In the present study, we have demonstrated the following: (1) MLN4924 causes dose- and time-dependent accumulation of TOP1 and blocks TOP1 ubiquitination; (2) MLN4924 effectively enhances the suppression of cell growth, migration, and apoptosis by 10-HCPT in HNSCC cells; and (3) the combination of MLN4924 and 10-HCPT may function via the NFKB1 pathway, which would partially explain the induction of apoptosis caused by NF-κB inactivation for IkBα accumulation. This evidence concerns the gene TOP1 and head and neck squamous cell carcinoma.