利用iPS技术研究复发T-细胞急性淋巴细胞白血病（T-ALL）细胞亚克隆中基因变异特征及其耐药复发机制

Children T-ALL Leukemia is a blood cancer with the high rates of relapse (40-50%). Although stem cell heterogeneity and gene mutations in T-ALL leukemia are thought to be the main reasons of its drug resistance and relapse, the molecular mechanisms of T-ALL replase remain elusive. Our preliminary data showed that compared with the primary T-ALL sample, a lot of gene mutations were found in 5 pair relapse samples after genome sequencing.It is hard to dissolve which one mutation is the major important for T-ALL relapse. iPSC technology is helpful to solve this. In general,an iPS cell lines is generated from a single cell, a couple of different iPS cell lines derived from one patient sample can stand for the leukemia stem cell heterogeneity. Using these different iPS cells is easy to address the relative of stem cell heterogeneity and gene mutations with drug resistance and relapse. Our data published have showed that iPSCs derived from mouse leukemia cells have the full developmental ability as ES cell to generate chimeric and iPS mice, and these mice will be died within one month with leukemia, indicating that the iPS cells derived from the leukemia cells will re-develop leukemia. iPS cells and their derived-differentiation cells will be the good materials to study the mechanism of drug resistance and relapse in vitro and in vivo. In this proposal, we will use iPS technology to establish a number of different iPS Cell lines from the same T-ALL patient sample.This in vitro patient-derived stable and sensitive platform is useful to dissolve the cell heterogeneity with drug resistance and relapse. Combined with DNA-Seq technology, it is good way to elucidate gene mutations and drug resistance. Moreover,it is very helpful to design patient's personal targeted drug treatment.

T细胞急性淋巴细胞白血病(T-ALL)复发率高、预后差，是临床亟待解决的科学难题之一。一般认为白血病的耐药复发与白血病细胞的基因异质性有关，但目前缺乏系统性的研究。我们预实验测序结果表明，与初发T-ALL标本相比，5对复发标本中基因突变数量都显著增加，这给判断主导耐药基因突变造成困难。我们拟利用iPSC技术解决这一难题：一个iPS细胞系来自同一个细胞，不同的iPS细胞系之间的基因异质性代表同一标本亚克隆之间的差异；而且iPS细胞分化后又会重现白血病细胞的药物敏感性和疾病特征。本项目将在前期测序结果的基础上，建立同一标本的多个iPS细胞系，检测iPS细胞系内存在的与耐药可能相关基因突变；再将iPS细胞分为不同的亚克隆群，比较不同白血病亚克隆群之间的耐药性与基因突变的关系，从而找到主导T-ALL耐药的特异基因突变及相关分子机制，为阐明T-ALL的耐药复发机制提供全新研究策略和手段。

T急性淋巴细胞白血病(T-ALL)复发率高、预后差，是临床亟待解决的科学难题之一。一般认为白血病的耐药复发与白血病细胞的基因异质性有关，值得深入研究。揭示白血病的耐药复发机制对临床治疗具有重要的指导意义。.通过本项目的研究，取得如下成果：1）建立系统分析T-ALL 白血病亚克隆基因突变与耐药性研究的全新技术平台。经过我们的努力，完成了技术的优化，文章相继在cellular reprogramming（IF=1.43）和stem cell research & therapy（IF=4.963）年发表2篇SCI论文； 2）在白血病耐药机制研究中，在白血病细胞系上研究发现PRPS2的突变通过影响PRPS1的结构，引起白血病细胞对6-MP和6-TG的耐药性。该部分研究结果在投稿过程中。3）在肿瘤发生发展机制方面相继发表标注文章7篇SCI论文，主要对胶质瘤以及朗格罕氏综合征等治病机制进行研究。我们对89例朗格罕氏综合征病人样本中测序分析，发现了一个新的融合基因PLEKHA6-NTRK3，对这个病人临床以及细胞学分析发现，该融合基因可以促进肿瘤细胞的增殖以及激活RAS-RAF-MEK-ERK信号通路。该部分内容已经在international Journal of cancer（IF=7.36）杂志上发表。在胶质瘤中，我们发现Trim家族中的Trim24对胶质瘤的发生发展起着重要作用。其是H3K23ac结合蛋白，我们研究发现其是STAT3的转录共激活因子，起着招募和稳定STAT3与染色质的结合，并且受EGFR调控，促进STAT3下游基因的表达，该部分数据已经发表在Nature communications（IF=12.124）和Cancer Research（IF=9.122）上。我们还发现Trim59的表达和胶质瘤的恶性程度成正相关；是受EGFR激素诱导调控，并且是通过SOX9来调控Trim59的表达的；并且发现Trim59可以和STAT3互作，并且定位在细胞核里，发现Y218/Q221这2个位点是Trim59-STAT3结合的关键位点。通过STAT3片段与Trim59点突变结合分析，也证明TRIM59促进STAT3活性是通过抑制STAT3和TC45的结合。该部分数据已经发表在Cancer Research（IF=9.122）杂志上等等。