RAS基因突变对脱氧胞苷激酶介导的ARA-C的敏感性影响及其分子机制研究

Cytarabine(Ara-C)is one of major conventional chemotherapy durgs for acute myeloid leukemia,but the dose gap between the regimens is nearly 100 folds. Treatment with low-dose Ara-C may fail to achieve remission while high-dose may associate with toxic side effects or complications.So far,the indicative molecular marker for distinguishing the low- or high-dose regimen is rare.The recent study showed that AML patients carrying mutRAS benefited from higher cytarabine doses more than wtRAS patient, and our preliminary work found that the cell line harbouring ras mutations may disable the up-regulation of dCK in low-dose cytarabine compared to the wild-type. Here we propose to establish a stable transfection cell line U937 with RAS mutation, then compare the differences of the sensitivity to Ara-C and expression and fuction levels of dCK between the mutant and wildtype cell.We will also study the transcriptional regulatory mechanisms by selecting potential transcription factors such as Sp1 and AP1 through ChIP and luciferase, and detect the phosphorylation levels of dCK to realize the post-translational modification.If permitted,this project is going to innovate the machanism of the difference in response to low- or high-dose Ara-C with RAS mutations, and provide theoretical and experimental basis for tumor individualized treatment and multi-drug resistance study.

阿糖胞苷(Ara-C)作为急性髓细胞白血病（AML）的常规化疗药物，它在临床上所使用的剂量相差近100倍。部分患者使用低剂量Ara-C可能导致治疗无效，而高剂量Ara-C则可能引起更多的并发症，但能提示选择高或低剂量Ara-C的分子标志物甚少。最近有研究证实RAS基因突变的AML患者受益于高剂量Ara-C，而我们发现RAS突变AML细胞与野生型相比在低剂量Ara-C处理时可能无法使脱氧胞苷激酶（dCK）表达上调。本项目拟在前期工作基础上：1）建立RAS突变稳定转染的U937细胞，比较转染前后RAS对Ara-C敏感性和dCK表达及功能的影响；2）运用报告基因检测等技术初步阐明Sp1和AP1等转录因子对dCK的转录调控机制，并通过检测dCK磷酸化探究其翻译后修饰的调控。本项目拟创新性地阐述RAS突变导致对高低剂量Ara-C反应差异的新机制，为AML个体化治疗及耐药机制研究提供理论和实验基础。

阿糖胞苷用于急性髓系白血病的治疗已经有超过40年的历史，但是小剂量阿糖胞苷的治疗作用机制仍未可知。在此项研究中，我满发现小剂量阿糖胞苷可能通过自噬产生治疗作用。通过对白血病细胞株U937及HEL的研究发现阿糖胞苷的治疗作用是基于时间及剂量依赖的；在低剂量处理24h后，我们在U937细胞株中发现其LC3及Beclin1表达升高，出现自噬现象并伴有Akt-mTOR通路活化。再加入自噬抑制剂3-MA及CQ后，自噬及分化均被逆转，Beclin1表达降低。综上所述，小剂量阿糖胞苷可能通过自噬在AML中起到治疗作用，Akt-mTOR通路在上述过程中起到关键作用。