Study On The Mechanism Of Drug Sensitivity Of Mutant DNA Polymerase Εpsilon And Defective NER Cell Models

With the development of society and the advancement of technology,human beings pay more and more attention to their own health problems.However,there are still many diseases that threaten people’s life and health.Cancer is one of them.If only the current standard treatment plan is adopted,most patients often cannot obtain the ideal treatment effect,so this requires more effective precision medicine.Precision medicine first requires accurate diagnosis.To implement accurate diagnosis,it is necessary to find effective biomarkers,and then design and develop corresponding effective targeted drugs for biomarkers.This paper mainly studies the key genes in the process of NER repair and proofreading after DNA damage,provides important theoretical and practical basis for finding biomarkers and developing targeted drugs.The main research contents and results are as follows:1.Study on Drugs Targeting POLE Mutant CellsBy analyzing the genome profile of endometrial cancer and colorectal cancer samples and corresponding cell lines,they were divided into DNA polymeraseε（DNA Polymeraseε,POLE）mutant and wild-type,and then to predict and verify using the Cancer Treatment Response Portal（CTRP）:The results showed that,compared with POLE wild-type cell lines（KLE,MFE280,SW948）,POLE mutant cell lines（HEC251,MFE296,HCC2998）were more sensitive to NAE inhibitor MLN4924 in vivo or in vitro.Compared with wild-type KLE,the KLE cell line overexpressing the POLE^P286R plasmid was more sensitive to MLN4924.In conclusion:The mutant DNA polymerase epsilon cells might be regard as a drug target site for MLN4924.2.Study on the Clearance Mechanism of Misfolded Protein Aggregates in POLE Mutant CellsThe POLE mutant and wild-type cell lines were identied after MLN4924 treatment by flow cytometry,cell thermal shift analysis and western blotting.The result showed that the number of cells in the G0/G1 phase of the two types of cell lines was significantly reduced;the BLVRA mutant（P99R）cell lines showed more protein instability characteristics than that of wild-type cell lines;the co-localization of the unstable mutant BLVRA and Nedd8could easily detected in over-expressing BLVRA mutant（P99R）cells;However,it was hard to detect in wild-type cells BLVRA expression.The result showed that Nedd8 could eliminate the expression of ubiquitin by co-immunoprecipitation and immunofluorescence.In conclusion:POLE high mutation might lead to the production of unstable mutant proteins,and these unstable mutant proteins can be eliminated by ubiquitination modification to maintain protein homeostasis.3.Study on the Sensitivity of POLE Mutant Cancer Cells to MLN4924 DrugsProteome stability in the POLE mutant（HCC2998）cell line was significantly lower than that of the wild-type cell line（SW948）by the cell thermal shift analysis（CETSA）.Proteome stability in a stable strain of mutant POLE^P286RMCF-10A showed similar result.Compared with the control group,sublethal drug-induced proteome instability cell lines（POLE wild-type）increased sensitivity to MLN4924;This sensitivity was reversed in the over-expressing HSPA8 cell line（POLE mutant）.ER stress markers was detected after MLN4924 treatment by Western blotting which indirectly implied the misfolded/unstable proteins were accumulated in the cells.In addition,the results showed that immunogenic cell death was observed such as surface calcium and ATP release.In conclusion:Protein instability in POLE mutant cancer cells increases the sensitivity to MLN4924.4.Study on the Determination of POLE Mutation by a Sample Combination MethodTo divide the multiple samples and to analyze several groups according to a specific combination,each sample was divided into two groups,and if and only if each sample was analyzed twice by Di-nucleotide pyrosequencing（detection limit 3%）.To judge the information of the specific sample according to the relative peak signal of pyrosequencing and combination rule.In conclusion:This method is suitable to detect a set of samples containing only a few positive samples（the best is one）,which provided rapid,accurate,and low-cost analysis for a large of samples.This combination method could quickly detect POLE mutations in clinical samples and determine the specific analysis information of a single sample.5.Study on the Establishment and Application of Defective NER Cell ModelFive genes（XPA,XPC,ERCC4/XPF,ERCC5/XPG and ERCC6/CBS）that play a key role in the nucleotide excision repair（NER）repair mechanism were selected,dNER scores of a panel of breast cancer cell lines were established according to dNER gene exprssion signature of kockdown-MCF-10A cell line.From each breast cancer subtypes,we selected an NER deficient（D）andNER intact（I）cell:Basic/triple negative HCC1806（D）and MDA-MB-231（I）,luminal MCF7（D）,MDA-MB-361（I）,HER2 type SKBR3（D）and MDA-MB-453（I）cell lines for functional analysis.The results showed that the defective NER cell line was sensitive to cisplatin drugs and ultraviolet radiation;The combination of GSK3 inhibitor and cisplatin increased the sensitivity to the intact NER cell line;We discovered that the novel drugs CDK9 inhibitor and myriocin were sensitive to the defective NER cell lines.In conclusion:Gene expression signatures could be used to predict defect nucleotide excision repair and new therapeutic drugs.In summary,this thesis established a DNA polymeraseεmutant cell model and found that POLE hypermutation could be used as a potential biomarker;This thesis successfully predicted and confirmed novel synthetic lethal drug that was sensitive to defective NER cell lines by constructing a defective nucleotide excision repair（dNER）cell model.Futhermore,a potential drug that had a synergistic effect with cisplatin had been discovered by using dNER cell model.The above two cell models could be used as a platform for drug screening,providing important theoretical and practical basis for preclinical screening of potential targeted drugs and precision treatments.