pevonedistat and Carcinoma--Squamous-Cell

Topics: Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cyclopentanes; Drug Screening Assays, Antitumor; Endopeptidases; Endosomal Sorting Complexes Required for Transport; F-Box Proteins; Gene Knockdown Techniques; Humans; Molecular Targeted Therapy; Nuclear Proteins; Pyrimidines; RNA, Small Interfering; Skin Neoplasms; Ubiquitin Thiolesterase; Ubiquitin-Activating Enzymes

Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation trienzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.

Topics: Amides; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cullin Proteins; Cyclopentanes; Drug Design; Fibroblasts; Glycine; Humans; Intracellular Signaling Peptides and Proteins; NEDD8 Protein; Protein Domains; Protein Interaction Mapping; Pyrazoles; Pyridones; Pyrimidines; Reactive Oxygen Species; Structure-Activity Relationship; Ubiquitin-Conjugating Enzymes

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure-activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1-UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1-UBE2M protein-protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Crystallography, X-Ray; Cullin Proteins; Drug Discovery; Drug Screening Assays, Antitumor; High-Throughput Screening Assays; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Models, Molecular; Molecular Conformation; NEDD8 Protein; Protein Binding; Proteins; Proto-Oncogene Proteins; Signal Transduction; Structure-Activity Relationship; Ubiquitin-Conjugating Enzymes

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.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclopentanes; Flow Cytometry; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; Lung Neoplasms; Molecular Targeted Therapy; NEDD8 Protein; Pyrimidines; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumor Stem Cell Assay; Ubiquitins

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective cytokine with potential anticancer activity and is currently under clinical testing. Head and neck squamous cell carcinoma (HNSCC), like other cancer types, exhibits varied sensitivity to TRAIL. MLN4924 is a newly developed investigational small molecule inhibitor of NEDD8-activating enzyme with potent anticancer activity. This study reveals a novel function of MLN4924 in synergizing with TRAIL to induce apoptosis in HNSCC cells. MLN4924 alone effectively inhibited the growth of HNSCC cells and induced apoptosis. When combined with TRAIL, synergistic effects on decreasing the survival and inducing apoptosis of HNSCC cells occurred. MLN4924 decreased c-FLIP levels without modulating death receptor 4 and death receptor 5 expression. Enforced expression of c-FLIP substantially attenuated MLN4924/TRAIL-induced apoptosis. Thus c-FLIP reduction plays an important role in mediating MLN4924/TRAIL-induced apoptosis. Moreover, MLN4924 decreased c-FLIP stability, increased c-FLIP ubiquitination, and facilitated c-FLIP degradation, suggesting that MLN4924 decreases c-FLIP levels through promoting its degradation. MLN4924 activated c-jun-NH(2)-kinase (JNK) signaling, evidenced by increased levels of phospho-c-Jun in MLN4924-treated cells. Chemical inhibition of JNK activation not only prevented MLN4924-induced c-FLIP reduction, but also inhibited MLN4924/TRAIL-induced apoptosis, suggesting that JNK activation mediates c-FLIP downregulation and subsequent enhancement of TRAIL-induced apoptosis by MLN4924. Because knockdown of NEDD8 failed to activate JNK signaling and downregulate c-FLIP, it is likely that MLN4924 reduces c-FLIP levels and enhances TRAIL-induced apoptosis independent of NEDD8 inhibition.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Squamous Cell; CASP8 and FADD-Like Apoptosis Regulating Protein; Cyclopentanes; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Synergism; Head and Neck Neoplasms; Humans; Proteolysis; Pyrimidines; Squamous Cell Carcinoma of Head and Neck; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Ubiquitin-Activating Enzymes; Ubiquitination; Up-Regulation