| BackgroundLung cancer is the malignancy with the highest morbidity and mortality rate in China.More than 70%of the patients lost the opportunity of surgery,and chemoradiotherapy becomes the main treatment option.For advanced non-squamous non-small cell lung cancer(NSCLC)without driver mutations,pemetrexed combined with platinum is the preferred treatment option.However,the primary resistance of pemetrexed and the lack of effective predictive markers are major clinical problems at present.Methylthioadenosine phosphorylase(MTAP)is the key enzyme in the salvage synthesis of purines.In vivo,purine can be synthesized by the de novo synthesis pathway under the action of dihydrofolate reductase(DHFR),and it can also be synthesized by the metabolism of methionine via the salvage synthesis pathways with the participation of MTAP.Pemetrexed is a novel multitargeted antifolate that inhibits DHFR blocking de novo synthesis of purines.Therefore,blocking DHFR completely blocks the purine synthesis pathways and interferes with DNA metabolism in MTAPdeficient cells.However,normal cells are not affected by pemetrexed due to the presence of MTAP.This metabolic difference constitutes the basis of anti-tumor.A phase Ⅱ clinical trial showed that for patients with urothelial cancer lacking MTAP,the disease control rate of pemetrexed treatment can reach 67%,which is much higher than the previous data of 8%-28%.However,the study of MTAP in the field of lung cancer is limited.In this study,we found that the high expression of MTAP in advanced lung adenocarcinoma was negatively correlated with the short-term efficacy of pemetrexed,and correlated with KRAS mutation.High expression of MTAP in NSCLC cell lines was common.However,the predictive value of MTAP expression to pemetrexed sensitivity was affected by KRAS mutation.Several studies have shown that MTAP and KRAS interact with each other,but the mechanism is unknown.Genomic analysis of NSCLC showed that KRAS mutation was higher in NSCLC expressing MTAP(p=0.003).Therefore,solving the resistance in MTAP-positive NSCLC with KRAS mutant has become the main clinical problem.Since there were no specific targeted drugs for KRAS mutations when the study was conducted,finding new treatments or combination treatments to bypass KRAS mutations is key to solving the problem.Based on previous studies,radiotherapy(RT)can not only kill tumor cells directly but also generate DNA fragments,promoting the release of IFNs and immune-related chemokine ILs by activating the cGAS-STING pathway.The direct metabolite of methionine,S-adenosylmethionine,is the main donor of DNA methylation,and DNA methylation is an important reason for radiotherapy resistance.Decitabine(DAC)is an inhibitor of DNA methyltransferase(DNMT).DNMT binds to DNA and makes DNA methylation replicate to the daughter chain during DNA replication,while DAC can irreversibly bind to DNMT to block DNA replication and directly inhibit tumor growth.Previous studies have also revealed that RT+DAC can promote T cell proliferation and cytotoxicity by promoting the expression of MHC class Ⅰ molecules and costimulatory signals,while generated a synergistic therapeutic effect.Therefore,NSCLC cell lines with MTAP+/-and KRAS mutation were selected and treated with RT± DAC to explore the mechanism and the feasibility of treatment modality on regulating the sensitivity to pemetrexed.The results showed that the combination therapy can promote the expression of type I IFN by activating the cGAS-STING pathways,and promote the expression of immune-related chemokines,improving the sensitivity of MTAP-positive cells with KRAS mutant to pemetrexed.This study puts forward new therapeutic targets,provides new ideas for the prediction of the curative effect of advanced lung adenocarcinoma and the improvement of the therapeutic effect and provides a direction for further immunotherapy combination and clinical trials.Part 1 Study on the Expression of MTAP and Pemetrexed Chemosensitivity in Lung AdenocarcinomaObjective:To evaluate the correlation between MTAP expression in advanced lung adenocarcinoma and the efficacy of pemetrexed plus platinum chemotherapy.Methods:The patients diagnosed with advanced lung adenocarcinoma via histopathology and imaging study between January 2013 and December 2018 were retrospectively reviewed.All patients were treated initially with the chemotherapy of pemetrexed and platinum.Immunohistochemistry(IHC)was performed to evaluate MTAP expression in the tumor tissue.The IHC score was graded 0-4,5-8,and 9-12,which was calculated based on the staining intensity and the percentages of positive staining tumor cells.MTAP-low expression was defined as IHC score 0-4,while MTAP-high expression was defined as IHC score 5-12.Response was evaluated according to RECIST v1.1.All statistical tests were two-sided,and p<0.05 was considered statistically significant for all analyses.Results:A total of 165 patients were reviewed,with the median age of 59 years.The follow-up ended in March 2019,with the median follow-up period of 32.6 months.MTAP-high expression was detected in 64(38.8%)patients.The results showed MTAP expression was not correlated with