Neddylation Inhibitor Research Articles

The Influence of Neddylation on the Mucosal Inflammatory Response

Recent work has revealed a central role for neddylation (the conjugation of a Nedd8 protein to Cullin‐proteins) in the induction of the NF‐κB pathway (via Cullin‐1) and stabilization of hypoxia‐inducible factor (HIF, via Cullin‐2) during inflammatory responses. Our recent work indicated that pharmacological stabilization of HIF with the neddylation inhibitor MLN4924 significantly abrogated colitis endpoints in mice. In the present studies, we addressed the contribution of Cullin‐1 neddylation and NF‐kB signaling to mucosal inflammatory responses in vitro and in vivo. Initial in vitro studies using T84 intestinal epithelial cells revealed that MLN4924 prominently induces the deneddylation of Cullin‐1. Parallel western blot analysis and luciferase reporter assays revealed MLN4924 as a potent inhibitor of intestinal epithelial NF‐kB. In vivo administration of MLN4924 (3mg/kg/day) in a TNBS colitis model significantly increased disease severity. Indeed, MLN4924 resulted in increased weight loss and colon shortening as well as increased mortality early in the inflammatory response (day 2). Histologic analysis of the colon revealed that neddylation inhibition results in increased tissue damage and significantly increased mucosal apoptosis as determined by TUNEL staining, particularly within the epithelium. These studies reveal that Cullin‐1 neddylation, and most particularly NF‐κB signaling, is protective during mucosal inflammatory responses. We conclude that Cullin neddylation fine‐tunes the mucosal inflammatory response in vitro and in vivo.

Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Targeting protein neddylation with an NEDD8-activating enzyme inhibitor MLN4924 induced apoptosis or senescence in human lymphoma cells

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.

Synaptic structure and function are altered by the neddylation inhibitor MLN4924.

The posttranslational modification of proteins by the ubiquitin-like small molecule NEDD8 has previously been shown to be vital in a number of cell signaling pathways. In particular, conjugation of NEDD8 (neddylation) serves to regulate protein ubiquitination through modifications to E3 ubiquitin ligases. Despite the prevalence of NEDD8 in neurons, very little work has been done to characterize the role of this modifier in these cells. Here, we use the recently developed NEDD8 Activating Enzyme (NAE) inhibitor MLN4924 and report evidence of a role for NEDD8 in regulating mammalian excitatory synapses. Application of this drug to dissociated rat hippocampal neurons caused reductions in synaptic strength, surface glutamate receptor levels, dendritic spine width, and spine density, suggesting that neddylation is involved in the maintenance of synapses.

NEDDylation promotes endothelial dysfunction: A role for HDAC2

Emerging evidence strongly supports a role for HDAC2 in the transcriptional regulation of endothelial genes and vascular function. We have recently demonstrated that HDAC2 reciprocally regulates the transcription of Arginase2, which is itself a critical modulator of endothelial function via eNOS. Moreover HDAC2 levels are decreased in response to the atherogenic stimulus OxLDL via a mechanism that is apparently dependent upon proteasomal degradation. NEDDylation is a post-translational protein modification that is tightly linked to ubiquitination and thereby protein degradation. We propose that changes in NEDDylation may modulate vascular endothelial function in part through alterations in the proteasomal degradation of HDAC2. In HAEC, OxLDL exposure augmented global protein NEDDylation. Pre-incubation of mouse aortic rings with the NEDDylation activating enzyme inhibitor, MLN4924, prevented OxLDL-induced endothelial dysfunction. In HAEC, MLN enhanced HDAC2 abundance, decreased expression and activity of Arginase2, and blocked OxLDL-mediated reduction of HDAC2. Additionally, HDAC2 was shown to be a substrate for NEDD8 conjugation and this interaction was potentiated by OxLDL. Further, HDAC2 levels were reciprocally regulated by ectopic expression of NEDD8 and the de-NEDDylating enzyme SENP8. Our findings indicate that the observed improvement in endothelial dysfunction with inhibition of NEDDylation activating enzyme is likely due to an HDAC2-dependent decrease in Arginase2. NEDDylation activating enzyme may therefore be a novel target in endothelial dysfunction and atherogenesis.

