Preclinical Study Of AZD3759 Combined With Radiation In The Treatment Of Brain Metastasis From EGFR Mutant NSCLC

Objectives:1.To determine whether AZD3759 combined with radiotherapy have synergistic effect and mechanism underneath.2.To determine whether cranial radiotherapy influence the blood brain barrier(BBB)penetration of AZD3759.3.To determine the antitumor efficacy of AZD3759 and radiotherapy in brain metastasis(BM)models in nude mice and the optimal treatment modality.Methods:1.MTS and colony formating assay(CFA)were performed to determine whether AZD3759 combined with radiotherapy have synergistic effect in EGFR mutant NSCLC(H226 and H2228)and EGFR activating mutations NSCLC(PC-9 and H3255),different concentrations of AZD3759 were dosed to test whether the concentration of AZD3759 influenced the efficacy of AZD3759 combined with radiotherapy.2.Immunofluoresence assay was performed to assess the percentage of DNA damage,flow cytometry assay was performed to assess the distribution of cell cycle and percentage of cell apoptosis.3.Expression of EGFR and downstream pathway(EGFR,pEGFR,AKT,pAKT,ERK1/2,pERK),rad51,DNA-PKcs,cdc2,pcdc2 Y15,cleaved PARP and cleaved caspase 3,expression of nuclear and cytoplasmic EGFR were detected in western blot;coimmunoprecipitation assay was performed to detected the binding of EGFR and DNA-PKcs in nucleus.4.Brain metastasis model was established through injection of tumor cell into the brain parenchyma of Nu/Nu mouse.In PK/PD study,concentration of AZD3759 in brain and blood were detected,expression of pEGFR,Ki 67 and cleaved caspase 3 in brain tumor were detected using immunohistochemistry assay.In efficacy study,tumor growth was monitored by measuring bioluminescence signals weekly,and tumor area at the end of in vivo efficacy study was detected using HE assay.Results:1.AZD3759 combined with radiation synergistically inhibit cell proliferation and survival in EGFR mutant NSCLC.5nM AZD3759 have radiosensitizing effect while lOnM AZD3759 combined with radiation have synergistic effect.2.AZD3759 inhibit the repair of radiation induced DNA damage,AZD3759 induced Gl arrest and abrogate the radiation induced G2/M arrest.AZD3759 combined with radiation dose dependently and time dependently increased cell apoptosis.3.AZD3759 supprcss the activily of non homologous end joining(NHEJ)and homologous recombination(HR)DSBs repair pathway through inhibit the expression of rad51 and DNA PKcs.abrogate G2/M arrest through inhibit the expression of p-cdc2.AZD3759 could translocate the EGFR/DNA-PKcs complex from nucleus to cytoplasm.4.Cranial radiation did not significantly influence the penetration of AZD3759 through BBB.AZD3759 combined with radiation synergistically enhanced antitumor efficacy in BM model in nude mice.Conclusions:1.AZD3759 combined with radiation have synergistic effect in EGFR mutatant NSCLC,which was influenced by the concentration of AZD3759.2.Mechamisms underlying the radiosensitizing effect of AZD3759 involved inhibiting cell proliferation and survival,inhibiting the repair of DNA damage.AZD3759 suppressed the repair of DNA damage through inhibiting the NHEJ and HR DSBs repair pathway,and abr-ogating the G2/M arrest.Besides.AZD3759 could inhibit the activity of nuclear DNA-PKcs through translocate the EGFR/DNA PKcs complex from nucleus to cytoplasm.3.Cranial radiation did not significantly influence the penetration of AZD3759 through BBB,the high BBB penetration of AZD3759 was essential for the synergistic effect in BM when combing AZD3759 with radiation.