Abstract 1730: TAS4464, a novel highly potent NEDD8 activating enzyme inhibitor, demonstrates antitumor activity in multiple myeloma through the inactivation of NF-κB pathways

Background: NEDD8 activating enzyme E1 (NAE) regulates NEDD8 conjugation in the ubiquitin-proteasome system. NAE activates cullin-RING ligases (CRL) and controls the degradation of their substrates, which play key roles in cell cycle progression, DNA replication and signal transduction via various pathways. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of multiple myeloma (MM). MM is highly treatable but rarely curable, and patients with refractory MM would benefit from additional therapeutic options. NF-κB is activated through two signaling cascades, canonical and non-canonical pathways, which are regulated by the CRL substrates, phosphorylated IκBα (p-IκBα) and phosphorylated p100 (p-p100), respectively. Here, we report on the possible use of an NAE inhibitor that we discovered, TAS4464, to treat MM. Materials and methods: TAS4464 was designed and synthesized at Taiho Pharmaceutical Co., Ltd. Human MM cell lines, KMS-11, KMS-12-BM, KMS-26, KMS-34, and MM.1S were used to evaluate the biological activities of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. Effects on the cell cycle were analyzed by using fluorescence-activated cell sorting. The effects of TAS4464 on NEDD8 conjugation and the levels of CRL substrates were evaluated by means of Western blotting. The transcription levels of NF-κB-targeted genes were assessed by using qRT-PCR. Activated p65 and RelB (subunits of NF-κB) were quantified by using commercial DNA-binding ELISA kits. The antitumor activity of TAS4464 was evaluated in an MM.1S xenograft model. Results: TAS4464 inhibited cullin neddylation, leading to cell-growth arrest and apoptosis in MM cell lines in the nanomolar range. These effects were not attenuated after stimulation with interleukin 6 or insulin-like growth factor 1 or in co-culture with stroma cells. TAS4464 led to the accumulation of p-IκBα and p-p100 with subsequent suppression both p65 and RelB activity, whereas bortezomib suppressed only RelB activity. Once-weekly intravenous administration of TAS4464 showed much stronger antitumor activity than that of bortezomib in an MM.1S xenograft model, with inhibited cullin neddylation and induced the cleaved forms of caspases 3 and 8 in the tumors.Conclusion: TAS4464 demonstrated marked antitumor activity in a human MM xenograft model. In addition, TAS4464 inhibited canonical as well as non-canonical pathways of NF-κB, whereas bortezomib inhi