RRM1调控核苷类DNMT抑制剂的DNA整合及在肺癌抗甲基化治疗中的差异性作用的机制研究

Lung cancer is ranked top one in cancer morbidity and mortality in China and worldwide usually with poor response to most classical therapy, thus it is utmost critical to explore new therapy and improve sensitivity to classical therapy. Promoter hypermethylation and hypomethylation throughout gene body have been long noteworthy in most cancers which include lung cancer as well. It has been reported that nucleotide analog demethylating agents, Azacytidine(AZA) and Decitabine(DAC), can be metabolized into 5’-aza-deoxycytidne triphosphate and then incorporated into genomic DNA where the 5’-aza group can covalently trap and inactivate DNA Methyltransferases (DNMTs) and therefore demethylation in offspring DNA chain. We have found the expression level of Ribonucleotide Reductase Module 1 (RRM1), but not RRM2 nor other deoxycytidine metabolic enzyme, is significantly correlated with the DNA incorporation rate in a panel of NCI 60 cell lines with special emphasis on human lung cancer cells. Our pilot data showed there is differential effect of RRM1 gene in DAC or AZA treatment: there is strong synergy between three RRM1 inhibitors, while these RRM1 inhibitors are antagonistic with AZA. Furthermore these differential effects between DAC or AZA with RRM1 inhibitors can be validated with rescue assay with deoxycytidine or cytidine respectively. We thus hypothesize that RRM1 gene is the rate-limiting enzyme in DAC/AZA metabolism and their therapeutic effect. In this proposal we plan to use shRNA lentiviral constructs to knockdown RRM1 and RRM1-ORF lentivirus to increase RRM1 expression and the holo-enzyme activity in several lung cancer cell lines. We will use our newly developed LC-MS/MS method to quantitate DAC/AZA incorporation, its differential effect in DNA demethylation and epigenetic therapeutic effects. We further plan to explore the rate-limiting and differential effect of RRM1 in cytotoxicity, DNMT1 protein trapping, cell cycle change, long-term clonogenic survival, which all will be counter proved by deoxycytidine or cytidine rescue assay.As a summary our projects will validate the rate-limiting and differential effects in epigenetic therapy of DAC and AZA. Our findings will comprise rational adjuncts to these epigenetic drugs ripe for further pre-clinical and clinical trial in lung cancer.

肺癌病人常存在异常甲基化。Decitabine（DAC）/Azacitidine(AZA)，可作为DNMT抑制剂逆转DNA甲基化。我们前期发现同一DAC浓度下不同肿瘤株中DAC DNA整合有明显差别，将之与cBio Portal相关代谢酶数据库比对发现RRM1与其关联；并测到RRM1抑制剂联合DAC起强协同作用，对AZA起强拮抗作用。据此提出RRM1可能是DAC/AZA的DAC DNA整合差异的关键分子。本课题拟建过表达及沉默RRM1稳转肺癌细胞系，再以LC-MS/MS法观察DAC/AZA的DAC DNA整合率，甲基化水平变化；观察去甲基化和抗肿瘤疗效变化；并以胞苷和脱氧胞苷挽救试验反证。阐明RRM1是否调控DAC/AZA的差异性DNA整合，从而影响去甲基化及抗癌效能，起限速酶作用。为开拓相关肺癌临床试验和指导个体化用药奠定基础。

肺癌病人常存在异常甲基化。Decitabine（DAC）/Azacitidine(AZA)，可作为DNMT抑制剂逆转DNA甲基化。我们前期发现同一DAC浓度下不同肿瘤株中DAC DNA整合有明显差别，将之与 Bio Portal相关代谢酶数据库比对发现RRM1与其关联；并测到RRM1抑制剂联合DAC起强协同作用，对AZA起强拮抗作用。据此提出RRM1可能是DAC/AZA的DAC DNA整合差异的关键分子。本课题建过干扰RRM1稳转肺癌细胞系，再以LC-MS/MS法观察DAC/AZA的DAC DNA整合率，甲基化水平变化；观察去甲基化和抗肿瘤疗效变化。本研究证实RRM1能差异性调控DNMT抑制剂的DNA整合，进而影响去甲基化效果及抗癌疗效，起关键限速酶作用。故而从基因功能和遗传学角度来阐明RRM1可作为DAC/AZA疗效的关键标记物，为开拓相关肺癌临床试验和指导肺癌个体化用药奠定科学基础。