Synthetic lethal targeting of DNA double‐strand break repair deficient cells by human apurinic/apyrimidinic endonuclease inhibitors

Abstract

An apurinic/apyrimidinic (AP) site is an obligatory cytotoxic intermediate in DNA Base Excision Repair (BER) that is processed by human AP endonuclease 1 (APE1). APE1 is essential for BER and an emerging drug target in cancer. We have isolated novel small molecule inhibitors of APE1. In the current study we have investigated the ability of APE1 inhibitors to induce synthetic lethality in a panel of DNA double strand break (DSB) repair deficient and proficient cells; a) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant (V-C8(Rev1)). b) Human cancer cells: BRCA1 deficient (MDA-MB-436), BRCA1 proficient (MCF-7), BRCA2 deficient (CAPAN-1 and HeLa SilenciX cells), BRCA2 proficient (PANC1 and control SilenciX cells). We also tested synthetic lethality (SL) in CH ovary cells expressing a dominant–negative form of APE1 (E8 cells) using ATM inhibitors and DNA-PKcs inhibitors (DSB inhibitors). APE1 inhibitors are synthetically lethal in BRCA and ATM deficient cells. APE1 inhibition resulted in accumulation of DNA DSBs and G2/M cell cycle arrest. Synthetic lethality was also demonstrated in CH cells expressing a dominant–negative form of APE1 treated with ATM or DNA-PKcs inhibitors. We conclude that APE1 is a promising synthetic lethality target in cancer.

Targeting XRCC1 Deficiency in Breast Cancer for Personalized Therapy

“…This assay was conducted as described previously (28). Briefly, subconfluent cells were exposed to DSB inhibitors [KU55933 (5 mmol/L)] or [NU7441 (1.5 mmol/L)].…”

“…Cells grown to subconfluence were treated with ATM inhibitor [KU55933 (5 mmol/L)] or DNA-PKCS inhibitor [NU7441 (1.5 mmol/L)] for 24 hours and collected by trypsinization and centrifugation (1,000 rpm for 5 minutes). Fluorescence-activated cell sorting (FACS) assay was conducted as described previously (28).…”

“…This assay was conducted as described previously (28). Primary antibodies used were mouse anti-XRCC1 (Thermo Fisher Scientific); rabbit anti-ATM (Cell Signaling Technology); and rabbit anti-DNA-PKcs (Novus Biologicals).…”

Investigating the 3' RNA Phosphodiesterase and 3'-5' Exoribonuclease Activities of APE1.

“…In this study, 5 '-radiolabeled Oligo IA-4 RNA substrate was used to examine the possible inhibitory effect of the small molecule inhibitors on 3'RNA phosphodiesterase activity of APE1 (Simeonov et al 2009, Sultana et al 2012, Zou et al 2008. As previously mentioned in chapter 2, WT APE1 was able to generate two cleavage products of higher intensity in Oligo IA-4RNA (Figure 1 IB) than in Oligo LA and Oligo IA-RNA.…”

“…Molecular modeling studies indicated that Myricetin docks onto the active site of APE1 but the rigid structural feature of the molecule lacks anchoring groups and therefore resulted in numerous possibilities for docking orientations without shape complementarity conflicts (Sultana et al 2012;Simeonov et al 2009). Myricetin is known to have numerous other targets besides APE1 in cells; therefore, it has not been considered a very promising agent with selectivity or specificity as an APE1 inhibitor (Simeonov et al 2009).…”