Abstract 3407: A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity.

Abstract

Abstract Centromere-associated protein-E (CENP-E) is a mitotic spindle motor protein belonging to the kinesin superfamily and controls chromosome alignment during metaphase by capturing the microtubule plus end at the kinetochore. Loss of CENP-E function has been reported to result in misaligned chromosomes at metaphase leading to spindle assembly checkpoint (SAC) activation. Here, we developed a novel small-molecule inhibitor of CENP-E (Compound-A), which targets an ATPase domain at the CENP-E N-terminal motor region. Enzymatic kinetics reveals that Compound-A is a slow-off rate type of ATP-competitive CENP-E inhibitor. Treatment of Hela cells with Compound-A induced chromosome misalignment during the SAC-dependent mitotic arrest, resulting in potent growth suppression by apoptosis. Furthermore, intraperitoneal administration of Compound-A displayed potent anti-tumor efficacy in colo205-xenograft mouse models (T/C=11 % at day 8). Given that SAC activation by CENP-E inhibition induced mitotic death, we next investigated whether SAC attenuation by BubR1 knockdown recovers cell viability in these cells. However, CENP-E inhibition was able to cause apoptosis after mitotic slippage in the BubR1-knockdown Hela cells, while inhibition of another mitotic kinesin Eg5, which controls centrosome separation during mitosis, induced polyploidy instead of apoptosis in these cells. Our data suggest that asymmetric chromosome segregation accelerated by CENP-E inhibition, but not polyploidy by Eg5 inhibition, is responsible for apoptosis after mitotic slippage. A comprehensive gene expression analysis of microarray comparisons revealed that p53 pathways are activated by CENP-E inhibition after mitotic slippage, and p53 knockdown suppressed post-mitotic caspase-3/7 activation by CENP-E inhibition. Furthermore, we found that both phosphorylation of at Ser-15 by ATM and ATR kinases and accumulation of p53 protein by unfolding protein response (UPR) were involved in post-mitotic p53 activation after mitotic slippage. In conclusion, we developed the novel CENP-E inhibitor, Compound-A, and CENP-E inhibition by Compound-A induced potent growth inhibition in both SAC-intact and SAC-defective cancer cells. In the latter case, p53 pathways play important roles in the induction of apoptosis. Our data demonstrate that SAC and p53 pathways complementally function to eliminate aberrant chromosome segregation accelerated by CENP-E inhibition. Thus, SAC activation and p53 protein accumulation are available for complementary PD biomarkers of the CENP-E inhibitors, and several molecules in the SAC and p53 pathways could be potential biomarkers to select sensitive tumors to the CENP-E inhibitors. Citation Format: Akihiro Ohashi, Momoko Ohori, Kenichi Iwai, Yusuke Nakayama, Tadahiro Nambu, Daisuke Morishita, Tomohiro Kawamoto, Maki Miyamoto, Takaharu Hirayama, Masanori Okaniwa, Hiroshi Banno, Tomoyasu Ishikawa, Hitoshi Kandori, Kentaro Iwata. A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3407. doi:10.1158/1538-7445.AM2013-3407