Abstract 2500: ON 01910. Na, a clinical stage anticancer mitotic inhibitor, produces prolonged hyperphosphorylation of RanGAP1•SUMO1 as a potential mechanism of G2/M arrest and apoptosis
Authors: Oussenko Irina , Holland James F. , Reddy E. Premkumar , Ohnuma Takao 
Published: February 2011

Abstract
INTRODUCTION: The benzyl styryl sulfone ON 01910. Na (abbreviated as 1910) is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell cultures. The compound is currently in Phase 1 and 2 trials. Available data show the drug produces three major abnormalities in tumor cell lines: (a) aberrant cell division, including irregular chromosomal segregation and cytokinesis; (b) G2/M arrest and apoptosis in many tumor cells; and (c) decreased expression of Cdc25C phosphatase. Early data suggested that 1910 was a PLK1 inhibitor. Subsequent studies did not confirm this, although 1910 was found to inhibit the PLK pathway. Precise mechanism of action continues to be investigated. METHODS and RESULTS: We assessed DNA damage checkpoints throughout the cell cycle and effects on signaling molecules upstream of Cdc25C following treatment of DU145 prostate cancer, Bel7404 hepatoma, U937 lymphoma or MOLT-3 ALL cells with 1910. Camptothecin (CPT) and doxorubicin (DOX) served as positive controls. Cell lysates after drug exposure for 4, 16 or 24 h were resolved on SDS-PAGE and analyzed by Western blot for Chk1, Chk2, ATM, RanGAP1•SUMO1 and their phosphorylated forms. CPT and DOX exposure resulted in activation/phosphorylation of DNA damage-responsive molecules Chk1, Chk2 and ATM by 4 h, whereas 1910 did not. However, hyperphosphorylation of RanGAP1•SUMO1 was observed within 4 h and sustained for more than 24 h during 1910 exposure. This was also seen with the anti-tubulin agent, nocodazole, but not with ON 01911, an inactive analog of 1910. Mild phosphorylation of Chk2 was observed only after 24 h exposure, suggesting that DNA damage response is not a primary effect of 1910. MOLT-3 cells, synchronized by double thymidine block, were released into medium supplemented with 1910, which resulted in peak accumulation of G2/M cells by 9-12 h. The G2/M cell fraction remained in plateau for more than 20 h. This timeframe correlated with the hyperphosphorylation of RanGAP1•SUMO1. Cleaved Lamin B, detected by 16 h and thereafter in 1910-containing release medium, confirmed an active apoptotic process during this period. MOLT-3 cells released into drug-free medium reached G2/M peak at 9-10 h and continued to cycle with synchronized transition into G1 at 15-16 h. RanGAP1•SUMO1 was not hyperphosphorylated and no cleaved Lamin B was detected. Tubulin polymerization assay revealed that 1910 and ON 01911 had little or no effect, whereas nocodazole inhibited the process. CONCLUSION: These findings show that 1910 is neither a direct DNA damage response inducer nor a tubulin toxin, and suggest that 1910 is an inhibitor of RanGAP1•SUMO1 phosphatase. Its mechanism of action appears to rely on prolonged hyperphosphorylation of RanGAP1•SUMO1, leading to G2/M arrest and induction of apoptosis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2500.