pevonedistat and Disease-Models--Animal

Topics: Alcoholism; Animals; Central Nervous System Depressants; Central Nervous System Sensitization; Cullin Proteins; Cyclopentanes; Disease Models, Animal; Enzyme Inhibitors; Ethanol; Hyperalgesia; Inflammation; Locomotion; Male; Mice; Mice, Inbred BALB C; NEDD8 Protein; Pyrimidines

Chronic pancreatitis (CP) is characterized by a wide range of irreversible fibro-inflammatory diseases with largely ambiguous pathogenesis. Although neddylation pathway has been implicated in regulating immune responses, whether the dysregulation of neddylation is involved in the progression of CP and how neddylation regulates the inflammatory microenvironment of CP have not yet been reported. Here, we demonstrate that global inactivation of neddylation pathway by MLN4924 significantly exacerbates chronic pancreatitis. The increased M2 macrophage infiltration, mediated by the upregulated chemokine (C-C motif) ligand 5 (CCL5), is responsible for the enhanced pancreatitis-promoting activity of MLN4924. Both CCL5 blockade and macrophage depletion contribute to alleviating pancreatic fibrosis and inflammation in MLN4924-treated CP mice. Mechanistic investigation identifies that inactivation of Cullin-RING ligases (CRLs) stabilizes cellular levels of hypoxia-inducible factor 1α (HIF-1α), which increases CCL5 expression by promoting CCL5 transactivation. Clinically, UBE2M expression remarkably decreases in human CP tissues compared with normal specimens and the levels of CCL5 and M2 marker CD163 are negatively correlated with UBE2M intensity, suggesting that neddylation is involved in the pathogenesis of pancreatitis. Hence, our studies reveal a neddylation-associated immunopathogenesis of chronic pancreatitis and provide new ideas for the disease treatment.

Topics: Animals; Cell Line; Chemokine CCL5; Chemotaxis; Cyclopentanes; Disease Models, Animal; Enzyme Inhibitors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Macrophages; Mice, Inbred C57BL; Pancreas; Pancreatitis, Chronic; Phenotype; Pyrimidines; Signal Transduction; Ubiquitin-Activating Enzymes; Ubiquitination

Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Genetic suppression or pharmacological inhibition of this pathway using MLN4924 caused cell death not only in mouse SCLC cell lines but also in patient-derived xenograft (PDX) models of pulmonary and extrapulmonary small cell carcinoma treated ex vivo or in vivo. A subset of PDX models were exceptionally sensitive to neddylation inhibition. Neddylation inhibition suppressed expression of major regulators of neuroendocrine cell state such as INSM1 and ASCL1, which a subset of SCLC rely upon for cell proliferation and survival. To identify potential mechanisms of resistance to neddylation inhibition, we performed a genome-scale CRISPR/Cas9 suppressor screen. Deletion of components of the COP9 signalosome strongly mitigated the effects of neddylation inhibition in small cell carcinoma, including the ability of MLN4924 to suppress neuroendocrine transcriptional program expression. This work identifies neddylation as a regulator of neuroendocrine cell state and potential therapeutic target for small cell carcinomas.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Small Cell; Cell Death; Cell Line, Tumor; COP9 Signalosome Complex; Cyclopentanes; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Lung Neoplasms; Mice; NEDD8 Protein; Neuroendocrine Cells; Proteins; Pyrimidines; Repressor Proteins; Sequence Deletion

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Neovascular age-related macular degeneration (nAMD), featured as choroidal neovascularization (CNV), can cause blindness in the elderly population. MLN4924, a highly selective small-molecule inhibitor of NEDD8 (neuronal precursor cell-expressed developmentally down-regulated protein 8)-activating enzyme (NAE), inhibits the proliferation, angiogenesis and inflammation of multiple cancers via up-regulating hedgehog pathway-regulated autophagy. MLN4924 intraperitoneal injection mitigated the leakage, area and volume of mouse laser-induced CNV lesion. Additionally, compared to CNV 7 d group, MLN4924 treated mouse retina-retinal pigment epithelium (RPE)-choroid complex showed decreased expression of hedgehog pathway-associated molecules patched 1 (PTCH1), smoothened (SMO), GLI family zinc finger 1 (GLI1) and GLI family zinc finger 2 (GLI2) with increased expression of autophagy-associated molecules sequestosome 1 (p62) and LC microtubule-associated protein 1 light chain 3 (LC3). Meanwhile, human choroidal endothelial cells (HCECs) exposed to hypoxia condition also showed decreased expression of hedgehog pathway-associated molecules and increased expression of autophagy-associated molecules. Compared to hypoxia + MLN4924 group, SMO agonist SAG up-regulated hedgehog pathway and down-regulated autophagy, whereas autophagy inhibitor PIK-III inhibited autophagy with no effect on hedgehog pathway, indicating that MLN4924 facilitated autophagy of HCECs via hindering hedgehog pathway under hypoxia condition. Finally, MLN4924 inhibited proliferation, migration and tube formation of HCECs via boosting hedgehog pathway-regulated autophagy. In summary, MLN4924 relieved the formation of mouse laser-induced CNV lesion might via up-regulating hedgehog pathway-regulated autophagy. The results provide a potential interfering strategy for nAMD targeting the autophagy of choroidal endothelial cells.

Topics: Animals; Autophagy; Biomarkers; Choroidal Neovascularization; Cyclopentanes; Disease Management; Disease Models, Animal; Disease Susceptibility; Endothelial Cells; Hedgehog Proteins; Lasers; Mice; Pyrimidines; Retinal Pigment Epithelium; Signal Transduction

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-1β, 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.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Line; Chemokine CXCL1; Cyclopentanes; Disease Models, Animal; Enzyme Inhibitors; Humans; Hypertension, Pulmonary; Interleukin-17; Interleukin-1beta; Interleukin-6; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pneumonia; Pulmonary Disease, Chronic Obstructive; Pyrimidines; TNF Receptor-Associated Factor 6; Ubiquitination

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.

