Abstract 2918: Molecular analysis of the mechanism of action of AsiDNATM brings new clues on DNA damage response regulation
Authors: Nathalie Berthault, Kozlac Maria, Sergey Alekseev, Pierre-Marie Girard, Marie Dutreix
Journal: Cancer Research (2019)

Abstract Purpose: Our laboratory succeeded at developing an original DNA repair inhibitor, called AsiDNATM, developed and tested in clinic by Onxeo. AsiDNATM is a small modified double-stranded DNA molecule vectorised by a covalently bound cholesterol. It acts by inhibiting DNA repair and sensitizes tumors to genotoxic treatments. Here we investigate the different steps involved in its activity. Experimental design: SK28, MRC-5 and MDA-MB-231 cells were treated by AsiDNATM. Cellular uptake of AsiDNATM was monitored by FACS and electron microscopy. AsiDNATM interacting proteins were identified by mass spectrometry in cell extracts pooled down with AsiDNA. Kinetics of activation of the two main targeted enzyme, DNA-PK and PARP, were monitored by measuring phosphorylation of H2AX and formation of poly-adenyl-ribose polymers (PAR) and NAD consumption. Inhibition of DNA repair enzymes recruitment at damage site (53BP1, XRCC4, BRCA1, Rad51 and NBS1) was analyzed by confocal microscopy. DNA damage were induced at different times after AsiDNATM addition. The role of PARP, DNA-PK and ATM enzymes in the recruitment inhibition was tested using specific inhibitors or deficient cells. DNA repair activity was analyzed by alcalin comet assay and survival curves were established. Transcriptomes were monitored 24h after AsiDNATMtreatment. Results: AsiDNATM enters the cells via cholesterol route using LDL receptors. It binds DNA-PK and PARP and triggers their activation for more than 24h. The levels of DNA-PK and PARP activation as well as NAD consumption were always higher than those induced by a 10 Gy irradiation. Survival rate to AsiDNATM alone was between 90% and 70%. No significant effect was observed on cell cycle or proliferation. Pretreatment of tumor cells with AsiDNATM inhibits DNA repair and increase cell death induced by irradiation. The earliest event observed after irradiation was a transitory phosphorylation of ATM(ser1981) disappearing after 2h. It was followed by the inhibition of 53BP1and XRCC4 recruitment in a PARP dependent manner. Then, concomitantly with γ-H2AX and PAR increase, recruitment of BRCA1 and RAD51 was inhibited in cells with high level of DNA-PK activation. This inhibition was dependent of DNA-PK and not of PARP or ATM. Transcriptome analysis showed large change in the expression of genes involved in metabolism. Conclusion: AsiDNATM inhibits NHEJ and HR double-strand break DNA repair by preventing the recruitment of key enzymes at break sites. The inhibition of NHEJ proteins recruitment is the earliest event and requires PARP activity. The inhibition of HR proteins appears lately and is dependent upon DNA-PK activation. PARP activation induces metabolism change that might participate to the antitumoral activity of AsiDNATM. These results highlight the unique mechanism of action of AsiDNATM through the activation of two complementary key enzymes involved in DNA damage response. Citation Format: Nathalie Berthault, Kozlac Maria, Sergey Alekseev, Pierre-Marie Girard, Marie Dutreix. Molecular analysis of the mechanism of action of AsiDNATM brings new clues on DNA damage response regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2918.