Abstract B136: NEDD8-activating enzyme inhibitor TAS4464 inhibits tumor growth in endometrial cancer mouse model accompanied by DNA damage response
Authors: Komiya Yu , Yoshimura Chihoko , Muraoka Hiromi , Ohkubo Shuichi , Nakagawa Fumio , Ochiiwa Hiroaki , Sootome Hiroshi , Mizutani Takashi , Matsuo Kenichi , Utsugi Teruhiro , Iwasawa Yoshikazu 
Published: August 2018

Abstract
Background: Patients with unresectable or metastatic endometrial cancer (EMC) have a poor prognosis. Despite the use of platinum-based chemotherapy in the treatment of EMC, more effective therapy is urgently needed for treatment of unresectable or metastatic cancers. We have shown that TAS4464, a highly selective and the most potent inhibitor of NEDD8-activating enzyme (NAE) to date, induced apoptosis in various cancer cell lines. Here, we evaluated the therapeutic potential of TAS4464 in a preclinical EMC xenograft model and delineated how NAE inhibition leads to antitumor activity in EMC. Materials and Methods: The effect of TAS4464 on cell growth was evaluated in twenty-four EMC cell lines. Cytotoxicity to patient-derived EMC cells was examined at Fukushima Medical University (Japan). The effects of TAS4464 on protein expression and phosphorylation status were evaluated by using Western blotting. To identify the factor responsible for TAS4464-induced DNA damage, RNAi knock-down experiment was performed. The antitumor activity of TAS4464 was evaluated in a HEC-59, EMC xenograft mouse model. Results: TAS4464 markedly inhibited cell growth and induced cell death in all of the human EMC cell lines examined. Evaluation of the cytotoxicity of TAS4464 in platinum-refractory patient-derived EMC cells revealed that TAS4464 inhibited growth in these cells. Treatment with TAS4464 at 100 nmol/L for 24 hours resulted in accumulation both in the S phase of the cell cycle and the fraction containing &gt;4N DNA in all the evaluated EMC cells such as HEC-1-B and HEC-59. Further treatment with TAS4464 for additional 48 hours induced sub-G1 accumulation. Because NAE inhibition inactivates cullin-RING ubiquitin ligases (CRLs), TAS4464 treatment induced substrates of CRLs related to cell cycle control such as c-Myc, p21, p27, and Wee1, and DNA replication such as CDT1 and CDC6 within 8 hours accompanied by phosphorylation of DNA checkpoint-related proteins such as RPA32 and CHK1. Phospho-γH2AX and cleaved-caspase 3 were observed at 48 hours treatment with TAS4464. These data indicate that TAS4464 induced single-strand DNA break first, and then caused double-strand DNA break and apoptosis in EMC cells. In RNAi knockdown among TAS4464-induced substrates of CRLs, CDT1 siRNA pretreatment dramatically suppressed appearance of TAS4464-meadiated &gt;4N DNA fraction and DNA double-strand breaks. These data suggest that induced-CDT1, a licensing factor for DNA replication, increase in &gt;4N DNA fraction by DNA rereplication and results in DNA damage in EMC cells. Once- or twice-a-week administration of TAS4464 at a dose of 100 mg/kg for 3 weeks resulted in tumor growth inhibition (TGI) of 79% and 87% in the HEC-59 xenograft model, respectively, compared with single-dose administration of carboplatin at a dose of 100 mg/kg that resulted in a TGI of only 67% with significant weight loss. Conclusion: TAS4464 was broadly active against EMC cell lines accompanied by DNA damage. TAS4464 also inhibited tumor growth in an EMC xenograft model. These results suggest that TAS4464 deserves further research and development as a novel anticancer agent for EMC.
Citation Format: Yu Komiya, Chihoko Yoshimura, Hiromi Muraoka, Shuichi Ohkubo, Fumio Nakagawa, Hiroaki Ochiiwa, Hiroshi Sootome, Takashi Mizutani, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. NEDD8-activating enzyme inhibitor TAS4464 inhibits tumor growth in endometrial cancer mouse model accompanied by DNA damage response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B136.