Nedd8 regulates inflammasome-dependent caspase-1 activation.

Caspase-1 is activated by the inflammasome complex to process cytokines like interleukin-1β (IL-1β). Pro-caspase-1 consists of three domains, CARD, p20, and p10. Association of pro-caspase-1 with the inflammasome results in initiation of its autocatalytic activity, culminating in self-cleavage that generates catalytically active subunits (p10 and p20). In the current study, we show that Nedd8 is required for efficient self-cleavage of pro-caspase-1 to generate its catalytically active subunits. Nedd8 silencing or treating cells with the neddylation inhibitor MLN4924 led to diminished caspase-1 processing and reduced IL-1β maturation following inflammasome activation. Coimmunoprecipitation and mass spectrometric analysis of 293 cells overexpressing pro-caspase-1 (and CARD) and Nedd8 suggested possible neddylation of caspase-1 CARD. Following inflammasome activation in primary macrophages, we observed colocalization of endogenous Nedd8 with caspase-1. Similarly, interaction of endogenous Nedd8 with caspase-1 CARD was detected in inflammasome-activated macrophages. Furthermore, enhanced autocatalytic activity of pro-caspase-1 was observed following Nedd8 overexpression in 293 cells, and such activity in inflammasome-activated macrophages was drastically diminished upon treatment of cells with MLN4924. Thus, our studies demonstrate a role of Nedd8 in regulating caspase-1 activation following inflammasome activation, presumably via augmenting autoprocessing/cleavage of pro-caspase-1 into its corresponding catalytically active subunits.

MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies

MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC.

The NEDD8-activating enzyme inhibitor MLN4924 disrupts nucleotide metabolism and augments the efficacy of cytarabine.

New therapies are urgently needed for patients with acute myelogenous leukemia (AML). The novel NEDDylation inhibitor MLN4924 (pevonedistat) has demonstrated significant preclinical antileukemic activity and preliminary efficacy in patients with AML in a phase I trial. On the basis of its antimyeloid and DNA-damaging properties, we investigated the ability of MLN4924 to augment conventional cytarabine (ara-C) therapy. The effects of MLN4924/ara-C on viability, clonogenic survival, apoptosis, DNA damage, and relevant pharmacodynamic targets were determined. The efficacy and pharmacodynamics of MLN4924/ara-C were assessed in an AML xenograft model. Cotreatment of AML cell lines and primary patient specimens with MLN4924 and ara-C led to diminished clonogenic survival, increased apoptosis, and synergistic levels of DNA damage. RNAi demonstrated that stabilization of CDT-1, an event previously shown to mediate the DNA-damaging effects of MLN4924, was not a key regulator of sensitivity to the MLN4924/ara-C combination. Global metabolic profiling revealed that MLN4924 disrupts nucleotide metabolism and depletes intracellular nucleotide pools in AML cells. Subsequent experiments showed that MLN4924 promoted increased incorporation of ara-C into the DNA of AML cells. This effect as well as the therapeutic benefit of the MLN4924/ara-C combination was antagonized by supplementation with the nucleotide building block ribose. Coadministration of MLN4924 and ara-C to mice bearing FLT3-ITD(+) AML xenografts stably inhibited disease progression and increased DNA damage in vivo. Our findings provide strong rationale for clinical investigation of the MLN4924/ara-C combination and establish a new link between therapeutic inhibition of NEDDylation and alterations in nucleotide metabolism. Clin Cancer Res; 21(2); 439-47. ©2014 AACR.

Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition

Neddylation is a ubiquitylation-like pathway that controls cell cycle and proliferation by covalently conjugating Nedd8 to specific targets. However, its role in neurons, nonreplicating postmitotic cells, remains unexplored. Here we report that Nedd8 conjugation increased during postnatal brain development and is active in mature synapses, where many proteins are neddylated. We show that neddylation controls spine development during neuronal maturation and spine stability in mature neurons. We found that neddylated PSD-95 was present in spines and that neddylation on Lys202 of PSD-95 is required for the proactive role of the scaffolding protein in spine maturation and synaptic transmission. Finally, we developed Nae1(CamKIIα-CreERT2) mice, in which neddylation is conditionally ablated in adult excitatory forebrain neurons. These mice showed synaptic loss, impaired neurotransmission and severe cognitive deficits. In summary, our results establish neddylation as an active post-translational modification in the synapse regulating the maturation, stability and function of dendritic spines.

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.

Protein neddylation: beyond cullin-RING ligases.

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.

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer.

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.

Therapeutic Effects of MLN4924 on Sclerodermatous Chronic Graft Versus Host Disease

▪Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for the treatment of a variety of hematological diseases. However, high incidences of graft versus host disease (GVHD) and tumor relapse remain as major hurdles to this approach. While recent advances in transplant preparation and prophylactic regimens have lowered the incidence of acute GVHD, chronic GVHD (cGVHD) still remains a major cause of long-term morbidity and mortality. Recent studies have suggested that post-translation modifications (PTMs) may serve as therapeutic targets during GVHD. PTMs of proteins are important pathways that confer biochemical alterations, such as acetylation, phosphorylation, and ubiquitination. Targeting PTMs by using small molecular inhibitors has demonstrated potent anti-tumor effects, as well as immune modulatory properties. We have previously shown that bortezomib, an inhibitor of PTMs pathways, can successfully ameliorate sclerodermatous cGVHD responses in both preclinical and clinical studies. Here we reported a new small molecular targeting agent, MLN4924, a selective inhibitor of NEDD8-activating enzyme, on the treatment of cGVHD.MLN4924 is a selective inhibitor of NEDD8-activating enzyme and has been shown to be well tolerated at higher doses and exhibit potent anti-tumor activity in murine models and in early clinical trials. Furthermore, MLN4924 can suppress dendritic cell functions, as well as their production of proinflammatory cytokines during allogeneic responses. Due to these findings, we sought to test the treatment effects of MLN4924 on cGVHD responses. To investigate this, we utilized a minor MHC mismatch murine model by transferring donor B10.D2 (H-2d) cells into BALB/c (H-2d) recipients. MLN4924 or vehicle control (10% 2-hydroxy-propyl-beta cyclodextrin) was injected intraperitoneally on day 20, the point at which skin sclerodermatous cGVHD is initiated. Compared with the vehicle groups, animals that received either bortezomib (0.1 mg/kg) or MLN4924 (20 mg/kg) developed significantly less cGVHD, as determined by clinical scores (2.34±0.34 versus 1.03±0.37 versus 0.8±0.14, respectively; P <0.01). Surprisingly, a preventive approach started from either day 0 or day 10 post-HSCT resulted in higher cGVHD scores compared to early therapeutic interventions, started from Day 20, (3.3 and 2.37± 0.18 versus 0.8±0.14, respectively). The temporally dependent efficacy of MLN4924 suggests that PTMs may play a suppressive role in the early development of cGVHD pathogenesis while worsening cGVHD response as a prevention treatment. Flow cytometric analysis revealed a significant reduction in the total number of macrophages and dendritic cells with MLN4924 treatment starting on day 20, while no significant changes were noticed in T cell subpopulations. Similarly, MLN4924 can successfully inhibit mixed lymphocyte reactions (MLR) in vitro. Importantly, a reduction in the total number of plasma cells, a major source of autoantibody production, was also observed. Together these data suggest that inhibition of neddylation with small molecules, such as MLN4924, can be used as a new approach for the treatment of patients with sclerodermatous cGVHD, while potentially maintaining graft versus tumor effects through preserving T cell populations. DisclosuresNo relevant conflicts of interest to declare.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

