Abstract C178: TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia
Authors: Ochiiwa Hiroaki , Yoshimura Chihoko , Muraoka Hiromi , Ishida Keiji , Haruma Tomonori , Tsuji Shingo , Hashimoto Akihiro , Mizutani Takashi , Ohkubo Shuichi , Matsuo Kenichi , Utsugi Teruhiro , Iwasawa Yoshikazu 
Published: May 2016

Abstract
BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by numerous genetic mutations and chromosomal abnormalities. Current chemotherapies have shown limited efficacy in AML patients, most of whom develop chemoresistance and relapse repeatedly. Because the expression of anti-apoptotic proteins reportedly correlates with poor prognosis in AML, the modulation of apoptosis-related proteins emerges as a promising treatment strategy.
Apoptosis occurs mainly via two signaling pathways. In the mitochondria-mediated intrinsic apoptotic pathway, the release of cytochrome c triggers caspase-9 activation, which subsequently activates effector caspases (e.g., caspase-3). In the death receptor-mediated extrinsic apoptotic pathway, the binding of death receptor ligands to their receptors activates caspase-8 and, in turn, effector caspases, as in the intrinsic pathway. NEDD8 activating enzyme (NAE) inhibitors, which act on the NEDD8 conjugation pathway, induce quantitative changes in the substrates of E3 cullin-RING ligases (CRLs) and cause apoptosis in AML.
Here, we describe the mechanism by which our highly potent and selective NAE inhibitor TAS4464 promotes apoptosis in AML.
MATERIALS AND METHODS: Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation, the protein levels of CRL substrates, and the activation of various caspases were evaluated by means of Western blotting. The transcription levels of the Noxa and c-FLIP genes were assessed by using qRT-PCR. The antitumor activity and in vivo pharmacodynamic activity of TAS4464 were evaluated in a THP-1 xenograft model.
RESULTS: TAS4464 led to cell growth arrest and cell death in AML cell lines with various genetic backgrounds, including the MLL-AF9 fusion gene and FLT3-ITD mutation. TAS4464 activated both caspase-9 (intrinsic apoptotic pathway) and caspase-8 (extrinsic apoptotic pathway); combined treatment with inhibitors of these caspases markedly diminished the TAS4464-associated induction of apoptosis.
By analyzing apoptosis-related factors in each apoptotic pathway, we found that TAS4464 increased the pro-apoptotic factor Noxa protein (intrinsic apoptotic pathway) and decreased the anti-apoptotic factor c-FLIP protein (extrinsic apoptotic pathway) with changes evident at the mRNA transcriptional level. In addition, treatment with TAS4464 led to accumulation of the CRL substrate c-Myc, and c-Myc knockdown by siRNA ablated the TAS4464-induced decrease in cFLIP protein.
Intravenous administration of TAS4464 on day 1, 3 and 5 for 3 weeks resulted in tumor growth inhibition (TGI) of 100% including complete response in human THP-1 xenograft model, compared with twice-weekly administration of cytarabine that resulted in a TGI of only 6%. Pharmacodynamics analysis revealed that TAS4464 inhibited cullin neddylation and induced activation of caspases in THP-1 xenografted tumor.
CONCLUSION: TAS4464 exerts a strong apoptosis-inducing effect in AML cell lines via both intrinsic and extrinsic apoptotic pathway by modulating apoptosis-related proteins. In addition, TAS4464 demonstrates marked antitumor activity in the THP-1 xenograft model. Given its potent preclinical activities, TAS4464 is a promising agent for treating AML.
Citation Format: Hiroaki Ochiiwa, Chihoko Yoshimura, Hiromi Muraoka, Keiji Ishida, Tomonori Haruma, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel NEDD8 activating enzyme inhibitor, potently induces cell death via both intrinsic and extrinsic apoptotic pathways in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C178.