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 this study, we have investigated the ability of APE1 inhibitors to induce synthetic lethality (SL) in a panel of DNA double-strand break (DSB) repair deficient and proficient cells; i) Chinese hamster (CH) cells: BRCA2 deficient (V-C8), ATM deficient (V-E5), wild type (V79) and BRCA2 revertant [V-C8(Rev1)]. ii) 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 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. SL 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 SL target in cancer.

Figure 1

Clonogenic survival assays for CH lung cells. Inhibitors were added at the indicated concentrations (see methods for details): A. Inhibitor-1, B. Inhibitor-2, and C. Inhibitor-3 with V-C8, V79 and V-C8(Rev1) cells. D. Inhibitor-1 with V-E5 and V79 cells. E. Methoxyamine induces synthetic lethality in V-C8 cells compared to V-79 and V-C8 (Rev1) cells. F. E3330 does not induce synthetic lethality in V-C8 cells. Survival of V-C8 cells is similar to V79 and V-C8 (Rev1) cells.

Figure 2

Clonogenic survival assays. Inhibitors were added at indicated concentrations (see methods for details). A. NU1025 induces synthetic lethality in V-C8 cells compared to V79 and V-C8 (Rev1) cells. B. Non-inhibitor-1 has no effect on V-C8 cells. Survival of V-C8 cells is similar to V79 and V-C8 (Rev1) cells. Clonogenic survival assays for T-REx CHO control cells and E8 cells. C. NU7441, D. KU55933, and E. Wortmannin. Results shown in panels C-E represent the average and standard deviation of six independent data points. F. Western blot (inset) confirming stable APE1 knock down (KO) in MDA-MB-231 treated with shRNA constructs compared to scramble and wild type cells. Clonogenic survival assays preformed with wortmannin at the indicated concentrations.

Figure 3

A. Aldehyde Reactive Probe (ARP) assay. In pre-treatment samples, background AP site level was low in all CH cell lines. At 2 hours and 4 hours of treatment with 10 μM of inhibitor-1, cells accumulated a significantly (p<0.001) higher level of AP sites. See text for details. B. Alkaline COMET assay. V-C8 cells demonstrated a higher mean tail moment compared to V79 and V-C8(Rev1) cells at 24 hours (p= 0.0008). C. Neutral COMET assay was performed as described in methods at various time points. Mean tail moment was higher in V-C8 cells at 24 hours (p=0.002) and 48 hours (p=0.003) compared to V-79 and V-C8-Rev1 cells. D. γH2AX immunocytochemistry. After 24 treatment with 10μM of inhibitor-1, γH2AX positive cells are more (p=0.04) in V-C8 cells compared to, V-C8 (Rev1) and V79 cells (UT= untreated, T = treated). E. FACS analyses. At 24 hours, V-C8 cells were shown to be arrested in G2/M phase compared to V-79 cells. F. Quantification of each stage of cell cycle including standard deviations is shown here. See text for details

Figure 4

A. Inhibitor-1 induces synthetic lethality in BRCA2 deficient SilenciX cells compared to BRCA2 proficient control SilenciX cells. B. Neutral COMET assay was performed as described in methods at various time points. Mean tail moment was higher in BRCA2 deficient SilenciX cells at 24 hours (p= 0.01) and 48 hours (p= 0.01) compared to BRCA2 proficient control SilenciX. C. γH2AX immunocytochemistry. After 24 hour treatment with 10μM of inhibitor-1, γH2AX positive cells are significantly more (p=0.02) in BRCA2 deficient SilenciX compared to BRCA2 proficient control SilenciX (UT= untreated, T = treated). D. Inhibitor-1 induces synthetic lethality in CAPAN1 cells compared to PANC1 cells. E. NU1025 induces similar synthetic lethality in CAPAN1 cells compared to PANC1 cells.

Figure 5

A. Neutral COMET assay was performed as described in methods at various time points. Mean tail moment was significantly higher in CAPAN1 cells at 24 hours (p=0.03) and 48 hours (p=0.05) compared to PANC1 cells. B. γH2AX immunocytochemistry. After 24 treatment with 10μM of inhibitor-1, γH2AX positive cells are more (p=0.05) in CAPAN1 cells compared to PANC1 cells(UT= untreated, T = treated). C. Inhibitor-1 induces synthetic lethality in MDA-MB-436 cells compared to MCF-7 cells. D. NU1025 induces similar synthetic lethality in MDA-MB-436 cells compared to MCF-7 cells. E. Neutral COMET assay was performed as described in methods at various time points. Mean tail moment was higher in MDA-MB-436 cells at 24 hours (p=0.002) and 48 hours (p=0.005) compared to MCF-7 cells. F. γH2AX immunocytochemistry. After 24 treatment with 10μM of inhibitor-1, γH2AX positive cells are more (p=0.05) in MDA-MB-436 cells compared to MCF-7 (UT= untreated, T = treated).