延胡索酸酶基因缺陷细胞的ADSL反转引起的核苷酸代谢异常

Loss of function of fumarate hydratase (FH) can lead to hereditary leiomyomatosis and renal cell cancer (HLRCC), but the mechanism still remains elusive. In our previous studies, the intermediates in purine de novo biosynthesis, such as SAICAR, adenylosuccinate and succinoadenosine are enormously augmented in FH knockout mice and FH-/- cells. The flux analysis shows that ADSL enzyme takes a reverse reaction, and the metabolomics study demonstrates that dNTP pool is significantly decreased that is required by DNA replication and damage repair. These data suggested that the loss of FH unswervingly causes the dysfunctional nucleotide biosynthesis. Its specific principle and consequence need to be answered. Based on the achieved data, we would use the metabolomics approaches combined with cellular biology to elaborate the mechanism underlying regulation and effect of reversal of ADSL on the purine biosynthesis, to explain the interaction between ADSL reversal and one-carbon metabolism and pyrimidine biosynthesis. The characteristic aspartate auxotroph jeopardizes further the reversal of ADSL on the metabolism of nucleotides. It is critical to understand by what mechanism the abnormal nucleotides metabolism will lay influence on the DNA replication, damage repair and genome integrity and stability. As a result, it will provide a new way to think that oncometabolite fumarate is necessarily responsible for cancer cell metabolic reprogramming. It will also provide new theory basis that the cause of FH loss leading to HLRCC and earlier clinical diagnostics.

延胡索酸酶基因（FH）的失活可导致遗传性平滑肌瘤病和肾细胞癌综合症（HLRCC），但其分子机制欠明。我们前期研究发现在FH缺陷小鼠和FH-/-细胞中，嘌呤从头合成的中间产物SAICAR, 腺苷酸基琥珀酸和琥珀酰腺苷显著升高。代谢流实验表明了其中的关键酶ADSL发生逆转反应，代谢组学实验发现了DNA复制和损伤修复所需的dNTP显著降低。以上结果提示FH缺陷可造成核苷酸代谢异常，其具体机制与影响亟待研究。本项目拟在前期工作的基础上，通过代谢组学和细胞生物学等手段阐明ADSL反转在嘌呤合成途径的作用和影响，探究天冬氨酸缺乏和ADSL反转的双重压力对核苷酸代谢的影响，揭示核苷酸代谢异常对DNA复制和基因组稳定性的影响，为致癌代谢物（oncometabolite）延胡索酸在FH缺陷细胞的代谢重编程调控提供新认识，为FH失活导致HLRCC的致病机理和临床诊断提供理论依据。

延胡索酸酶（FH）是三羧酸循环的一个关键酶，催化一分子延胡索酸和水可逆地转化成苹果酸。FH大部分存在于线粒体中，参与三羧酸循环和呼吸链电子传递；还有一小部分存在于细胞浆内，主要参与氨基酸/尿素循环代谢。线粒体生产了细胞大约90%的ATP和还原产物，因此被认为是真核细胞唯一的能量工厂。FH基因突变会导致三羧酸循环的部分停滞和线粒体功能障碍，进一步会引发癌症，但是其机制仍然不清楚。功能缺失（Loss Of Function）的种系杂合突变FH会导致遗传性平滑肌瘤病和肾细胞癌综合征(hereditary leiomyomatosis and renal cell cancer ，HLRCC)，突变位点位于染色体1q42上。目前文献报道最多的是FH R190H和E319Q突变。我们发现了13个未报到的全新突变。HLRCC是一种常染色体显性遗传性肾癌，在临床上，罹患疾病的个体主要为皮肤平滑肌瘤，约占患者总数的76%，有单发或者多发性肿瘤，患者平均年龄25岁。女性患者几乎都有子宫平滑肌瘤，年龄18-52岁不等。另外，在10%-16% 的HLRCC患者中会出现II型乳头状肾癌的并发症，加重了肿瘤的侵袭性和早期转移。在我国，肾癌是泌尿生殖系统中致死的第一大肿瘤，占成人恶性肿瘤的2%~3%，占小儿恶性肿瘤的20%左右，有遗传性特征的肾癌占所有肾癌的5%。我们发现并验证了FH缺陷导致的ADSL发转在嘌呤合成途径中的重要机制，发现其代谢中间体在病人血浆中有异常的升高，对FH缺陷细胞的DNA合成有显著的负调控。因此，本项目的研究意义，不仅对治疗遗传性肾癌有重要意义，还可以更好地认识代谢与肿瘤发生发展以及转移的分子生物学基础，为早期诊断、早期干预和精准治疗提供新的思路。