Abstract 3770: MLN4924, a neddylation inhibitor, abolishes chemoresistance of bladder cancer stem-like cells through suppression of stemness

Abstract Introduction: Bladder urothelial carcinoma (UC) ranks fourth in incidence among cancers in men and eighth in women in the United States, however, the chemotherapy response rate is only about half. The cancer stem cells (CSCs), which are believed as the driving force of tumor progression, have related to chemoresistance. Here we present the finding of CSC-like cells from human UC cell line NTUB1, and investigate the effect of MLN4924, a neddylation inhibitor on conquering chemoresistance in human UC. Materials &amp; methods: The CSC-like cells were enriched from its parental cell line NTUB1 by cisplatin selection consecutively for at least one year. The properties of CSC-like cells were demonstrated by colony formation assay, side population assay, and cell viability assay. The expression levels of specific stemness-related markers, EMT-related markers and cell death-related molecules were revealed by western blot. The ability of migration was analyzed by wound-healing assay and transwell assay. The MLN4924-induced apoptosis on CSC-like cells were determined by flow cytometry with annexin V and propidium iodide (PI) staining. Gene knockdown was performed with gene-specific siRNA. The in vitro findings were further confirmed in vivo via xenograft mouse model. Results: The CSC-like cells exhibited multiple drug resistance, highly expression level of stemness markers as well as EMT markers, and highly mobility. The treatment of MLN4924 repressed the expression of stemness markers and EMT markers and reduced cell mobility; meanwhile, it caused significant cell death in CSC-like cells. The knockdown of stemness markers diminished the drug resistance of CSC-like cells. And in the end, MLN4924 significantly inhibit tumor growth in xenograft models bladder cancer stem-like cells. Conclusion: Our data suggested that the drug resistance in UC may conferred by CSC-like cells via up-regulation of stemness markers, and the treatment of MLN4924 could abolish drug resistance through repression of stemness markers. This could provide a new remedy for bladder cancer treatment. Citation Format: Kuan-Lin Kuo, I-Lin Ho, Yeong-Shiau Pu, Yu-Chieh Tsai, Tsung-Hsien Shih, Kuo-How Huang. MLN4924, a neddylation inhibitor, abolishes chemoresistance of bladder cancer stem-like cells through suppression of stemness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3770. doi:10.1158/1538-7445.AM2014-3770

Abstract 1028: Anti-angiogenic activity of MLN4924 suppresses the growth of urothelial carcinoma via enhancing vascular normalization

