Combined Inhibition Of ATR And WEE1 As A Novel Therapeutic Strategy In Triple Negative Breast Cancer

Background and ObjectiveTriple negative breast cancer(TNBC),characterized by lacking estrogen receptor and progesterone receptor,as well as human epidermal growth factor receptor 2,has been a huge challenge due to the absence of endocrine therapy and effective target therapy.While conventional chemotherapy is the mainstay treatment of TNBC patients,toxicity with these agents is hard to tolerate,and improvement in prognosis of patients remains negligible.Accordingly,there is an urgent need for identification of novel cancer therapies for this malignant disease.Over half of TNBCs harbor deficient p53 signaling,leading to an inactive G1/S checkpoint.Thereby,TNBC relies more on the G2/M checkpoint to respond to DNA damage.Tyrosine kinase WEE1 and ATR play the crucial role in the G2/M checkpoint and DNA damage response(DDR).Our research first studies the effect between ATR inhibitor AZD6738 and WEE1 inhibitor AZD1775 in TNBC.We demonstrate that the synergy between the two inhibitors by a series of in vitro and in vivo experiments.Furthermore,we perform immunoblotting,immunofluorescence and mouse xenograft model to explore the mechanisms of the synergy.Together,our results might be an innovative and potent targeted therapy for TNBC patients.Materials and Methods1.We treated seven TNBC cell lines with AZD1775,and the cell viability were determined by CCK8.MDA-231 and Hs578t cells were treated with 0.1-0.5 ?M of AZD1775 for 1 and 3 h,and then western blot was used to test activation of WEE 1 targets.2.MDA-231 and Hs578t cells were treated with AZD1775 in combination with ATR inhibitor(AZD6738),ATM inhibitor(KU-60019)or DNA-PKcs inhibitor(NU7441).Cell viability was detected by CCK8.3.AZD6738 was used to treat seven TNBC cell lines,and the cell viability was determined by CCK8.After treating MDA-231 and Hs578t cells by either AZD1775 and AZD6738,or both,we performed CCK8 and colony-forming array to detect cell proliferation and flow cytometry to define apoptotic cells.The CI values of AZD1775 and AZD6738 were calculated by CompuSyn software.4.MDA-231,Hs578t,MCF7 and MCF10A cells were treated with AZD1775 and AZD6738,and the dose ratio of AZD1775/AZD6738 was 1:5 or 5:1.Cell viability was investigated by CCK8 assay.The CI values were analyzed by CompuSyn software.MDA-231,Hs578t,MCF7 and MCF10A cells were treated with AZD1775,AZD6738 or both.Prop iodide(PI)staining was carried out to detect the cell-cycle profile using flow cytometry.5.MDA-231 or Hs578t cells were treated with AZD1775 with or without AZD6738.After treatment,western blot was performed to detect DDR moleculars,pHH3,and cleaved-caspase 3;flow cytometry was used to identify the population of cells positive for yH2AX;nuclear expression of yH2AX,RAD51 and pRPA32(S4/S8)were determined by immunofluorescence.6.MDA-231 or Hs578t cells were treated with AZD1775 with or without AZD6738.And then,cells were probed with anti-pHH3 or anti-?H2AX antibodies by flow cytometry;staining for p-HH3,a-tubulin together with DAPI was by immunofluorescence to show mitotic and nuclear phenotypes.7.CDK1/2 inhibitor roscovitine or CDK4/6 inhibitor palbociclib were used to treat MDA-231 and Hs578t cells,after which cells were treated with DMSO,or both AZD1775 and AZD6738.Cell viability was detected by CCK8 and cells after treatment were probed with anti-?H2AX and anti-RAD51 antibodies by immunofluorescence.Cells were pretreated with roscovitine or palbociclib,after which cells were treated by AZD1775 and AZD6738 and cell viability was determined by CCK8.The dose ratio of AZD1775/AZD6738 was 1:5 or 5:1.The CI values were analyzed by CompuSyn software.8.MDA-231 and Hs578t cells were treated by cisplatin with AZD1775,or AZD6738 or in combination.Cell viability was detected by CCK8 assay.The CI values were analyzed by CompuSyn software.Expression of RAD51,yH2AX and pHH3 of MDA-231 and Hs578t exposed to cisplatin alone,or in combination with AZD1775 or AZD6738,or both,were determined by western blot and immunofluorescence.9.After treatment with vehicle or BRCA1 siRNA,MDA-231 cells were treated with AZD1775 or AZD67398 or cisplatin with the combination of AZD1775 and AZD6738 or veliparib with the combination of AZD1775 and AZD6738.MDA-231 was transfected with lentivirus encoding mutational BRCA1,after which cells were treated with cisplatin or veliparib in combination with AZD1775 and AZD6738.Cell viability was determined by CCK8 assay.10.Mice bearing MDA-231 xenografts were dosed with AZD1775 or AZD6738 or both.Tumors were taken after last drug administration.Body weight and