Therapeutic Regimens For Advanced NSCLC With Wild-type Driver Genes And The Joint Inhibitory Effect Of Two Targets On A549 Cells

Background and objective Lung cancer has the highest incidence and mobility among all the kinds of cancers in our country.It is a serious threat to public health.Non small cell lung cancer（NSCLC）is a major type of lung cancer.For those advanced NSCLC patients who have driver gene mutations,molecular targeted therapies are first line therapy.But for those patients who have wild type or unknown status of driver genes,platinum-based chemotherapy remains the mainstay for the treatment.There are several randomized controlled trials（RCTs）have demonstrated improved outcomes for those patients with the use of maintenance therapy,which can be defined as the continued administration of one or more of the drugs used in first-line regimen or the selection of another non-cross-resistant agent after a specified number of first-line treatment cycles once disease stabilization or maximum tumor response has been achieved.In fact,for patients with advanced NSCLC without driver gene mutations,NCCN（National Comprehensive Cancer Network）have recommended maintenance therapy for nonprogressing patients after a specified number of first-line treatment cycles as standard treatment.However,several first-line treatment schemes and various maintenance therapies are available for selection.The optimal combination of first-line and maintenance treatment schemes remains to be established.In this study,we systematically identified all available RCTs that compared different first-line therapies combined with maintenance therapies and conducted a comprehensive Bayesian network meta-analysis of the effectiveness and safety of the regimens for patients with different types of wild-type or unknown driver genes.The different first-line therapies combined with maintenance therapies based on their efficacy and tolerability have been ranked.In addition,we also evaluated and presented the quality of the evidence for the primary outcome using the Grading of Recommendations Assessment,Development and Evaluation（GRADE）approach.Molecular targeted agents have the advantage of high effectiveness and low toxicity.Thus,finding new molecular targets and developing new molecular targeted agents have been the research hotspots for the treatment of patients with wild type driver genes.NQO1 is an oxidordeuctase and MTH1 is a hydrolase.Both of them are high expression in many types of cancers while low expression in normal tissues.Because of the characteristic,many researchers have investigated the potential of them to become new therapy targets and their bioactivatable drugs to become new molecular targeted agents respectively.But the expression situations of the two proteins in patients with advanced lung adenocarcinoma with wild type EGFR and the relationship between the expressions of the two proteins and patient prognosis remains unknown.In addition,the modest treatment effect and high toxicity of NQO1 bioactivatable drug β-Lapachone showed by a large amount of experimental laboratory data have limited its further application and development.Therefore,we used biological information technology to investigate the relation between the expressions of NQO1 and MTH1 and prognosis in patients with advanced lung adenocarcinoma with wild type EGFR and to understand basically the possible cell signaling pathways involved by NQO1 and MTH1 firstly.Then,based on the main functions of NQO1 and MTH1 and the main mechanisms of their corresponding targeted drugs,we proposed that the NQO1 bioactivatable drug β-lap can have synergetic anti-lung adenocarcinoma cells effect with MTH1 inhibitor TH588.Finally,this study use a series of experiments to validate that NQO1 bioactivatable drugβ-lap and MTH1 inhibitor TH588 indeed can produce synergistic effect to inhibit the proliferation of lung adenocarcinoma cells and to investigate the mechanisms involved.Methods（1）Bayesian network meta-analysis: In this study,we searched the three main English databases and International Clinical Trials Registry Platform.Then,we included all RCTs that include advanced NSCLC patients with wild type or unknown status of driver genes receiving first-line therapy combined with maintenance therapy in at least one treatment arm and randomized patients at onset of first-line therapy.The Cochrane risk of bias tool was used to evaluate the risk of bias of the included RCTs.Bayesian network meta-analysis and single arm meta analysis were used to assess the effectiveness and safety of all the combination regimens of first line therapies and maintenance treatments.We also evaluated and presented the quality of the evidence for primary outcomes using the Grading of Recommendations Assessment,Development and Evaluation（GRADE）approach.