Abstract 1417 Molecular mechanisms of APE2 in DNA repair and DNA damage response pathways

Objective: Genome of cells is often challenged by insults from endogenous resources or environmental toxins, leading to different types of DNA damages including oxidized and methylated bases, Apurinic/apyrimidinic (AP) sites, DNA single-strand breaks (SSBs), and double-strand breaks (DSBs). Unrepaired AP sites, SSBs and DSBs impair DNA replication and transcription machineries, leading to genome instability and associated diseases such as cancer and neurodegenerative disorders. Cells have evolved various DNA repair and DNA damage response (DDR) pathways to mitigate various DNA damage for genome integrity maintenance. However, it remains unclear how distinct DNA repair and DDR pathways collaborate and/or crosstalk for the repair and signaling of DNA damage. AP endonuclease 2 (APE2, APEX2, APN2) emerges as an essential regulator protein in genomic and epigenomic integrity. APE2 is recently identified as a synthetic lethal target in BRCA1/BRCA2-deficient cancer cells. However, it remains unclear whether and how APE2 exactly function in genome integrity. The objective of this study is to identify and characterize the roles and mechanisms of APE2 in DNA repair and DDR pathways. Methods: Cutting-edge biochemical, cell biology, molecular biology, and computational biology approaches in Xenopus egg extracts, in vitro reconstitution systems, and cultured mammalian cells are utilized in our series of studies on APE2 biology and functions in genome integrity. Results: My lab is the first to demonstrate that the ATR-Chk1 DDR pathway is activated in response to hydrogen peroxide-induced oxidative stress in Xenopus egg extracts. Notably, APE2 plays a critical role in the ATR DDR pathway in oxidative stress. APE2 resects the oxidative DNA damage in the 3' to 5' direction and recruits Chk1 to activated ATR for activation. We further demonstrated that APE2 Zf-GRF binds with ssDNA, but not dsDNA, and regulates APE2's exonuclease activity in oxidative stress response. We have also developed a defined site-specific SSB structures that can trigger ATR-Chk1 DDR pathway activation in a eukaryotic cell-free system. We have demonstrated that APE2 interacts with PCNA through two distinct modes and that APE2 is essential for SSB repair and ATR signaling in response to defined SSB structures in Xenopus egg extracts. We recently demonstrated that APE1 and its exonuclease is required for the recruitment of APE2 to SSB sites and SSB repair and signaling. Our series studies in SSB repair and response prompted us to propose a two-step APE1/APE2-mediated mechanism in "SSB end resection" for genome integrity maintenance. Our computational analysis has revealed that APE2 genomic alterations occur at ∼17% frequency in 14 cancer types, and APE2 expression is upregulated in tumor tissue compared with matched normal tissue in several cancer types. APE2 is a critical regulator of the ATR DDR in pancreatic cancer cells, and can be targeted for cancer therapeutics in pancreatic cancer cells by its first in class inhibitor Celastrol. Furthermore, APE2 upregulation by interstrand-crosslinking agent cisplatin provokes mitochondrial deficiency and acute kidney injury. APE2's catalytic function and synthetic lethality has drawn attention as a cancer therapy target. Taken together, our series studies on APE2 had demonstrated the distinct coupling DNA repair and ATR DDR pathways by APE2 under various stress conditions and have provided novel insights into genome integrity and cancer therapeutics. The Yan lab was supported by the grants from the NIH/NCI (R01CA225637, R03CA270663, and R01CA251141), NIH/NIEHS (R21ES032966), and NIH/NIGMS (R15GM101571 and R15GM114713), and funds from UNC Charlotte.