gender(p=0.41),age(p=0.11),smoking history(p=0.23),clinical T stage(p=0.34),clinical N stage(p=0.70),and EGFR mutation(p=0.41).In the whole cohorts,no patients achieved complete response.The partial response rate in the MTAP-low group was 64.4%versus 46.9%in the MTAPhigh group.Therefore,the objective response rate(ORR)was significantly worse in the MTAP-high group(p=0.035),as well as disease control rate(79.7%vs.92.1%,p=0.03).Subgroup analyses showed that ORR in the MTAP-high group was significantly lower than that in the MTAP-low group for patients with EGFR-wild type(42.1%vs 73.5%,p=0.04).For patients with KRAS mutations,ORR in the MTAPhigh group was also significantly lower than that in the MTAP-low group(27.3%vs.70.0%,p=0.01).In addition,for patients with MTAP high expression,the ORR in patients with KRAS mutations was significantly lower than that in patients with KRASwild type(27.3%vs.57.1%,p=0.02).Conclusion:The results show that the low expression of MTAP in advanced lung adenocarcinoma is positively correlated with the efficacy of pemetrexed combined with platinum chemotherapy.The sensitivity of patients with MTAP high-expression to pemetrexed was affected by KRAS mutation.Part 2 Effect of MTAP Expression on the Sensitivity of Pemetrexed and Improvement of Therapeutic Sensitivity of MTAP-positive Lung Cancer CellsObjective:1.To evaluate the MTAP expression in lung cancer cell lines,and to investigate the predictive value of MTAP expression on the sensitivity to pemetrexed.2.To assess whether radiation(RT)± DAC could modulate the sensitivity to pemetrexed in MTAP-positive lung cancer cell lines.Methods:In Chapter 1,the background expression of MTAP was detected by Western Blot to evaluate the distribution of MTAP in lung cancer cell lines.Cytotoxicity tests were performed by CCK8 to detect half maximal inhibitory concentration(IC50)to evaluate the sensitivity of pemetrexed in different lung cancer cell lines with different MTAP expression or different pathological types.At the same time,MTAP gene was transfected into A549 or suppressed in H1944 by constructing plasmids through lentiviral packaging.The changes in sensitivity of A549 and H1944 to the treatment of pemetrexed were then evaluated after importing or silencing MTAP.In Chapter 2,the MTAP+/-cell lines with KRAS mutation were treated with RT alone or RT combined with DAC to assess the changes in sensitivity to pemetrexed and the effect of combination treatment on cell proliferation.Results:(1)In Chapter 1,a total of 37 cell lines were used in this study,including one mesothelioma.The majority of cell lines were adenocarcinoma(56.8%,21/37)and squamous cell carcinoma(18.9%,7/37).Among them,MTAP expression was observed in 77.8%(28/36)of cell lines,including 53.6%(15/28)adenocarcinoma and 17.9%(5/28)squamous cell carcinoma.The sensitivity of pemetrexed was performed in 16 cell lines which were selected randomly.The results showed IC50 of cell lines was cross-over regardless of the status of MTAP deficiency or not,or the pathological type.In lung adenocarcinoma cell lines with MTAP expression,partial cells were sensitive for pemetrexed but some are not.However,for A549 introduced with MTAP gene,the sensitivity to pemetrexed significantly decreased compared to A549 with MTAP deficiency.Moreover,after the introduction of MTAP,the proliferation rate of A549 cells was significantly higher than that of A549 primary cells.In addition,IC50 of H1944 to pemetrexed significantly reduced after the silence of the MTAP gene,which means the sensitivity of H1944 to pemetrexed significantly increased.N Those results suggested that there was a correlation between MTAP expression and the sensitivity of lung cancer cells to pemetrexed,but it was affected by potential unknown factors.(2)Based on the information of American Type Culture Collection,driver gene mutations(EGFR,ALK&KRAS)may mask the predictive value of MTAP expression on the efficacy of pemetrexed.However,since only 3 of the 16 cell lines tested in this study were without driver gene mutations,it is impossible to evaluate whether MTAP expression could predict the chemosensitivity of pemetrexed in cell lines without driver gene mutations,as well as the comparison the sensitivity difference of pemetrexed in cell lines with gene mutations or not.Targeted drugs could be directly selected in patients with sensitive driver gene mutations.However,the special spatial structure of KRAS makes it difficult to develop targeted drugs directly tarrgeting KRAS mutations.KRAS may be co-mutated with CDKN2A,which in turn affects MTAP expression.However,there is a lack of relevant studies in the field of lung cancer.Furthermore,it is unclear whether KRAS dominates regulation independently of MTAP genes or whether KRAS influences efficacy of treatment by regulating MTAP gene changes leading to activation of new signaling pathways.Moreover,the main purpose of this study is to solve the problem of treatment resistance in patients with MTAP expression.Consequently,exploring new therapeutic modalities to bypass KRAS mutations becomes a way to solve clinical problems.S-adenosylmethionine,a metabolite of methionine,is a crucial donor of DNA methylation.Decitabine