Synthetic Lethal Interactions Between PARP Inhibition And Alantolactone-induced Oxidative DNA Damage In Cancer Cells

DNA damage response?DDR?and DNA repair pathways are promising therapeutic targets against diverse types of cancer.The poly?ADP-ribose?polymerases?PARPs?,mainly PARP1 and PARP2,are DNA damage sensors that catalyze the transfer of the ADP moiety from NAD⁺to target proteins,thereby activating the repair of single-or double-strand DNA break?SSB,DSB?or regulating DNA replication fork stability and restart.Thus,PARP1/2 play critical roles in the maintenance of genomic stability and normal cellular processes.PARP inhibitors?PARPis?are the first class of clinically approved anti-cancer drugs designed to exploit the concept of synthetic lethality.When used alone,PARP inhibitors selectively kill BRCA-mutant and hence homologous recombination?HR?-deficient ovarian and breast cancer cells.In addition,it has long been known that inhibition of PARP1/2 sensitizes cells to DNA-damaging genotoxic agents.However,clinical trials designed to test the use of PARPis in combination with DNA damaging chemotherapy have been unsuccessful,largely due to normal tissue toxicity.Besides,most breast cancers,and nearly all other types of cancer,have normal BRCA1 and BRCA2;and even among BRCA1/2-mutant tumors,responses to PARPi are heterogeneous and all initially responsive cancers eventually develop PARPi resistance.Therefore,it is important to gain a deeper understanding of the mechanisms underlying the actions of PARP inhibitors and to develop drug combinations using synthetic lethal principle to broaden the therapeutic potential of and overcome resistance to PARPis.The natural compound alantolactone?ATL?,the major medicinal compound of Radix Inulae extract,has a wide range of pharmacological activities,such as antitumor,anti-bacterial and insecticidal effects.In addition,ATL has been reported to enhance ROS accumulation and induce oxidative DNA damage selectively in cancer cells by inhibiting thioredoxin reductase-1?TrxR-1?.One of the major oxidative DNA damage products,8-oxoguanine?8-oxoG?,is quickly repaired by 8-oxoG-specific DNA glycosylase?OGG1?-mediated base excision repair?BER?pathway,during which,generation of SSB is an intermediate step.Hence,PARP1/2 are required for the repair of oxidative DNA lesions by BER and it has been shown that PARP inhibition sensitizes cells to oxidative stress,thereby raising the possibility of using PARPis in combination with pro-oxidative agents to yield cancer-specific synergistic lethality.In this study,we investigated the effects and mechanisms of action of combining ATL and PARPis.First,we showed that a non-cytotoxic dose of ATL induced a rapid increase in ROS levels specifically in cancer cells.The ATL-induced,cancer-specific ROS increase was followed by NAC-suppressible accumulation of 8-oxoG,DNA strand breaks and poly?ADP-ribose??PAR?,indicating generation of oxidative DNA damage and activation of PARP1/2.These results demonstrated that nontoxic doses of ATL induced oxidative DNA damage selectively in cancer cells,which subsequently activated PARP1/2.Second,we showed that nontoxic doses of olaparib in combination with nontoxic ATL resulted in potent synergistic cytotoxicity specifically in cancer cells.DR-GFP reporter assay and RAD51 immunostaining demonstrated that the strong synergism between ATL and olaparib was produced through mechanisms other than inducing HR deficiency.Thus,the combination of ATL and olaparib has a broad-spectrum anticancer effect irrespective of HR status.The toxicity was directly related to ATL-induced oxidative damage as it was significantly reduced by NAC.OGG1 depletion or inhibition significantly reduced the synergistic cytotoxicity,revealing that the lethality resulted primarily from the repair of oxidized DNA bases.Remarkably,our results showed that the non-trapping PARPi veliparib failed to synergize with ATL to cause cytotoxicity and depletion of PARP1 did not sensitize cells to ATL,on the contrary,the synergistic toxicity between ATL and olaparib was abolished in the absence of PARP1,demonstrating that the lethality associated with the ATL and olaparib combination was dependent on the formation of DNA-PARP complex.Further studies demonstrated that intense replication stress and DDR signaling were induced in S phase cancer cells specifically by the ATL and olaparib combination,which resulted in extensive replication fork stalling and formation of DNA double-strand breaks.Thus,after treatment with ATL and olaparib,the cancer cells were gradually blocked in the advanced G₂ stage and subsequently activated intrinsic apoptosis pathway.In vivo,the combination effectively induced regression of tumor xenografts.In summary,here we show that combining the PARPi olaparib with the highly tumor-specific DNA damaging agent,ATL,results in synergistic lethality at nontoxic doses of both drugs.The combination exploits a cancer vulnerability driven by the high levels of intrinsic oxidative pressure in cancer cells.On the other hand,olaparib inhibits the abnormally activated DNA repair activity that cancer cells rely on for survival.Normal tissues are spared due to their lower basal ROS output.Our results further reveal that synthetic lethality between ATL and olaparib results from the oxidative DNA damage specifically induced by ATL in cancer cells and the DNA-PARP complex caused by olaparib at the sites of DNA damage.Our study takes advantage of the biochemical and metabolic difference between cancer and normal cells to design effective and safe drug combinations.Thus,combining ATL with olaparib provides a promising therapeutic strategy that may offer safe and effective ways to broaden the therapeutic potential of PARP inhibitors.