Abstract 3847: Knockout of DNMT1 using CRISPR gene-editing technology confers resistance to DNMT inhibitors in human breast cancer cells

Abstract

Abstract DNA methyltransferase (DNMT) 1 plays an important role for DNA replication and cell proliferation, and it is implicated in the maintenance and propagation of DNA methylation patterns. Targeting DNMT represents a promising approach for epigenetic therapy in cancer. To better elucidate the function of DNMT1 on the treatment effect of DNMT inhibitors in cancer cells derived from human solid tumors, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas9) to edit DNMT1 gene in MCF-7 breast carcinoma cells. The co-transfection of the DNMT1 target sequence that directs to exon 1 of the gene in pCas-guide vector and a donor DNA vector effectively knocked down DNMT1 protein in MCF-7 cells as detected by Western blot. After 96h of drug exposure, IC50s of azacitidine (5-azacytidine) and decitabine (5-aza-2’-deoxycytidine) were all higher than 10 µM in DNMT1 knockout (DNMT1-/-) cells assessed by MTT; in contrast, IC50s were 1.44 µM and 10 µM in parental (DNMT1+/+) cells. As for the cellular survival in the presence of drugs, a remarkable increase in the colony formation was observed in DNMT1-/- cells compared to DNMT1+/+ cells. The annexin-V/propidium iodide test by flow cytometry provided evidence of lower percentages of necroptosis in the DNMT1-/- cells than DNMT1+/+ cells when the cells were exposed to these inhibitors for 48 hours at 5 µM (7.12 ± 5.24 vs. 39.0 ± 2.65 by azacitidine, p = 4.11 × 10-5, and 5.77 ± 4.19 vs. 20.0 ± 6.66 by decitabine, p = 4.11 × 10-5); in addition, there were large fractions of viable cells in the DNMT1-/- cells versus DNMT1+/+ cells (85.86 ± 6.39 vs. 56.86 ± 4.00 for azacitidine treatment, p = 4.11 × 10-5 and 87.4 ± 5.56 vs. 76.2 ± 9.36 for decitabine, p = 5.60 × 10-3). The findings demonstrate that knockout of DNMT1 using CRISPR/Cas9 method leads to an increased resistance to azacitidine and decitabine, in corroboration with our previous data in DNMT1 knockout HCT116 colorectal cancer cells. Therefore, DNMT1 is critical to antitumor activity of DNMT inhibitors in human solid tumor cell lines. Citation Format: Angelo B. Laranjeira, Erich Huang, Larry Rubinstein, Dat Nguyen, Sherry X. Yang. Knockout of DNMT1 using CRISPR gene-editing technology confers resistance to DNMT inhibitors in human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3847.