tumor volumes were evaluated every 3 days.Apoptosis of the tumor tissues in different groups was detected by TUNEL assay.Western blot and immunofluorescence assay were utilized to analyze yH2AX,pPRA32(S4/S8)and pHH3 expression of tumor tissues.Results1.CCK8 data showed that TNBC cells were highly sensitive to AZD1775,as the majority of AZD1775 IC50S in these cells were below 1 ?M.Western blot results indicated that AZD1775 rapidly induced a time and dose dependent increase of DDR proteins,cleaved-caspased-3,and the mitosis marker pHH3 expression.2.The inhibition of ATR by AZD6738 significantly enhanced the AZD1775-caused cell viability loss in TNBC cells,compared to the inhibition of ATM by KU-60019 or DNA-PKcs by NU7441.3.Most TNBC cells were also sensitive to AZD6738,and the majority of AZD6738 IC50S in these cells were below 3 ?M.CCK8 and colony formation found that AZD6738 sensitized cells to AZD 1775 in TNBC cells,and drug combination between AZD1775 and AZD6738 was markedly synergistic because most CI values were<1 in TNBC cells.Apoptosis assays also showed that the combined treatment triggered more significant cell apoptosis compared to AZD 1775 or AZD6738 alone in MDA-231 and Hs578t.4.CI values at different effect levels in MDA-231 and Hs578t cells were all<1,while almost Cl values in MCF7 and MCF10A were>1.Flow cytometry results showed that the combinational AZD1775 and AZD6738 led to a slight S phase arrest in TNBC cells and induced a distinct G1 arrest in MCF7 and MCF10A cells.5.Western blot suggested that AZD6738 suppressed AZD1775-induced CHK1 phosphorylation and increased yH2AX,pHH3 and cleaved caspase-3 expression in TNBC cells.The results of flow cytometry and immunofluorescence showed that the combined treatment caused extremely enhanced expression of yH2AX and the replication stress marker pRPA32(S4/S8)compared to AZD1775 or AZD6738 alone in MDA-231 and Hs578t.6.Flow cytometer results showed that AZD 1775 and AZD6738 in combination significantly increased the number of the pHH3-positive cells and yH2AX-positive cells at the same time.Immunofluorescence images showed that there was a striking increase in mitotic cells following combined exposure,in comparison with single-drug treatment,and combination treatment induced multiple mitotic abnormalities and abnormal nuclear morphology characterized by gross micronuclei.7.Cell viability results showed that roscovitine and palbociclib antagonized the cytotoxic effect of the combined AZD1775 and AZD6738 treatment in TNBC cells and increased the CI values.Immunofluorescence results showed that both roscovitine and palbociclib dicseased the increase of yH2AX expression induced by co-treatment.8.The CCK8 assay revealed that low doses of AZD1775 or AZD6738 did not enhance the effect of cisplatin in TNBC cells significantly,but dual exposure to both drugs led to significant sensitivity to cisplatin.Immunofluorescence and western blot data indicated that inhibition of AZD1775 and AZD6738 strikingly repressed the cisplatin-induced RAD51 expression,resulting in more yH2AX accumulation and pushing more cells into mitosis.9.BRCA1 depletion did substantially sensitize the cells to AZD6738.A combination of AZD1775 and AZD6738 further sensitized BRCA1-deficient cells to cisplatin and veliparib,although this enhancement was smaller than that of control cells.10.Combined treatment induced a significant reduction in tumor growth compared to single drug treatments in MDA-231 xenograft model.And the single drug or combination treatments did not cause body weight loss.Immunofluorescence and western blot confirmed that expression of ?H2AX,pRPA32(S4/S8)and pHH3 increased in the combination group and AZD6738 inhibited AZD1775-induced pCHK1.The TUNEL results of the tumor tissues showed compared to the single drugs,combinational AZD1775 and AZD6738 remarkably increased cell apoptosis.Conclusions1.AZD1775 can inhibit cell proliferation in TNBC and induced DNA damage and DDR.We demonstrate the synergy between AZD1775 and AZD6738 for the first time.2.The mechanisms of synergy of AZD1775 and AZD6738 are as follows:1.AZD6738 inhibits DDR activated by AZD1775 and enhances DNA damage and replication stress,resulting in cell apoptosis.2.AZD6738 increases AZD1775-induced forced mitotic entry,causing mitotic catastrophe.3 CDKs activity is required for the above processes.3.Combinational AZD1775 and AZD6738 sensitized the TNBC cells to cisplatin.The sensitivity to AZD6738,combinational treatment with cisplatin,and combinational treatment with PARP inhibitor in BRCA1-deficient TNBC cells were further enhanced.All these highlight its clinical advantages in TNBC patients.