Synergistic Lethality Between Auranofin-induced Oxidative DNA Damage And ATR Inhibition In Cancer Cells

ATR-mediated DNA damage response(DDR)promotes cell survival by upregulating DNA repair and regulating cell cycle checkpoints or induces cellular senescence or apoptosis if the damage is irreparable.For cancer cells,their inherent DNA replication stress and genomic instability,combined with treatment-induced DNA damage,make them critically dependent on ATR-mediated DDR.As such,inhibiting ATR represents a promising anticancer strategy.However,normal tissue toxicity significantly limits the combined use of ATR inhibitors with DNA damaging radio-or chemotherapies in the clinic.Alterations in metabolism lead to high internal oxidative pressure and limited spare antioxidant capacity in cancer cells,making them more sensitive to further increase in oxidative stress than normal cells.By stimulating ROS generation or inhibiting antioxidant pathways,it may be possible to produce toxic levels of ROS selectively in cancer cells to generate oxidative DNA damage and therefore synergistic anticancer effects with ATR inhibitors.Thus,cancer-specific oxidative DNA damage caused by elevated ROS may become an alternative to DNA damaging radio-or chemotherapy to be combined with ATR inhibitors,thus avoiding the impact on normal cells.Auranofin,a therapeutic drug for rheumatoid arthritis,is an inhibitor of the thioredoxin reductase(Trx R)and increases intracellular ROS levels through Trx R inhibition.Because it has displayed certain antitumor activity,combined with its well-characterized safety and pharmacokinetic profiles,there is an increasing interest in repurposing auranofin as an anticancer drug in recent years.The aim of this study is to investigate whether the combination of auranofin and ATR inhibitors produces cancer-specific cytotoxicity and synergistic anticancer effects.Firstly,we examined the cytotoxicity of auranofin in a panel of diverse human tumor cell lines and selected a noncytotoxic dose of auranofin to treat cancer and noncancerous cells.We found that the sublethal concentration of auranofin specifically caused a significant increase in ROS levels in cancer cells and induced8-oxoG and single-strand DNA break(SSB)production,replication fork stalling,ATR-Chk1 pathway and S cell cycle checkpoint activation.The ROS inhibitor N-acetyl-L-cysteine(NAC)blocked this phenomenon.These studies showed that sublethal auranofin induced an increase in ROS and extensive oxidative DNA damage,resulting in vigorous activation of the ATR-Chk1 pathway and S cell cycle checkpoint in cancer cells to promote cell survival.Next,we combined auranofin with the ATR inhibitor VE822 in cancer and noncancerous cells.The study found that auranofin sensitized cancer cells to VE822 to yield synergistic cytotoxicity at nontoxic doses of both drugs without affecting the survival of noncancerous cells.In addition,combination of other pro-oxidative DNA damaging drugs with ATR inhibitors similarly demonstrated good synergistic anticancer effects.Consistent with this,auranofin also had a significant synergistic anticancer effect with Chk1 inhibitors.Further experiments revealed that the addition of VE822 blocked Chk1 phosphorylation induced by auranofin,abolished S cell cycle arrest and restarted stalled replication forks,and caused a large number of double stranded DNA breaks(DSBs)in cancer cells,leading to catastrophic nuclear disintegration and cell death.In addition,we validated the combined anti-tumor effect of auranofin and VE822 in vivo through tumor xenograft experiments.The use of either drug alone had no effect on the growth of the tumor xenograft compared with the vehicle control,whereas co-administration of auranofin and VE822 completely inhibited the growth and induced substantial regressions of the tumor.Immunohistochemical staining of tumor tissues showed that only the groups treated by the combination of auranofin and VE822 revealed intense γH2AX and TUNEL staining,demonstrating significant DNA replication stress and apoptosis.There was no significant change in body weight in the treatment groups compared with the controls,and hematoxylin-eosin(HE)staining of vital organs did not show any histological changes in all groups,indicating that the combination of auranofin and VE822 was well tolerated.In conclusion,we showed that subtoxic concentrations of auranofin induced oxidative DNA damage selectively in cancer cells which sensitized them to ATR inhibition,on the other hand,VE822 enhanced the cancer cell toxicity of oxidative DNA damage by preventing DNA repair and the protective response of cells.Therefore,the combination of auranofin with VE822 produced DNA replication catastrophe and significant synergistic cytotoxic effects.In vivo,auranofin and VE822 coadministration enabled complete regressions of tumor xenografts,while each drug alone had no effect.Our data suggest that cancer-specific synergistic lethality is attainable by ATR inhibition in combination with specific pro-oxidative agents.The combination exploits an oxidative stress-driven cancer vulnerability to achieve selective targeting of cancer and may open new routes to broaden the therapeutic potential of ATR inhibitors.