Abstract 1050: Extended treatment with azacitidine maintains low DNMT1 levels and DNA methylation in MDA-MB-231 cells in vitro and in vivo

Abstract

Background: Azacitidine (AZA) is approved for treatment of patients with myelodysplastic syndromes or WHO-defined acute myeloid leukemia with multi-lineage dysplasia (<30% blasts). The drug is administered subcutaneously or intravenously once a day during the first 7 days of a 28-day cycle. Clinical trials investigating the use of AZA in solid tumors have been reported, although response rates were poor, possibly due to suboptimal dose and schedule. An oral formulation of AZA is currently being evaluated in a Phase 1 clinical trial in solid tumors, using continuous dosing of single agent oral AZA or intermittent dosing (14 days on, 7 days off) in combination with chemotherapeutics. It is postulated that extended AZA dosing schedules may be optimal for maintaining DNA hypomethylation and inducing cell death, and thus may cause responses in patients with solid tumors. Purpose: To better understand the effects of short-term vs. extended treatment with AZA on pharmacodynamic markers. Methods: The effects of AZA dose and schedule (short-term vs. extended) on pharmacodynamic markers such as DNMT1 depletion, DNA methylation, and apoptosis were evaluated in MDA-MB-231 breast cancer cells in vitro and in vivo. For in vitro experiments, MDA-MB-231 cells were treated daily with 0.1 or 0.3μM AZA for up to 12 days, and harvested at various times during treatment, as well as up to 12 days following treatment. For in vivo studies, MDA-MB-231 tumor-bearing mice were dosed (ip) with 1 or 3mg/kg AZA daily for 3, 7, 14, 21, or 28 days and tumors were harvested during and at several time points after the dosing period. DNA and cell lysates were prepared (from cell pellets or xenograft tumors) for DNA methylation analysis (LINE-1 or EpiTech Methyl qPCR assay) and DNMT1/cleaved-PARP western blotting, respectively. Results: In both in vitro and in vivo studies, AZA caused a rapid (by 8 hours post in vivo dose), dose-dependent depletion of DNMT1 protein; when AZA treatment was halted, DNMT1 protein levels returned to basal levels within 3-4 days. Consistent with these results, AZA in vitro and in vivo caused a dose-dependent decrease in DNA methylation (LINE-1 and gene-specific) and further reduction in DNA methylation with additional days of AZA dosing. In vitro, DNA methylation returned to basal levels upon AZA removal (within 8 days); the kinetics of DNA re-methylation was slower in more hypomethylated DNA. Lastly, apoptosis (PARP cleavage) was not observed in tumors from mice until 14 or 21 days of dosing with 3mg/kg or 1mg/kg AZA, respectively. Studies are underway in other xenograft models to support these findings. Conclusions: Extended AZA treatment maintains low DNMT1 levels and DNA methylation, and induces cell death. These results provide a strong rationale for the use of extended AZA dosing schedules in the clinic. 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 1050. doi:1538-7445.AM2012-1050