Abstract 3016: APOBEC3 enzymes induce damage to the cellular genome during DNA replication
Authors: Green Abby M. , Landry Sebastien , Evans James P. , Shalhout Sophia , Bhagwat Ashok S. , Weitzman Matthew D. 
Published: August 2015

The APOBEC3/AID family of cytidine deaminase enzymes are powerful DNA editors that convert cytidine to uracil. APOBEC3 (A3) enzymes provide immune defense by mutating and inactivating viral genomes. We have previously shown that A3 enzymes are also capable of causing cellular DNA damage. Recent bioinformatics analyses of breast cancer and other solid tumor genomes identify a mutational pattern consistent with A3 activity. The mutational signature of A3 enzymes includes a predominance of C-to-T transitions, mutated cytidines within a TC context, and clustered hypermutation. We identified the mutational hallmarks of A3 activity in a subset of pediatric acute lymphoblastic leukemia (ALL) genomes. To understand the circumstances under which A3 enzymes cause damage to cellular DNA, we examined the effect of A3 overexpression using a doxycycline-inducible system in human cancer cell lines. A3 enzymes edit single-stranded DNA (ssDNA), thus we hypothesize that A3 enzymes act on the cellular genome when ssDNA is exposed during DNA replication. APOBEC3A (A3A) is the most potent of the seven A3 enzymes and induces phosphorylation of histone-variant H2AX (γH2AX), indicating double-stranded DNA breaks (DSBs). We examined the DNA damage response pathways activated by A3A expression and found that both Ataxia-Telangiectasia Mutated (ATM) kinase and ATM-Related (ATR) kinase signaling occurs when A3A is expressed. As expected from these findings, we show that cycling cells induced to express A3A arrest in S phase. ATR signaling is activated in response to DNA damage such as ssDNA breaks, and replication fork stress. To assess whether A3A acts on the cellular genome during DNA replication, we induced replication stress by treating cells with hydroxyurea (HU). We found that A3A expression increases DNA damage signaling in HU-treated cells. Additionally, we examined γH2AX and quantified genomic uracil throughout the cell cycle in cells induced to express A3A. Cells that expressed A3A and were arrested in G1 phase by isoleucine depletion had decreased cellular DNA damage and less genomic uracil as compared to those in S phase. These observations suggest that cells undergoing DNA replication are particularly vulnerable to deamination by A3A. Overall, these data support the hypothesis that A3 enzymes contribute to genomic instability and may contribute to malignant transformation. A3 enzymes may be responsible for the clustered cytidine mutations identified in pediatric leukemia genomes. This research is the first investigation into the association between A3 enzymes and pediatric cancer, and provides insight into the impact of A3A on replicating DNA.

Citation Format: Abby M. Green, Sebastien Landry, James P. Evans, Sophia Shalhout, Ashok S. Bhagwat, Matthew D. Weitzman. APOBEC3 enzymes induce damage to the cellular genome during DNA replication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2015-3016