Abstract Introduction: Cullin-RING ligases (CRLs), the cullin-scaffold RING-finger domain containing E3, needs Nedd8 modification, neddylation, for the complete E3 activity, which promote the ubiquitination of specific substrate's degradation and regulate numerous biological processes. MLN4924, a neddylation inhibitor, was repoted to interfere in the neddylation of cullins and subsequently inactivate CRLs, leading to cell cycle arrest and apoptosis of tumors. Thus, we hereby present the detailed mechanism regarding the anti-angiogenesis of MLN4924 on suppressing the growth of urothelial carcinoma (UC) via inducing vascular normalization. Materials &amp; methods: Human umbilical vein endothelial cells (HUVECs) were used to investigate the anti-angiogenic functions of MLN4924 in vitro, including WST-1 assay for evaluating proliferation, Transwell with or without Matrigel-coating for testing invasion and migration, gelatin zymography for assaying the matrix metalloproteinase activity, propidium iodide (PI) staining for investigating cell cycle progression, PI and annexin V co-staining for exploring cell death, Matrigel tube formation for examining angiogenic activity, and Western blotting for checking protein levels in cell cycle, apoptosis, and angiogenesis-associated signaling pathways. Besides, Matrigel plug assay was used to studying angiogenesis in vivo, and UC xenograft model was applied to research the tumor growth and tumor vascularity. Results: Our results showed that MLN4924 suppressed HUVEC's proliferation, migration, invasion, MMP 2 activation, and angiogenic tube formation. MLN4924 decreased angiogenic receptor VEGFR 2 and Tie2 expressions and inhibited VEGFR 2, ERK1/2, STAT 1 and 3, and AKT phosphorylations. MLN4924 induced the G2/M cell cycle arrest, which increased p21 and p27 accumulations and p53 phosphorylation but diminished histone 3 phosphorylation. Additionally, MLN4924 enhanced PARP, caspase -3, and -7 cleavages, which caused apoptosis. Furthermore, MLN4924 inhibited VEGF-induced angiogenesis in Matrigel-plug assay and repressed UC growth. CD31 and α-SMA immuno-staining was used for revealing the tumor vascularity, which showed that the microvessel density had no significantly difference between MLN4924-treated tumors and control tumors. However, the higher maturation vasculatures were found in MLN4924-treated tumors than control tumors. Conclusion: Our data suggest that MLN4924 inhibits the angiogenic phenotype of endothelial cells for enhancing tumor vascular normalization, which alleviates the hypoxic stress for slowing tumor growth. In addition, due to MLN4924 resulting in vascular normalization, MLN4924 may be able to synergize with conventional chemotherapies for treating UC. Therefore, exploring the function of MLN4924 in angiogenesis may result in a new perspective on targeting tumors and angiogenesis-dependent diseases. Note: This abstract was not presented at the meeting. Citation Format: Kuo-How Huang, Yeong-Shiau Pu, Yu-Chieh Tsai, Kuan-Lin Kuo, I-Lin Ho, Ju-Ton Hsieh, Tsung-Hsien Shih. Anti-angiogenic activity of MLN4924 suppresses the growth of urothelial carcinoma via enhancing vascular normalization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1028. doi:10.1158/1538-7445.AM2014-1028

Overactivated Neddylation Pathway as a Therapeutic Target in Lung Cancer

A number of oncoproteins and tumor suppressors are known to be neddylated, but whether the neddylation pathway is entirely activated in human cancer remains unexplored. NEDD8-activating enzyme (NAE) (E1) and NEDD8-conjugating enzyme (E2) expression and global-protein neddylation were examined by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction analysis. Cell proliferation, clonogenic survival, migration, and motility in vitro, as well as tumor formation and metastasis in vivo, were determined upon neddylation inhibition by MLN4924, an investigational NEDD8-activating enzyme inhibitor. Survival was analyzed with Kaplan-Meier methods and compared by the log-rank test. All statistical tests were two-sided. The entire neddylation pathway, including NEDD8-activating enzyme E1, NEDD8-conjugating enzyme E2, and global-protein neddylation, is overactivated in both lung adenocarcinoma and squamous-cell carcinoma. Compared with lung adenocarcinoma patients with low expression, those with high expression had worse overall survival (NEDD8-activating enzyme E1 subunit 1 [NAE1]: hazard ratio [HR] = 2.07, 95% confidence interval [CI] = 0.95 to 4.52, P = .07; ubiquitin-conjugating enzyme E2M (UBC12): HR = 13.26, 95% CI = 1.77 to 99.35, P = .01; global protein neddylation: HR = 3.74, 95% CI = 1.65 to 8.47, P = .002). Moreover, inhibition of neddylation by the NAE inhibitor MLN4924 statistically significantly suppressed proliferation, survival, migration, and motility of lung cancer cells in vitro and tumor formation and metastasis in vivo. At the molecular level, MLN4924 inactivated Cullin-RING E3 ligases, led to accumulation of tumor-suppressive Cullin-RING E3 ligase substrates and induced phorbol-12-myristate-13-acetate-induced protein 1 (NOXA)-dependent apoptosis or cellular senescence. Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Activation of histone deacetylase 2: a novel strategy for reversing vascular dysfunction in atherogenesis (671.9)

