CENP-E Inhibitor Research Articles

Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than are diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.

HPV16 E6 Induces Chromosomal Instability due to Misaligned Chromosomes Caused by E6AP-Dependent Degradation of the Mitotic Kinesin CENP-E

<h3>Purpose/Objective(s)</h3> Chromosome missegregation over multiple cell cycles, termed chromosomal instability (CIN), is common in tumors and often leads to aneuploidy, a hallmark of cancer. High risk human papillomavirus (HPV) causes anogenital and head and neck cancers (HNC). HPV has been shown to cause certain types of CIN, but the mechanisms are unclear and this has not been studied in HNC. Here we describe a specific, novel mechanism of CIN induced by the HPV16 oncoprotein E6, in a physiologically relevant HNC model and in patient tumors. <h3>Materials/Methods</h3> Normal oral keratinocytes (NOKs) were transduced with retrovirus expressing LXSN control vector, HPV16 E6, E7 or E6+E7 and were kept under selection to achieve stable cell lines. CIN was quantified by assessing mitotic defects using immunofluorescence microscopy or H&E-stained slides in the NOKs, HNC cell lines, patient-derived xenografts (PDX), and in patient HNC tumors. Quantitative immunofluorescence and western blotting were used to quantify CENP-E levels. Tetracycline (tet) inducible GFP-CENP-E was introduced into NOKs expressing the Tet repressor using the PiggyBac transposon system. p53 and E6AP null NOKs were created using CRISPR. Significant differences were determined using a two-tailed student's t-test. <h3>Results</h3> HPV+ HNC cell lines have a significant increase specifically in misaligned chromosomes during mitosis, as compared to HPV- cell lines (20% v. 5% of cells, n=4 cell lines each). Using NOKs expressing individual HPV oncogenes, we found that these defects are specifically induced by the HPV oncogene E6 rather than E7. Further studies revealed that HPV16 E6 causes the specific degradation of CENP-E, a mitotic kinesin that plays a critical role in chromosome congression during mitosis. Tetracycline induced over-expression of GFP-CENP-E in LXSN- and E6-expressing NOKs revealed appropriate localization to kinetochores in LXSN cells. Strikingly, E6 mediated degradation of CENP-E was so efficient that CENP-E levels (total and at kinetochores) did not increase even after tet-inducible expression. Degradation of CENP-E occurred post-translationally in a proteasome dependent manner. Proteasome inhibition in E6-expressing NOKs rescued CENP-E protein levels at the kinetochore and rescued the misaligned chromosome phenotype. Furthermore, E6-mediated CENP-E degradation required the ubiquitin ligase E6AP, as evidenced by CENP-E protein restoration and rescue of chromosome alignment in E6AP null NOKs expressing E6. Finally, a significantly increased incidence of misaligned chromosomes was confirmed in HPV+ tumors from HNC patients and HPV+ PDXs. <h3>Conclusion</h3> This study reveals a novel, specific mechanism by which HPV16 E6 induces CIN in HNC, which may play a role in HPV-induced tumor initiation, progression and radiation response. Additionally, there is exciting potential for synergy with a CENP-E inhibitor, currently in clinical trials, and radiation to increase CIN beyond a viable threshold to increase cell death.

Abstract 1030: CENP-E inhibition generates micronucleus formation activating the cGAS-STING pathway in cancer cells

Abstract Background: Micronuclear formation has been reported to activate innate immune response in tumor through the cGAS-STING pathway, which monitors cytosolic DNA primarily to detect bacteria or virus infection. We have developed a novel small molecule inhibitor targeting CENP-E, termed Cmpd-A, which intensively generate micronucleus formation in a broad range of cancer cells, causing a potent antiproliferation by aneuploid-mediated cellular stresses. In this study, using Cmpd-A as a tool compound, we aim to determine that micronucleus formation induced by CENP-E inhibitor could activate the cGAS-STING pathway in cancer cells, which may potentially induce an immunological conversion from cold to hot in cancer cells. Method: We generated micronuclear formation in cancer cells by Cmpd-A treatment. Phosphorylations of TBK1 and IRF3, indexes of the cGAS-STING pathway activation, were determined by immunoblotting. Gene set enrichment analysis of RNA-seq data was performed, comparing DMSO (control) and Cmpd-A treatment. Reporter assays for IRF and NF-kB were also performed. Results: Immunoblotting revealed that phosphorylations of TBK1 and IRF3 were remarkably upregulated in Cmpd-A-treated cells concordantly with micronuclear formation. Transcriptome analysis revealed that the down-stream genes in the cGAS-STING pathway (innate immune pathways) are elevated: IL6, IP10, CXCL1, IL8, TNF, IFNA families, and IFNB1. The Gene Set Enrichment Analysis (GSEA) also demonstrated that innate immune-associated pathways were significantly enriched in Cmpd-A-treated cells: cytosolic DNA sensing pathway, cytokine-cytokine receptor interaction pathway, and Toll-like receptor signaling pathway. Furthermore, the IRF-Lucia and NF-kB-SEAP reporter activity assays revealed that both IRF and NF-kB were significantly activated in Cmpd-A-treated cells in a time-dependent manner. Conclusions: The CENP-E inhibitor generates micronucleus formation to activate the cGAS-STING pathway in cancer cells. These findings suggest the CENP-E inhibitor could potentially induce the immunological conversion from cold to hot in cancer cells. Citation Format: Yumi Hakozaki, Yukie Kashima, Tomoko Y. Morita, Kosuke Tanaka, Susumu S. Kobayashi, Akihiro Ohashi. CENP-E inhibition generates micronucleus formation activating the cGAS-STING pathway in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1030.

Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells

The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations—misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.

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 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

Abstract B173: Synergistic interaction between CENP-E inhibitor GSK923295 and MEK inhibitor GSK1120212

Abstract Centromere protein-E (CENP-E) is a mitotic kinesin that is required during mitosis for metaphase chromosome alignment. GSK923295 (′295) is a potent and specific inhibitor of human CENP-E currently being developed as a next generation antimitotic drug for the treatment of cancer. It has shown a broad spectrum of anti-cancer activity in vitro and in vivo. A siRNA library screen targeting the ‘druggable genome’ identified multiple MAPK pathway genes as sensitizers to ′295, including BRAF and ERK1. Since the MEK protein plays an important role in the MAPK pathway, combination studies between GSK MEK inhibitor GSK1120212 (′212) and ′295 were performed in cancer cell lines from different tissue origins, and the combination effect was evaluated based on three different criteria: Excess over Single Highest Agent (EOHSA), Bliss, and Combination Index (CI).. Synergistic response was observed in 5/6 colon cancer cell lines, 5/15 lung cancer cell lines, and 6/8 pancreas cancer cell lines. Additionally, the combination of ′212 and ′295 treatment induced a much stronger apoptotic response than either drug alone in two colon cancer cell lines. These data suggest that combining MEK and CENP-E inhibitors might improve clinical efficacy over either as a single agent in selected tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B173.