The Influence And Mechanism Of DNA Damage Repair Protein FEN1 On The Biological Effects Of Chronic Myeloid Leukemia Cells

Objective: Chronic myeloid leukemia(CML)is a myeloid proliferative tumor characterized by the formation of the Philadelphia chromosome and the production of the BCR-ABL fusion protein,with significantly enhanced tyrosine kinase activity.Imatinib(IM)is the first line of clinical treatment for patients with CML,but more than 25% of patients with CML are resistant to IM,resulting in failure of treatment,and for patients with CML in the emergency phase,IM is not effective.For a long time,DNA damage has been considered as one of the causes of cancer.Inappropriate DNA repair may lead to malignant transformation of cells by inhibiting the inactivation of tumor factors or the activation of oncogenes.In patients with CML,the expression of BCR-ABL increases the level of Reactive Oxygen Species(ROS),resulting in DNA double-strand breaks(DSBs).CML cells are prone to rely on a highly mutagenic alternative end-joining(Alt-EJ)to cope with enhanced abnormal DSBs.Flap endonuclease 1(FEN1),as a candidate protein of Alt-EJ,was over-expressed in a variety of solid tumors.But the expression of FEN1 in CML cells is unknown.Therefore,this study aims to detect the expression level of FEN1 in CML,explore the biological effect of targeting FEN1 on CML cells and verify whether FEN1 is involved in the Alt-EJ pathway.Methods: 1.RT-q PCR and Western blot were used to detect the expression of FEN1 in CML cell lines and bone marrow samples of CML patients,Western blot was used to determine whether the expression of FEN1 was affected by BCR-ABL.2.The FEN1 in K562 and K562/G01 cells were stable knocked down by lentivirus,and its biological effects were detected by related experiments.3.Western blot and immunofluorescence were used to detect the level of γH2AX.Adopt the method of layered extraction and Western blot under the condition of the induction DSBs to analysis the layer distribution spectrum of FEN1 and the classic proteins that participate in c-NHEJ way and Alt-EJ pathways.Plasmid-based end-joining assay was used to detect the effect of FEN1 on the repair pathways of DSBs.Then,the interaction between FEN1 and other repair proteins in DSBS repair were detected by Co-IP assay.Results: 1.We found the overexpression of FEN1 in both CML cell lines and CML patients’ bone marrow,western blot showed that the FEN1 expression was not affected by the downregulated p-BCR-ABL.2.Knockdown of FEN1 alone or in combination with NU7441 inhibit the proliferation of CML cells.Knockdown of FEN1 induces apoptosis of CML cells and enhances the sensitivity of IM on K562/G01 cells.FEN1 knockdown CML cells accumulate more unrepaired DSBs.3.CML cells present more DSBs formation and deceased classical nonhomologous end-joining(c-NHEJ)core proteins expression.Our results have shown that after DSBs,c-NHEJ factors(KU70 and KU80)shifted to the extraction-resistant fraction IV,yet some Alt-EJ factors(LIG1 and LIG3)remained soluble.The activity of Alt-EJ pathway was decreased after the expression of FEN1 was knocked down,which indicated FEN1 is participating in the error-prone Alt-EJ pathway.Co-IP and Western blot showed that FEN1 participates in the Alt-EJ pathway through PCNA/FEN1/LIG1 complex.Conclusion: we reported the FEN1’s involvement in the Alt-EJ repair of CML.Inhibition of FEN1 showed a positive effect on CML treatment in both IM-sensitive and resistant cells.FEN1 is a promising target in the CML therapeutic option.