Abstract LB192: Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene

Abstract

Abstract DNA methyltransferase (DNMT) 1 is an enzyme responsible for maintaining DNA methylation pattern during DNA replication. However, its role on the methylation of tumor suppressor genes and DNA damage in relation to antitumor activity of DNMT inhibitors remains controversial. Here, we investigated the effects of DNMT inhibitors in human colorectal and breast cancer cell lines and animal models with intact (DNMT1+/+) and disrupted DNMT1 (DNMT1-/-) gene. Treatment with 5 µM of 4’-thio-5-aza-2’-deoxycytidine (aza-TdC), decitabine and azacytidine all inhibited DNMT1 expression and cell growth (94.0%/97.1%, 39.9%/58.6% and 69.2/91.3), and induced apoptosis (66.9%/62.5%, 30.9%/23.8% and 66.2/43.1%) in DNMT1+/+ HCT116/MCF7 cells; in contrast, the DNMT inhibitors had less/little activities in DNMT1-/- HCT116/MCF7 cells (P<0.001 each). γH2AX and G2/M cell-cycle arrest were induced by the inhibitors and greater degree of induction was produced by aza-TdC than decitabine in DNMT1+/+ cells (P<0.001), with activation of checkpoint kinase 1 and upregulation of p21. Both demethylation and increased acetylation of CDKN2A and CDKN2B gene promoters together with re-expression of p16INK4A and p15INK4B proteins were detected in DNMT1-/- versusDNMT1+/+ (demethylation only) cells upon exposure to decitabine for 72h. Compared with saline treatment, tumor growth was significantly inhibited by aza-TdC (90%; n=18 mice) and decitabine (62%; n=18) in DNMT1+/+ colorectal xenograft tumors, alongside decreasing expression of DNMT1 (51% and 44%) and an increase in p21 (85% and 48%). Similarly, tumor growth delays (86%; n=15 and 57%; n=14) were associated with the reduction of DNMT1 and augmentation of p21 in bladder PDX tumors. The findings suggest that DNMT inhibitors induce DNA damage and higher anti-tumor activity of aza-TdC is proportional to a greater magnitude of induction of DNA damage, p21 and cell cycle arrest without reactivating the expression of p16INK4A and p15INK4B. The gene re-expression coupled to demethylation and acetylation corresponds to the less anti-tumor activity by decitabine in DNMT1-/- cells. Thus, it is the DNA damage effects, rather than re-expression of the tumor suppressors, that are associated with antitumor activity of the DNMT inhibitors in cancer cells and/or xenograft and PDX tumors. Citation Format: Angelo B. Laranjeira, Melinda Hollingshead, Dat Nguyen, Robert Kinders, Michael Difilippantonio, James H. Doroshow, Sherry X. Yang. Demethylation, DNA damage and antitumor activity of DNA methyltransferase inhibitors in models with intact and disrupted DNMT1 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB192.