Although they are targeted by therapies for cancer, the implications of epigenetic modifications of histone tails by Histone Deacetylases (HDACs) for vascular homeostasis and cardiovascular diseases including atherosclerosis, diabetes and hypertension are unclear. The use of HDAC inhibitors in the treatment of cardiovascular disease is controversial, since the global HDAC inhibitor Trichostatin (TSA) was shown to accelerate atheroma formation in LDL‐receptor knockout mice.We have demonstrated that TSA causes impairment (~40%) of endothelium‐dependent vascular relaxation in isolated vascular rings. Using class‐specific biochemical HDAC inhibitors and siRNA‐mediated gene knockdown, we have identified HDAC2 as a specific, critical regulator of homeostasis in the vascular endothelium. We have also identified Arginase 2 (Arg2) as a major transcriptional target for HDAC2 that contributes to intimal dysfunction, by showing that vascular dysfunction mediated by HDAC inhibition was abolished by either pharmacologic inhibiton or genetic knockout of Arg2. Finally, inhibition of neddylation, a post‐translational modification that enhances ubiquitin‐mediated proteosomal degradation, increased the abundance of HDAC2 and caused decreased Arg2 activity. Hence, we hypothesize that atherogenic stimuli such as OxLDL and inflammation downregulate HDAC2 expression in vascular EC through NEDD8‐mediated enhancement of the proteosomal degradation of HDAC2. This reduction in HDAC2 results in the following cascade: increased Arg2 expression in EC, decreased L‐arginine concentration, impaired NO bioavailability via eNOS substrate depletion, and increased ROS. These events culminate in EC dysfunction and atherogenesis. In conclusion, activation of HDAC2 by pharmacologic agents or small molecule activators could serve as a potent strategy in an atheroprotective therapeutic regimen.Grant Funding Source: NIH

Reactive oxygen species/neddylation is a novel pathway of transforming growth factor‐β‐induced smooth muscle cell differentiation (952.2)

Smooth muscle cell (SMC) differentiation is an essential process in cardiovascular development. SMCs can be differentiated from pluripotent mesenchymal progenitors by transforming Growth Factor‐β (TGF‐β) while ubiquitin‐mediated protein degradation blocks SMC differentiation. Neural precursor cell‐Expressed Developmentally Down‐regulated 8 (NEDD8), a member in ubiquitin family, promotes protein stability and prevents protein degradation by conjugating to target proteins. We found that NEDD8/neddylation is essential for TGF‐β‐induced SMC differentiation. NEDD8 protein is increased during TGF‐β‐induced SMC differentiation and conjugates to smooth muscle α‐actin (α‐SMA). Blockade of neddylation by neddylation inhibitor (MLN‐4924) or NEDD8 short hairpin interfering RNA significantly inhibited the expression of α‐SMA and calponin (CNN) in TGF‐β‐treated cells. We found that reactive oxygen species (ROS) attenuation by SOD2 overexpression blocked TGF‐β‐induced NEDD8 conjugation to α‐SMA. SOD2 appeared to block neddylation through inhibiting expression of Casitas B‐lineage lymphoma (c‐Cbl), suggesting that ROS/c‐Cbl mediates TGF‐β‐mediated neddylation of α‐SMA and CNN. NEDD8/neddylation appears to enhance α‐SMA and CNN stability and prevent proteasome‐mediated degradation because administration of proteasome inhibitor (MG‐132) significantly inhibited α‐SMA and CNN protein degradation in neddylation‐depleted cells. Taken together, our study demonstrates that NEDD8‐mediated neddylation through ROS/c‐Cbl is a novel molecular event critical for TGF‐β‐induced SMC differentiation.Grant Funding Source: NIH‐R01

520 MLN4924, a neddylation inhibitor, synergistically enhances cisplatin-induced cytotoxicity in human urothelial carcinoma

520 MLN4924, a neddylation inhibitor, synergistically enhances cisplatin-induced cytotoxicity in human urothelial carcinoma