Abstract 3579: APOBEC3 activity sensitizes cells to ATR-Chk1 pathway inhibition

Abstract

Abstract There are six functional members of the APOBEC3 family of enzymes (A3A-A3C, A3F-A3H), which catalyze the conversion of cytosine to uracil in single-stranded DNA. APOBEC3 enzymes are best described for their ability to deaminate viral and LINE element DNA, thus restricting infection and retrotransposition. However, recent analyses of human tumor sequences reveal mutational signatures consistent with APOBEC3 activity. These signatures indicate the capacity for APOBEC3 enzymes to cause mutations and breaks in cellular DNA and further, their potential to contribute to cellular transformation or clonal evolution of cancer. We have previously shown that APOBEC3 enzymes are capable of causing cellular DNA damage and activation of the DNA damage checkpoint via Ataxia-Telangiectasia Mutated (ATM) kinase signaling. To investigate additional cellular mechanisms that respond to APOBEC3-induced DNA damage, we examined the impact of A3A expression on protein kinases involved in DNA replication checkpoint signaling. Here, we show that A3A expression can also cause cell cycle arrest through activation of the DNA replication checkpoint as indicated by ATM-Related (ATR) kinase phosphorylation of Checkpoint Kinase 1 (Chk1) on S345 and S317. Based on these results, we hypothesized that inhibition of DNA replication checkpoint kinases in tumors with upregulated APOBEC3 enzymes would lead to uncontrolled replication, accumulation of DNA damage, and ultimately cell death. To study the impact of ATR inhibition on cells in which A3A is active, we generated cell lines with inducible A3A expression. We introduced a doxycycline-inducible A3A gene into a human fibroblast cell line and treated the cells with a selective ATR inhibitor (VE822). Using a colony formation assay, we show that ATR inhibition restricts cell growth in the context of A3A expression. We also found that A3A expression led to decreased cell viability following treatment with an ATR inhibitor. We examined the mechanism of cell death and found that ATR inhibition in cells induced to express A3A resulted in increased Annexin V staining, indicative of apoptosis. To confirm that these findings represent effects specific to the DNA replication checkpoint mediated by ATR activation and phosphorylation of Chk1, we employed siRNA knockdown of Chk1. We found that inhibition of Chk1 in the context of A3A expression also resulted in reduced cell growth, decreased viability, and apoptosis compared to uninduced cells. These observations suggest that DNA replication checkpoint signaling is critical for protection of the cellular genome from A3A-induced DNA damage. Upregulation of APOBEC3 activity in tumor cells provides an opportunity for targeted therapy. Thus, the ATR-Chk1 pathway may represent a target for therapeutic intervention in tumors in which APOBEC3 enzymes are active. Citation Format: Konstantin Budagyan, Matthew D. Weitzman, Abby M. Green. APOBEC3 activity sensitizes cells to ATR-Chk1 pathway inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3579.