Abstract 5081: Disruption of DNA methyltransferase (DNMT) 1 confers resistance to DNMT inhibitors in human colorectal cancer cells

Abstract

Abstract DNA methyltransferase (DNMT) 1 is responsible for the maintenance and propagation of DNA methylation pattern, and plays an important role in cell survival and proliferation. Thus, targeting DNMT represents a promising approach for cancer treatment. However, the role of DNMT1 in treatment efficacy with DNMT inhibitors remains controversial. The aim of this study was to investigate potential alterations of DNMT inhibitor-induced cytotoxicity and cellular survival after disruption/knockout of DNMT1 in cancer cells. The human colorectal cancer cell line HCT116 and its isogenic DNMT1 knockout pair were treated with increasing concentrations of 4’-thio-5-aza-2’-deoxycytidine (aza-TdCyd), a novel DNMT inhibitor, and 5-aza-2’-deoxycytidine (aza-dCyd). Cytotoxic effects and cell survival were evaluated by MTT and clonogenic assays. Apoptosis was measured by annexin-V/propidium iodide test, and expression of DNMT1, DNMT3A and DNMT3B was detected by western blot. After 96h of drug exposure, IC50s of aza-TdCyd and aza-dCyd were 0.031µM and 0.28 µM, respectively, in HCT116 cells. In contrast, DNMT1 knockout cells were remarkably resistant to both drugs with IC50s all higher than 10 µM. The treatments resulted in a significant increase in apoptosis in the parental line versus its knockout counterpart. As for the cell survival, IC50s were ~0.12 µM for aza-TdCyd and ~4.62 µM for aza-dCyd in HCT116 cells, compared to ~43.77 µM and ~62.09 µM in DNMT1 knockout cells. As such, disruption of DNMT1 caused over 300-fold and 10-fold increase in resistance to aza-TdCyd and aza-dCyd. Moreover, we observed a G2/M arrest produced by aza-TdCyd versus aza-dCyd in the parental cells, and no apparent alteration in cell cycle phase distribution in DNMT1 knockout cells. Notably, aza-TdCyd was more potent than aza-dCyd in depleting DNMT3A and/or DNMT1 in both cell lines after 48h of treatment. Our findings demonstrate that disruption of DNMT1 leads to a significantly increased resistance to aza-TdCyd and aza-dCyd, suggesting that DNMT1 is critical to the antitumor activity of DNMT inhibitors. Aza-TdCyd is much more potent than aza-dCyd for both inhibition of DNMTs and growth of cancer cells. Citation Format: Angelo B. Laranjeira, Dat Nguyen, Erich Huang, James H. Doroshow, Sherry X. Yang. Disruption of DNA methyltransferase (DNMT) 1 confers resistance to DNMT inhibitors in human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2017-5081