can inhibit DNA methylation by inhibiting DNA methylation transferase.Previous studies also have shown that radiation(RT)combined DAC have synergistic therapeutic effects.Therefore,this study further explores the role of RT±DAC in improving treatment resistance in MTAP-positive cell lines.In Chapter 2,two cell lines(A549 with MTAP deficiency,H1944 with positive MTAP)with KRAS mutations and resistant to pemetrexed were selected for the study.First,IC50 of tumor cells to pemetrexed were examined in 4 groups(control,RT alone,DAC alone and RT+DAC group).The results showed that for A549,there was no significant difference in IC50 between the 4 groups(all p>0.05).While in H1944,RT+DAC significantly improve the sensitivity of H1944 to pemetrexed compared to the control group(282.4 ± 11.5 nM vs.574.9±26.4 nM,p=0.001),while IC50 in RT alone or DAC alone slightly decreased compared to the control group,but there was no statistical difference(all p>0.05).In addition,after pretreatment of the 4 groups of cells with pemetrexed,the three experimental groups were treated with RT,DAC and RT+DAC,respectively,and then the absorbance values of each group were examined daily.The results showed that there was no significant difference in A549 cell line in the absorbance of the 4 groups,indicating that RT alone or DAC alone and RT+DAC failed to modulate the sensitivity of A549 to pemetrexed.In contrast,in the H1944 cell line,the absorbance of the RT+DAC group was significantly lower than that of the control group,indicating that RT+DAC improved the sensitivity of H1944 to pemetrexed.Colony formation assay showed that for A549,the numbers of clone formation in pemetrexed(Pem)+DAC+RT was not significant different compared to that in Pem alone,Pem+DAC and Pem+RT group;however,for H1944 cell line,the clone formation numbers were significantly higher in Pem+RT+DAC compared to that in the remaining three groups.Conclusion:MTAP expression was common in lung cancer cell lines,and its expression was to some extent correlated to the sensitivity of pemetrexed.The predictive value for pemetrexed sensitivity may be affected by driver-gene mutations,particularly KRAS mutation.Radiation plus DAC could improve the sensitivity of MTAP-positive cancer cells to pemetrexed but failed in cells with MTAP deficiency.Part 3 Mechanism of Radiotherapy Combined with Decitabine to Improve the Treatment Sensitivity of MTAP-positive Lung Adenocarcinoma Through Activation of cGAS-STINGObjective:To investigate whether RT+DAC can promote type I interferon(IFN)expression by activating the cGAS-STING pathways to enhance the therapeutic sensitivity in MTAP-positive and KRAS mutated lung cancer cell lines.Further to explore whether cGAS-STING activation could promote immune-related chemokine expression,potentially activating immune responses and providing a theoretical basis for combination immunotherapy.Methods:MTAP(+)or MTAP(-)cell lines with KRAS mutated were selected for RT ±DAC treatment.The expression of cGAS-STING signaling pathway related molecules such as TBK1 and IRF3,and IFN expression were detected by RT-PCR.Meanwhile,the expression of IL-1,IL-6,IL-8 and CXCL10 were detected by RT-PCR.Westernblot was used to detect the expression of cGAS-STING and STING.Results:A549 MTAP(-)and H1944 MTAP(+)cell lines with KRAS mutation were selected for the study.Both cell lines were first treated with RT alone,and the results showed that cGAS,STING,TBK1,IRF3 and IRF7 were elevated upon 12Gy in H1944,whereas no significant elevation upon either 2Gy or 12Gy in A549(IRF3 expression was slightly elevated upon 12Gy).Similarly,IFN-α and IFN-β expression were elevated in H1944 irradiated with 12Gy,but no change in IFN-a and IFN-β expression was observed in A549 irradiated with either 2Gy or 12Gy.IL-1,IL-6,IL-8 and CXCL10 were not elevated in A549 irradiated either 2Gy or 12Gy.Only IL-1α and IL1β expression was elevated in H1944 irradiated with 12Gy,while 2Gy did not cause elevated expression of ILs in H1944.After RT+DAC treatment,no activation of cGAS-STING pathway was observed in A549 either by 2Gy or 12Gy combined with DAC,but IFN-α and IFN-β expression was elevated upon 12Gy,and ILs expression was not significantly changed.However,in H1944,no matter treated with 2Gy or 12Gy plus DAC,cGAS,STING,TBK1,IRF3 and IRF7 were significantly elevated,IFN-a and IFN-β expression was also elevated.Furthermore,IL-1,IL-6,IL-8 and CXCL 10 expression were also significantly elevated.After the inhibition of cGAS expression with G150,cGAS,STING,IFN-α and IFN-β expression did not change significantly in H1944 treated with RT+DAC.Further analysis of the background expression of cGAS-STING in both cell lines revealed that cGAS and STING were negative in A549 and positive in H1944.Conclusion:Radiotherapy combined with DAC can promote type Ⅰ IFN expression to kill tumor cells with MTAP expression by activating the cGAS-STING pathway.The background expression of cGAS-STING may be critical.RT+DAC can also promote ILs expression and potentially activate the host innate and adaptive immunity,providing a theoretical basis for combined immunotherapy in further study. |
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