pevonedistat and Colorectal-Neoplasms

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Topics: Colorectal Neoplasms; Humans; Ligases; Proteasome Endopeptidase Complex; RNA-Binding Proteins; Topoisomerase I Inhibitors; Ubiquitin; Ubiquitin-Protein Ligases

Deficient DNA mismatch repair (dMMR) induces a hypermutator phenotype that can lead to tumorigenesis; however, the functional impact of the high mutation burden resulting from this phenotype remains poorly explored. Here, we demonstrate that dMMR-induced destabilizing mutations lead to proteome instability in dMMR tumors, resulting in an abundance of misfolded protein aggregates. To compensate, dMMR cells utilize a Nedd8-mediated degradation pathway to facilitate clearance of misfolded proteins. Blockade of this Nedd8 clearance pathway with MLN4924 causes accumulation of misfolded protein aggregates, ultimately inducing immunogenic cell death in dMMR cancer cells. To leverage this immunogenic cell death, we combined MLN4924 treatment with PD1 inhibition and found the combination was synergistic, significantly improving efficacy over either treatment alone.

Topics: Animals; Cell Line, Tumor; Colorectal Neoplasms; Cyclopentanes; DNA Mismatch Repair; Endometrial Neoplasms; Female; HCT116 Cells; Humans; Immunotherapy; Mice, Inbred C57BL; Mice, Transgenic; Microsatellite Instability; Mutation; NEDD8 Protein; Programmed Cell Death 1 Receptor; Protein Stability; Proteome; Pyrimidines; Xenograft Model Antitumor Assays

NEDD8-activating enzyme (NAE) is an essential player of the NEDD8 conjugation pathway that regulates protein degradation. Meanwhile, drug repurposing is a cost-efficient strategy to identify new therapeutic uses for existing scaffolds. In this report, mitoxantrone (1) was repurposed as an inhibitor of NAE by virtual screening of an FDA-approved drug database. Compound 1 inhibited NAE activity in cell-free and cell-based systems with high selectivity and was competitive with ATP. Furthermore, compound 1 induced apoptosis of colorectal adenocarcinoma cancer cells through inhibiting the degradation of the neddylation substrate p53.

Topics: Adenocarcinoma; Apoptosis; Caco-2 Cells; Cell Line; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Discovery; Drug Evaluation, Preclinical; Enzyme Inhibitors; Humans; Mitoxantrone; Models, Molecular; Molecular Structure; NEDD8 Protein; Structure-Activity Relationship; Tumor Suppressor Protein p53; Ubiquitin-Activating Enzymes

Oxaliplatin-based chemotherapy is a primary treatment for patients with metastatic colorectal cancer (CRC); however, its efficacy is limited. Therefore, novel therapeutic agents are urgently required. MLN4924 is a first‑in‑class inhibitor of neural precursor cell expressed, developmentally downregulated 8 (NEDD8)‑activating enzyme E1, and has entered various phase‑I/II clinical trials for cancer therapy due to its significant anticancer efficacy. The aim of the present study was to examine the synergistic effect and underlying mechanisms of MLN4924 and oxaliplatin combined treatment for CRC. It was demonstrated that MLN4924 treatment induced the DNA damage response (DDR) by inactivating cullin‑ring ubiquitin ligases, subsequently leading to cell cycle disturbance and apoptosis in CRC cells. MLN4924 treatment increased the oxaliplatin‑induced DDR, G2 cell cycle arrest and apoptosis. Protein expression levels of phosphorylated checkpoint kinase 2 (p‑CHK2), p21 and p53, which are well‑known functional proteins involved in G2 cell cycle arrest, were assessed. p‑CHK2 protein expression levels were increased following combined treatment with MLN4924 and oxaliplatin, whereas p21/p53 protein expression levels were not. In conclusion, MLN4924 treatment may sensitize CRC cells to oxaliplatin treatment by inducing the DDR and increasing protein expression levels of p‑CHK2, leading to G2 cell cycle arrest and apoptosis. Therefore, combined MLN4924 and oxaliplatin‑based chemotherapy may be a potential therapeutic strategy for the treatment of CRC.

Topics: Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Cyclopentanes; DNA Damage; G2 Phase Cell Cycle Checkpoints; Humans; NEDD8 Protein; Neoplasm Proteins; Organoplatinum Compounds; Pyridines; Pyrimidines

