between ACT1 and tumor necrosis factor receptor-associated factor proteins (TRAFs), thereby inhibiting TRAF6 ubiquitination. Taken together, our data uncover a previously uncharacterized inhibitory effect of MLN4924 on the IL-17A-mediated inflammatory response; this phenomenon may facilitate the development of MLN4924 into an effective small-molecule drug for the treatment of pulmonary inflammatory diseases.

Transient inhibition of neddylation at neonatal stage evokes reversible cardiomyopathy and predisposes the heart to isoproterenol-induced heart failure.

Alterations in perinatal conditions (such as preterm birth) is linked to adult health and disease, in particular, the cardiovascular system. Neddylation, a novel posttranslational modification through which the ubiquitin-like protein NEDD8 is conjugated to protein substrates, has emerged as an important mechanism regulating embryonic cardiac chamber maturation. However, the importance of neddylation in postpartum cardiac development has not been investigated. Here, we aimed to determine whether transient, postnatal inhibition of neddylation has immediate and prolonged impact on the structure and function of the neonatal and adult hearts. Sprague-Dawley pups were given three intraperitoneal injections of MLN4924 (MLN), a specific neddylation inhibitor, at postnatal days (P)1, 3, and 5. Cardiac structure and function were temporally assessed during aging and after 2 wk of isoproterenol (ISO) infusion in adulthood. MLN treatment resulted in modest reduction of neddylated proteins in neonatal hearts. The MLN-treated rats developed cardiac hypertrophy and dysfunction by P7, which was accompanied by significantly reduced cardiomyocyte proliferation. At 3 mo of age, cardiac contractile function was restored in MLN-treated rats, but MLN-treated hearts displayed hypertrophic phenotype. Whereas ISO infusion triggered compensatory cardiac hypertrophy without impairing cardiac contractility in the control rats, the MLN-treated rats displayed a similar degree of hypertrophy, which quickly progressed to decompensation with ventricular wall thinning, chamber dilatation, and reduced ejection fraction as well as exacerbated pathological cardiac remodeling. Our findings suggest that neddylation is required for postnatal cardiac development and that perturbation of neddylation during development predisposes adult hearts to cardiac failure under stress conditions. NEW & NOTEWORTHY Our study demonstrates that perinatal perturbation of neddylation induces cardiomyopathy, impairs postnatal cardiac development, and increases susceptibility to catecholamine-induced cardiac dysfunction. The results reveal a previously unappreciated role of neddylation in postnatal cardiac maturation and call for close monitoring for the potential cardiotoxicity of MLN4924 (pevonedistat) and other agents that modify neddylation, especially in pregnant women and preadolescents.

Disruption of protein neddylation with MLN4924 attenuates paclitaxel-induced apoptosis and microtubule polymerization in ovarian cancer cells.

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.