Abstract 2776: Inhibition of Nek2 by novel small molecules affects proteasome activity

Abstract

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Nek2 is a serine/threonine kinase that has been associated with centrosome function and cell cycle progression. Nek2 overexpression has been reported in several tumor types including breast and lung cancers. We have previously shown that elevated expression of Nek2 in clinical samples of multiple myeloma (MM) correlates with bortezomib resistance. Furthermore, the overexpression of Nek2 in MM cell lines decreases sensitivity to bortezomib and knockdown with RNAi sensitizes bortezomib resistant cells. Due to the proteaseome inhibitory activity of bortezomib, we hypothesized that Nek2 overexpression may increase proteasome activity. 26S proteasomes were isolated by ultracentrifugation from stably transfected Hela cells (+Nek2 or +GFP) and Nek2 was shown to be involved in the 26S proteasome complex by western blot. Proteasome activity assays were performed both biochemically and in cell culture demonstrating that proteasome activity in Hela+Nek2 is significantly higher than in Hela+GFP. Furthermore, the isolated 26S proteasomes were incubated with novel Nek2 inhibitors (HCI-2184 and HCI-2389) resulting in a significant reduction in proteasome function when used as single agents or in combination with bortezomib. HCI-2184, a reversible inhibitor of Nek2, demonstrated an IC50 value in the range of 13-38 nM, and HCI-2389, an irreversible inhibitor of Nek2, showed an IC50 value between 8-26 nM. Similarly, both compounds significantly increased the efficacy of bortezomib in inhibiting the proteasome activity. These results were confirmed in multiple cancer cell lines, including ARP1 (human MM cell line), H299 (human lung cancer cell line) and K28 (mouse Leydig tumor cell line). The effect of inhibiting Nek2 on cell cycle was also investigated. The expression levels of cyclinB1 and cdc2, proteins degraded by the proteasome for cells to exit mitosis, were increased in HCI-2389 treated Hela+Nek2 and to a lesser extent Hela+GFP treated cells. While Nek2 overexpression leads to the down-regulation of cyclinB1 and cdc2, HCI-2389 treatment successfully rescued this effect. Flow cytometry data showed that both treatments of HCI-2184 and HCI-2389 significantly arrested Hela+Nek2 and Hela+GFP in G2/M phase after 24 hours at concentration as low as 10nM. In conclusion, we have discovered a novel biological function of Nek2 related to elevated proteasome activity. We also demonstrated that our novel Nek2 inhibitors efficiently inhibit Nek2 function, resulting in reduced proteasome activity. These Nek2 inhibitors have the potential to be applied clinically for treatment of the MM patients resistant to bortezomib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2776. doi:1538-7445.AM2012-2776