The NEDD8 conjugation pathway modulates the ubiquitination and activity of a wide range of intracellular proteins, and its blockade by pevonedistat is emerging as a promising therapeutic approach in various cancer settings. However, systematic characterization of pevonedistat efficacy in specific tumor types and definition of response predictors are still missing.. We investigated in vitro sensitivity to pevonedistat in 122 colorectal cancer (CRC) cell lines by an ATP-based proliferation assay and evaluated apoptosis and DNA content by flow cytometry. Associations between pevonedistat sensitivity and CRC molecular features were assessed by Student's t test. A 184-gene transcriptional predictor was generated in cell lines and applied to 87 metastatic CRC samples for which patient-derived xenografts (PDXs) were available. In vivo reponse to pevonedistat was assessed in PDX models (≥5 mice per group). All statistical tests were two-sided.. Sixteen (13.1%) cell lines displayed a marked response to pevonedistat, featuring DNA re-replication, proliferative block, and increased apoptosis. Pevonedistat sensitivity did not statistically significantly correlate with microsatellite instability or mutations in KRAS or BRAF and was functionally associated with low EGFR pathway activity. While ineffective on predicted resistant PDXs, in vivo administration of pevonedistat statistically significantly impaired growth of five out of six predicted sensitive models (P < .01). In samples from CRC patients, transcriptional prediction of pevonedistat sensitivity was associated with poor prognosis after surgery (hazard ratio [HR] = 2.49, 95% confidence interval [CI] = 1.34 to 4.62, P = .003) and early progression under cetuximab treatment (HR = 3.59, 95% CI = 1.60 to 8.04, P < .001). Histological and immunohistochemical analyses revealed that the pevonedistat sensitivity signature captures transcriptional traits of poor differentiation and high-grade mucinous adenocarcinoma.. These results highlight NEDD8-pathway inhibition by pevonedistat as a potentially effective treatment for poorly differentiated, clinically aggressive CRC.

Topics: Adenocarcinoma, Mucinous; Animals; Antineoplastic Agents; Apoptosis; Cadherins; CDX2 Transcription Factor; Cell Line, Tumor; Cell Proliferation; Cetuximab; Colorectal Neoplasms; Cyclopentanes; Disease-Free Survival; DNA Replication; Drug Resistance, Neoplasm; Female; Homeodomain Proteins; Humans; Keratin-20; Mice; NEDD8 Protein; Neoplasm Grading; Neoplasm Transplantation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Pyrimidines; Signal Transduction; Transcriptome; Ubiquitins

The COP9 signalosome (CSN) is a multi-protein complex that is highly conserved in eukaryotes. Due to its regulatory impact on processes such as cell cycle, DNA damage response and apoptosis, the CSN is essential for mammalian cells. One of the best-studied functions of the CSN is the deNEDDylation of cullin-RING ligases (CRLs) via its catalytically active subunit CSN5/JAB1, thereby triggering the degradation of various target proteins. CSN5 was found to be overexpressed in many human cancer entities, including colon adenocarcinoma. Overactivation of WNT signaling is known as a key step in colon cancer development. Recently, we found that depletion of CSN5 in colorectal cancer (CRC) cells affects WNT signaling by downregulation of β-catenin. To investigate changes in gene expression associated with the CSN5 knockdown, we performed a microarray using cDNA from the CRC cell line SW480. We found the WNT ligand WNT6 and the WNT inhibitors DKK1 and DKK4 differentially regulated in CSN5 knockdown cells. DKK1 expression and DKK1 protein levels depended on CSN5 in different CRC cell lines. In addition, DKK1 secretion was increased following CSN5 knockdown, affecting WNT signaling in SW480 cells. Consequently, blocking of secreted DKK1 in cell-conditioned media abolished β-catenin downregulation in SW480 cells, while treatment with recombinant DKK1 mimicked the CSN5 knockdown effect. Furthermore, knockdown of DKK1 was able to rescue the proliferative deficiency of CSN5 knockdown cells. We conclude that downregulation of WNT signaling in colorectal cancer cells resulting from CSN5 knockdown is mediated, at least in part, by elevated DKK1 secretion. Moreover, experiments with the NEDDylation inhibitor MLN-4924 indicated that DKK1 expression is regulated by a so far unidentified repressor, the stability of which could be controlled by a CSN-regulated CRL.

Topics: beta Catenin; Cell Line, Tumor; Colorectal Neoplasms; COP9 Signalosome Complex; Cyclopentanes; Down-Regulation; HCT116 Cells; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Peptide Hydrolases; Pyrimidines; Recombinant Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Wnt Proteins; Wnt Signaling Pathway

Colorectal cancer is the third most frequently diagnosed cancer and the combination of radiation with capecitabine has been shown to achieve only 15% to 25% of pathologic complete response. This study aimed to investigate the effect of MLN4924, a potent small molecule inhibitor of SKP1-Cullin-F-box proteins E3 ubiquitin ligases, as a novel radiosensitizing agent in colorectal cancer cells. Indeed, we found that MLN4924 effectively sensitized colorectal cancer cells to radiation with a sensitivity-enhancement ratio of 1.61 for HT-29 cells and 1.35 for HCT-116 cells. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest, apoptosis, and DNA damage response through accumulation of p27. Knockdown of p27 via small interfering RNA partially inhibited MLN4924-induced radiosensitization, indicating a causal role played by p27. Our study suggested that MLN4924 could be further developed as a novel radiosensitizing agent against colorectal cancer.

Topics: Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p27; Cyclopentanes; DNA Damage; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; NEDD8 Protein; Pyrimidines; Radiation-Sensitizing Agents; RNA, Small Interfering; Ubiquitins; X-Rays

MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA rereplication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, was found to be sufficient to induce DNA rereplication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence seems to be dependent on induction of p53 and its downstream effector p21(Waf1), we found that p53(-/-) and p21(-/-) cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, might be more important for the antiproliferative effect of MLN4924. Furthermore, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.

Topics: Apoptosis; Cell Cycle Proteins; Cellular Senescence; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclopentanes; DNA Replication; HCT116 Cells; Humans; Molecular Targeted Therapy; NEDD8 Protein; Pyrimidines; S Phase; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Ubiquitins