Abstract 3777: TAS4464, a novel and highly potent NEDD8 activating enzyme (NAE) inhibitor, causes apoptosis of sarcomas via cell cycle dysregulation
Authors: Fujita Hidenori , Fujioka Yayoi , Ishida Keiji , Yoshimura Chihoko , Hashimoto Akihiro , Tsuji Shingo , Mizutani Takashi , Okubo Shuichi , Matsuo Kenichi , Utsugi Teruhiro , Iwasawa Yoshikazu 
Published: July 2016

Abstract
Background
Soft-tissue sarcomas (STSs) are heterogeneous tumors that comprise approximately 1% of adult cancers. Patients with advanced STS have a poor prognosis because few chemotherapy options are available.
NAE catalyzes the first step in the NEDD8 conjugation (neddylation) pathway. Because it activates cullin-RING ligase complexes (CRLs) and thus is essential in cancer cell homeostasis, NAE is a promising target for cancer therapy. Here, we investigated the potency of the NAE inhibitor TAS4464 for various STS cell lines.
Material and methods
Cytotoxicity was evaluated through ATP-Based assay. The effects of TAS4464 on NEDD8 conjugation and CRL substrates were evaluated by Western analysis. Cell cycle progression was analyzed by using flow cytometry. Small interfering RNAs (siRNAs) were lipofected into a clear cell sarcoma (CCS) line (SU-CCS-1). The antitumor activity of intravenous TAS4464 was evaluated in xenograft models of the aforementioned CCS and 2 rhadomyosarcoma (RMS) lines (SJCRH30 and RD).
Results
TAS4464 suppressed cell growth and induced cell death in various STS cell lines at lower concentrations than did an investigational NAE inhibitor, MLN4924, and a conventional STS treatment agent, doxorubicin (DXR). Notably, the GI50 values of TAS4464 were less than 10 nM in the tested RMS and CCS cell lines. TAS4464 treatment led to the elimination of cullin neddylation, accumulation of CRL substrate proteins (CDT1, p27, and p21), S phase arrest, and ultimately apoptosis in these STS cell lines. Furthermore, knockdown of CRL substrate proteins by siRNAs markedly attenuated TAS4464-induced cytotoxicity in STS cells. These results suggest that TAS4464-induced cell death was triggered by cell cycle dysregulation based on accumulation of CRL substrate proteins following NAE inhibition.
TAS4464 also led to a decrease in cullin neddylation and accumulation of CRL substrate proteins in RMS and CCS lines subcutaneous xenografts. Weekly administration of TAS4464 (100 mg/kg, IV) completely suppressed tumor growth in mice bearing subcutaneous RD and SJCRH30 xenografts. The effects of TAS4464 significantly exceeded that of DXR and MLN4924. Furthermore, TAS4464 induced tumor regression of ∼50% in the pazopanib, a drug approved for STS, insensitive SU-CCS-1 xenograft model.
Conclusion
TAS4464 prominently inhibited cell growth and induced apoptosis in RMS and CSC cells through cell cycle dysregulation and demonstrated impressive antitumor activities in STS xenograft models that respond poorly to DXR and pazopanib. Therefore, TAS4464 may become a valuable therapeutic option for patients with advanced STS.
Citation Format: Hidenori Fujita, Yayoi Fujioka, Keiji Ishida, Chihoko Yoshimura, Akihiro Hashimoto, Shingo Tsuji, Takashi Mizutani, Shuichi Okubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent NEDD8 activating enzyme (NAE) inhibitor, causes apoptosis of sarcomas via cell cycle dysregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3777.