Topics: Animals; Animals, Newborn; Cell Proliferation; Cells, Cultured; Cyclopentanes; Disease Models, Animal; Heart Failure; Hypertrophy, Left Ventricular; Isoproterenol; Myocytes, Cardiac; NEDD8 Protein; Pyrimidines; Rats, Sprague-Dawley; Ubiquitin-Conjugating Enzymes; Ubiquitination; Ventricular Function, Left; Ventricular Remodeling

Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin-like posttranslational modification of neddylation, that conjugates Nedd8 (neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation- and CCl. Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694-709).

Topics: Aging; Analysis of Variance; Animals; Apoptosis; Biopsy, Needle; Cell Proliferation; Cell Survival; Cells, Cultured; Chemokine CCL4; Chemokines; Cyclopentanes; Disease Models, Animal; Hepatic Stellate Cells; Humans; Immunohistochemistry; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; NEDD8 Protein; Pyrimidines; Random Allocation; Signal Transduction; Ubiquitins

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.. 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.. 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.. 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.

Topics: Activating Transcription Factor 4; Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Biomarkers; Caspase 8; Cell Line, Tumor; Cyclopentanes; Disease Models, Animal; Esophageal Neoplasms; Gene Silencing; Humans; Mice; Models, Biological; Prognosis; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA Interference; Signal Transduction; Transcription Factor CHOP; Xenograft Model Antitumor Assays

Neddylation, a newly identified post-translational modification, is significant for the activity and stability of target proteins. The exact role of neddylation in the pathogenesis of inflammatory bowel disease, specifically those mediated by dendritic cells (DCs), was still rarely reported. Here, we showed that inhibition of neddylation protected mice from mucosal inflammation. Targeting neddylation also inhibited DC maturation characterized by reduced cytokine production, down-regulated costimulatory molecules and suppressed capacity in allogeneic T cell stimulation. Additionally, inactivation of neddylation promotes caspase dependent apoptosis of DCs. These phenomena were attributed to the inactivation of mTOR, which was caused by Cullin-1 deneddylation induced Deptor accumulation. Together, our findings revealed that neddylation inhibition suppressed DC functions through mTOR signaling pathway and provided a potential therapeutic opportunity in inflammatory bowel diseases.

Topics: Animals; Apoptosis; Caspase 3; Caspase 7; Cell Differentiation; Cells, Cultured; Cullin Proteins; Cyclopentanes; Cytokines; Dendritic Cells; Disease Models, Animal; Down-Regulation; Enzyme Inhibitors; Humans; Inflammatory Bowel Diseases; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Protein Processing, Post-Translational; Pyrimidines; Signal Transduction; TOR Serine-Threonine Kinases

The cullin-based CRL4-CDT2 ubiquitin ligase is emerging as a master regulator of cell proliferation. CRL4-CDT2 prevents re-initiation of DNA replication during the same cell cycle "rereplication" through targeted degradation of CDT1, SET8 and p21 during S-phase of the cell cycle. We show that CDT2 is overexpressed in cutaneous melanoma and predicts poor overall and disease-free survival. CDT2 ablation inhibited a panel of melanoma cell lines through the induction of SET8- and p21-dependent DNA rereplication and senescence. Pevonedistat (MLN4924), a specific inhibitor of the NEDD8 activating enzyme (NAE), inhibits the activity of cullin E3 ligases, thereby stabilizing a vast number of cullin substrates and resulting in cancer cell inhibition in vitro and tumor suppression in nude mice. We demonstrate that pevonedistat is effective at inhibiting the proliferation of melanoma cell lines in vitro through the induction of rereplication-dependent permanent growth arrest as well as through a transient, non-rereplication-dependent mechanism. CRISPR/Cas9-mediated heterozygous deletion of CDKN1A (encoding p21) or SET8 in melanoma cells demonstrated that the rereplication-mediated cytotoxicity of pevonedistat is mediated through preventing the degradation of p21 and SET8 and is essential for melanoma suppression in nude mice. By contrast, pevonedistat-induced transient growth suppression was independent of p21 or SET8, and insufficient to inhibit tumor growth in vivo. Pevonedistat additionally synergized with the BRAF kinase inhibitor PLX4720 to inhibit BRAF melanoma, and suppressed PLX4720-resistant melanoma cells. These findings demonstrate that the CRL4-CDT2-SET8/p21 degradation axis is the primary target of inhibition by pevonedistat in melanoma and suggest that a broad patient population may benefit from pevonedistat therapy.. The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4(CDT2) by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4(CDT2) substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4(CDT2) therapeutically.

Topics: Animals; Antineoplastic Agents; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Cyclopentanes; Disease Models, Animal; Female; Gene Expression; Gene Silencing; Genes, ras; Histone-Lysine N-Methyltransferase; Humans; Kaplan-Meier Estimate; Melanoma; Mice; Mutation; Nuclear Proteins; Prognosis; Protein Stability; Proto-Oncogene Proteins B-raf; Pyrimidines; RNA Interference; Ubiquitin-Protein Ligases; Ubiquitination; Xenograft Model Antitumor Assays

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.

Topics: Animals; Cell Line; Cullin Proteins; Cyclopentanes; Disease Models, Animal; Endopeptidases; Gene Knockdown Techniques; HeLa Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammatory Bowel Diseases; Intestinal Mucosa; Metabolic Networks and Pathways; Mice, Inbred C57BL; NEDD8 Protein; Protease Inhibitors; Protein Stability; Pyrimidines; Ubiquitins