Abstract C177: TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway
Authors: Muraoka Hiromi , Yoshimura Chihoko , Tsuji Shingo , Hashimoto Akihiro , Mizutani Takashi , Ohkubo Shuichi , Matsuo Kenichi , Utsugi Teruhiro , Iwasawa Yoshikazu 
Published: May 2016

Abstract
BACKGROUND: NEDD8 activating enzyme (NAE) regulates NEDD8 conjugation in neddylation pathway, a part of the ubiquitin-proteasome system, and controls cell cycle progression, DNA replication, and signal transduction via activation of cullin-RING ligases (CRLs) and subsequent degradation of their substrates. Through one such pathway, the constitutively active nuclear factor κB (NF-κB) triggers the progression of some types of hematological cancer. Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) remains the least curable form of DLBCL despite recent therapeutic advances. Because either phosphatidylinositol-3 kinase (PI3K)/AKT signaling or NF-κB signaling constitutively activated by B cell receptor signaling and MYD88 are reported to promote cancer cell growth and survival in ABC-DLBCL, the key kinases in these pathways [PI3Kδ, Bruton's tyrosine kinase (BTK), and Interleukin-1 receptor-associated kinases (IRAK)] are considered to be potential targets for ABC-DLBCL therapy. We previously reported an NAE inhibitor, TAS4464, that could inactivate NF-κB by accumulating phosphorylated IκBα (p-IκBα). Here, we report on the possible application of TAS4464 to treat ABC-DLBCL that is insensitive to kinase inhibitors.
MATERIALS AND METHODS: Human ABC-DLBCL cell lines, OCI-LY10, SU-DHL-2, and TMD8 were used to evaluate the biological activity of TAS4464. Intracellular ATP levels were measured to assess in vitro cell growth. The effects of TAS4464 on NEDD8 conjugation and levels of CRLs substrates were evaluated by Western blot analysis. The levels of NF-κB-targeted gene transcripts were assessed by qRT-PCR. The antitumor activities of TAS4464 were evaluated in both subcutaneous and systemic xenograft model of TMD8.
RESULTS: TAS4464 led to growth arrest and death in ABC-DLBCL cell lines in the nanomolar range through inhibition of cullin neddylation. Similar effects were also observed in a cell line insensitive to the kinase inhibitors that inhibit divergent activator of NF-κB. qRT-PCR analysis revealed TAS4464 inhibited the NF-κB pathway in all ABC-DLBCL cell lines tested through accumulation of p-IκBα, which directly inhibit NF-κB activity. Intravenous administration of TAS4464 showed strong antitumor activity in a TMD8 xenograft model, with inhibited cullin neddylation and activation of caspases 3 and 8 in the tumors. Futhermore, in a TMD8 systemic model, intravenous administration of TAS4464 prolonged mice survival more than two-fold. CONCLUSION: TAS4464 leads to cell growth through inhibition of NF-κB pathway regardless of the activation signaling pathway of NF-κB pathway. Though NF-kB activation signaling is complicated, the downstream inhibition of NF-κB pathway by TAS4464 indicated the potential for treatment of ABC-DLBCL in which NF-kB was dominant for their survival. In addition, TAS4464 demonstrates marked antitumor activity and survival benefit in a human ABC-DLBCL model. Therefore, TAS4464 may be a valuable addition to current options for chemotherapy in ABC-DLBCL.
Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Shingo Tsuji, Akihiro Hashimoto, Takashi Mizutani, Shuichi Ohkubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. TAS4464, a novel and highly potent inhibitor of NEDD8 activating enzyme, overcomes insensitivity to BTK, PI3Kδ, and IRAK4 inhibitors in activated B-cell like diffuse large B-cell lymphoma via inactivation of the NF-κB pathway. [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 C177.