（2）The relation between expression situations of NQO1 and MTH1 in lung adenocarcinoma and patients with wild type EGFR and prognosis and possible cell signaling pathways involved by NQO1 and MTH1: Lung adenocarcinoma RNAseq data and their corresponding clinical information from TCGA and GEO databases was collected and standardized.The biological information technology was used to analyse the relation between the expression situations of NQO1 and MTH1 and prognosis in patients with any stage of lung adenocarcinoma and patients with advanced EGFR wild type lung adenocarcinoma.In addition,the possible cell signaling pathways involved by NQO1 and MTH1 were also investigated.（3）To explore the synergetic anti-lung adenocarcinoma cells effect of NQO1 bioactivatable drug and MTH1 inhibitor and the mechanisms: A549（EGFR WT）cells were treated by NQO1 bioactivatable drug β-lap and MTH1 inhibitor TH588.Then,the survival situations of cells were observed.We also used comet assay,immunofluorescence,flow cytometry and Western blot to explore the mechanisms and cell signaling pathways involved by the combination treatments.Results:（1）Network meta analysis: 42 RCTs were included and analysed.All the RCTs contains 17109 advanced NSCLC patients.Among all the patients,12568-14561 were patients with wild type or unknown status of driver genes.The types of driver genes mainly included EGFR,ALK and KRAS.All the RCTs included 47 combination regimens of first line therapies and maintenance treatments,48 first line regimens and35 maintenance treatments.Only 5 RCTs did not report whether they were multicenter or single center research.All the rest studies were multicenter.14 studies included patients from single countries,most of them were from China and America.All the rest studies were carried out in multiple countries.The average age or median age of patients included in all the RCTs was over 55.The mean value or median value of follow up time was between 8.9 months and 49 months.The results of network meta-analysis showed that for KRAS-patients,based on the current evidence,compared with chemotherapy only combined with Atezolizumab or Bevacizumab and continued administration of Atezolizumab or Bevacizumab,the combination of chemotherapy and Atezolizumab and Bevacizumab and continued administration of Atezolizumab and Bevacizumab can improve the PFS.But the quality of evidence was low.In addition,based on the current evidence,there was no significant difference in OS among all the treatment regimens.For EGFR/ALK-patients,chemotherapy combined with Pembrolizumab and continue to use Pemetrexed and Pembrolizumab was the optimal treatment regimen for non-squamous cell carcinoma without EGFR or ALK mutation.The efficacy of this regimen was superior than chemotherapy（HR=0.34,95%CI:0.18-0.67,moderate quality）and most other regimens.In addition,this regimen can improve patients’ outcomes irrespective of the expression of PD-L1.For patients with squamous cell cancer,there was only limited data.Based on those data,the results showed that compared to chemotherapy,Nivolumab combined with Ipilimumab was the optimal treatment（HR=0.49,95%CI:0.30-0.79）,but the quality of evidence was low.For those patients with wild type or unknown status of EGFR/ALK,there was no difference in OS among all the treatment arms.But for the influence on PFS,compared to chemotherapy,chemotherapy combined with Atezolizumab and continued administration of Atezolizumab was better（HR=0.71,95%CI:0.6-0.85,moderate quality）.（2）Bioinformatics analysis: The results of bioinformatics analysis showed that compared to normal tissues,NQO1 and MTH1 were highly expressed in advanced wild type EGFR lung adenocarcinoma samples and the higher expression of NQO1 was related to the poorer prognosis.But there was no statistically significance between the expression of MTH1 and prognosis.In addition,we understood preliminarily possible cell signaling pathways involved by NQO1 and MTH1 which included Reactive Oxygen Species（ROS）,apoptosis,DNA replication,DNA repair and P53 pathway.（3）The synergetic anti-lung adenocarcinoma cells effect of NQO1 bioactivatable drug and MTH1 inhibitor and the mechanisms:NQO1 bioactivatable drug β-lap can produce a synergetic effect with MTH1 inhibitor TH588.They can kill A549 cells and inhibit the proliferation of the cells in an NQO1 dependent manner.The combination treatment can induce G2/M arrest through P53/P21/CDC2 pathway.Finally,they can cause the apoptosis and programmed necrosis of A549 cells.Conclusions For advance non-squamous NSCLC patients without EGFR/ALK mutations,chemotherapy combined with Pembrolizumab and continue to use Pemetrexed and Pembrolizumab as maintenance treatment was the optimal treatment regimen.The efficacy of this regimen was superior than chemotherapy and most other regimens.In addition,this regimen can improve patients’ outcomes irrespective of the expression of PD-L1.MTH1 and NQO1 are potential therapeutic target for lung adenocarcinoma patients without EGFR mutation.NQO1 bioactivatable drug β-lap can produce a synergetic effect with MTH1 inhibitor TH588 to kill A549 cells and inhibit the proliferation of the cells in an NQO1 dependent manner.Finally,the combination treatment can induce apoptosis and necrosis.