核苷类DNMT抑制剂DAC在肺癌抗甲基化治疗中对RRM1基因的依赖性及机制研究

肺癌常异常甲基化，抗甲基化治疗前景可期。核苷酸还原酶(RNR)是连接RNA和DNA代谢途径的限速酶；大亚基RRM1作为RNR的核苷酸结合位点，决定其酶学特异性和活性。我们先前证实降低RRM1水平能提高核苷酸类抗甲基化药物Decitabine(DAC)的DNA整合率，从而协同杀伤肺癌细胞，并且细胞内游离脱氧三磷酸胞苷（dCTP）含量显著降低，其毒性只能被正常脱氧胞苷挽救。据此推论DAC在肺癌抗甲基化治疗中对RRM1存在依赖性机制。本课题拟用RNR酶学反应，LC-MS/MS定量测定，甲基化基因分析等方法，在核苷酸代谢、RRM1遗传操控肺癌细胞、小鼠模型和人肺癌组织四层面，阐明DAC代谢中产物aza-dCDP、aza-dCTP是否能特异性抑制RRM1酶促反应、特异性抑制dCTP生成、提高DAC在DNA的整合率，从而增强抗甲基化这一RRM1抑制和DAC的协同新机制，为开拓该领域临床研究奠定基础。

Lung cancers often have aberrant methylation, and demethylation is a promising new therapy for lung cancer patients. Ribonucleotide reductase (RNR) is the rate-limiting enzyme that connects RNA and DNA metabolic pathways. RRM1 is the big subunit of RNR; it harbors the nucleotide recognition and binding sites which determines the specificity and activity of the holo-enzyme of RNR. We have proved in previous studies that lowering RRM1 level increased decitabine (DAC) incorporating into genomic DNA, exhibited synergistic cytotoxicity with DAC in vitro and in vivo, dramatically decreased the free dCTP content, all of which can only be rescued by supplying cells with deoxycytidine (dC). Our findings pose a novel mechanism that demethylation therapy of DAC in lung cancer is highly dependent on RRM1. We try to further explore these novel findings comprehensively throughout in vitro nucleotide metabolism pathways, lung cancer cell lines with genetic manipulation of RRM1, mouse models, and human lung cancer clinical samples by employing RNR enzymatic reaction and LC-MS/MS methods and genomic methylome assays. We will further elucidate the specificity and efficiency of the intermediate metabolites of DAC, aza-dCDP and aza-dCTP, in inhibiting RRM1 enzymatic activity, reducing dCTP production, DAC incorporating into genomic DNA, and hence for the epigenetic effects. Demethylation of RRM1i and DAC is ripe for further pre-clinical and clinical trial.