Abstract 5447: A novel inhibitor of DNA methylation

Abstract We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC50. Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself. 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 5447.