Background: Patients with high-risk and relapsed neuroblastoma have a poor event free survival (EFS). Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, provide great possibilities for advancements in cure. Pevonedistat blocks the degradation of proteins that would normally be degraded by the 26S proteasome, the major mechanism of protein removal in the cell. Pevonedistat is more specific than previous proteasome inhibitors because it blocks the degradation of Cullin-RING ligases (CRL), narrowing the targets to only a handful of key regulatory proteins important in cell survival.
Methods: MTT assays were used to test the efficacy of pevonedistat. Flow-cytometry was used to determine cell-cycle arrest, presence of DNA rereplication, and apoptosis after staining with Annexin V/PI. Whole cell extracts were analyzed by immunoblotting from cells treated with pevonedistat for proteins targeted in neddylation. Orthotopic xenografts of human neuroblastoma were generated. Mice were randomized in three treatment groups of ten mice each: vehicle control, pevonedistat 50 mg/kg, and MLN4924 100 mg/kg. All drugs were given i.p.daily 6 days/week for 2 weeks. After 4 weeks tumor weights were compared using Kruskal-Wallis analysis.
Results: The IC50 of pevonedistat in neuroblastoma cell lines was 200-500 nM. and unaffected by N-myc or p53 status. After treatment with pevonedistat, p53 mutant neuroblastoma cell lines undergo rereplication and cell-cycle arrest in G2-M, while p53 wild-type cell lines exhibit cell-cycle arrest in G0-G1. In treated cells proteins involved in DNA rereplication are increased in p53 mutant compared to wild-type cell lines. Control mice had an average tumor weight of 1.6mg+0.8mg versus mice treated with pevonedistat 0.5mg+0.4mg (p<0.05).
Conclusion: Neddylation affects proteins involved in DNA replication. The mechanism of action of pevonedistat in neuroblastoma cell lines appears to be p53 dependent. In our neuroblastoma xenograft experiments there was a significant decrease in tumor weight between control and pevonedistat treated mice.