Study On The Synergistic Effect And Mechanism Of PARP Inhibitor And GSK3 Inhibitor

Poly(ADP-ribose)polymerase(PARP)inhibitors are a class of drugs used to treat tumors with defects in homologous recombination repair through synthetic lethal mechanism.At present,four PARP inhibitors(PARPi)have been successively marketed for the treatment of advanced ovarian cancer,breast cancer,pancreatic cancer and prostate cancer,marking the clinical confirmation of PARP as an anti-tumor target and the theory of synthetic lethality.However,while PARPi produce good therapeutic effects,their limitations in the application of tumors with homologous recombination repair defect and the emergence of drug resistance hinder their further clinical development.In order to broaden the scope of application of PARPi and overcome their drug resistance in the process of clinical application.This study used a high-throughput method to detect the combined effects of PARPi and 99 anti-tumor drugs,and screened to obtain glycogen synthase kinase 3(GSK3)inhibitors,which has a significant synergistic effect with PARPi.After that,we verified the synergistic effects of PARPi and GSK3 inhibitors(GSK3i)in vitro and in vivo and explored the mechanism of action in depth.Firstly,we performed high-throughput screening of 99 anticancer drugs against 50 targets in combination with PARPi(olaparib and niraparib)to identify potential therapeutic combinations in HCC1937 and HCT-15 cell lines.Strikingly,a strong synergistic effect of GSK3i(CHIR99021 HCl and LY2090314)and PARPi(olaparib and niraparib)was observed in HCT-15 cells.In vitro proliferation inhibition experiments showed that in HCT-15 cells,the five PARPi including simmiparib,olaparib,niraparib,rucaparib,and talazoparib had a certain degree sensitization effect when combined with LY2090314 or CHIR99021 HCl which had no significant effect on cell proliferation inhibition.In particular,when simmiparib was combined with 5μM LY2090314,the sensitization folds were as high as 4463 times.Enhanced G2/M arrest and apoptotic cell death induced by the combination of simmiparib and LY2090314 were also observed.Moreover,we found that GSK3 i strongly synergized with simmiparib in all the 6 colorectal cancer(CRC)cells with diverse genetic backgrounds(CI < 0.6).As GSK3i(LY2090324 and CHIR99021 HCl)block both GSK3α and GSK3βactivity,we next generated GSK3α null and GSK3β null cells using CRISPR/Cas9 technique to investigate their role in the synergistic effect.The results showed that depletion of GSK3β,but not GSK3α,sensitized CRC cells to PARPi.Similarly,GSK3βinhibition or depletion significantly increased the cellular sensitivity to topoisomerase I inhibitor CPT-11,and hydroxyurea(HU).PARPi,CPT-11,and HU are known to selectively kill proliferating cancer cells by causing replication-dependent DNA double-strand breaks(DSBs).Indeed,GSK3βinhibition and knockout could significantly enhance the ability of PARPi to accumulate p-Chk1,p-RPA32 and γ-H2 AX.In addition,PARPi combined with GSK3 i could significantly increase the incidence of mitotic anaphase bridges and abnormal spindles in tumor cells.Collectively,this combination could contribute to replication-fork pressure,accumulation of DNA damage and mitotic catastrophe.The results of DSBs repair report system and RAD51 foci immunofluorescence experiment showed that GSK3β inhibition and knockout could significantly inhibit the process of homologous recombination(HR)repair,but had no obvious effect on the non-homologous end joining.Mechanistic study showed that inhibition and knockout of GSK3β could significantly down-regulate the m RNA and protein levels of BRCA1.In addition,overexpression of wild-type GSK3β in parental cells and reconstitution of wild-type GSK3β in GSK3β knockout cells could significantly up-regulate the protein expression of BRCA1.The previous study has shown that Wnt3a/GSK3β/Slug/Snail axis regulated BRCA1 m RNA expression in breast cancer.However,the functional impact of Slug/Snail-dependent BRCA1 repression remained unclear.We further confirmed that Slug and Snail played a pivotal role in Wnt3a/GSK3β-dependent BRCA1 expression in HCT-15 cells.Moreover,our results advanced the current understanding of the role of GSK3β in HR repair,and also revealed a possibility of using GSK3 i to sensitize PARPi,thereby expanding the benefits of PARPi to CRC.We further revealed that the synergistic effect of GSK3 i and PARPi depended on the levels of BRCA1.The BRCA1-deficient UWB1.289 cells exhibited much lower synergistic effect than its BRCA1 rescue cell line UWB1.289 +BRCA1(CI values: 0.68,0.87 vs.0.30,0.38).Additionally,the synergistic effect was significant reduced in the HCT-15 and RKO cells upon BRCA1 knockdown.Finally,we demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without apparent toxicity.The results showed that the combination of simmiparib and LY2090314 could synergistically inhibit the growth of subcutaneous xenograft tumors in nude mice of HCT-15 and RKO cells,with the T/C ratio of 20.82% and 45.67%,respectively.The bodyweight of the mice had no significant change in all the treated groups.Consistent with the in vitro data,LY2090314 significantly reduced the protein levels of BRCA1,and combined with simmiparib increased protein levels of γ-H2 AX and splicing Caspase3 in tumor tissue.Importantly,simmiparib significantly inhibited the growth of HCT-15 GSK3βknockout tumor xenografts,but not the parental tumor xenografts.In summary,we found out that GSK3 i had a significant synergistic effect with PARPi through a high-throughput screening method,and confirmed their combinational effect using a variety of methods in multiple CRC cells and nude mice subcutaneously tumor models.In terms of the mechanism of action,GSK3β inhibition and knockout could significantly down-regulate BRCA1 m RNA and protein levels in a Snail/Slug dependent manner,thereby mediated HR repair defect,and ultimately sensitized PARPi.Based on the above experiments,we reveal the obvious synergistic effects and their mechanism of PARPi and GSK3 i,which provides the experimental data for the development of clinical trials of this type of combination and a new selectively targeted therapeutic strategy for patients with CRC.