靶向DNA甲基化阅读器治疗T-ALL的效应及分子机制研究

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with poor prognosis and high relapse rate, but still lack of high efficient and low toxic drugs. Recently, next generation sequencing showed that T-ALL patients were characterized by distinct DNA methylation signatures that exhibited significant correlation with gene expression profiles. It indicated that DNA methylation might exert a key role in T-ALL leukemogenesis. The repressive effects of DNA methylation on gene expression are mediated by methyl-CpG binding proteins (MBD). Compared to traditional DNA methylation inhibitors, targeting MBD2 which was the reader of specific DNA methylation would create attractive new approaches for abnormal DNA methylation in tumors. Our previous study also provided a clue to this hypothesis. Therefore, we assume that MBD2 might play an important role in the pathogenesis of T-ALL and potentially serve as an excellent novel therapeutic target. In this study, we will employ MBD2 knock-out mice models to investigate the gene expression changes in T lymphocyte development made by MBD2. Additionally, we will study the impact of MBD2 on T-ALL by adopting a retrovirus-induced mouse model. Then, by means of gene expression profiling and bioinformatic tools, we will explore the DNA methylated genes or pathways which play an essential role in T lymphocyte development and leukemogenesis. The key genes regulated by DNA methylation will also be validated in clinical specimens. This study will decode the epigenetic mechanism and provide novel therapeutic targets for T-ALL leukemia.

T-ALL是一种高度侵袭且易复发的白血病，目前尚缺乏高效低毒的靶向药物。新一代测序技术显示T-ALL患者具有独特的甲基化图谱，提示DNA甲基化可能在T-ALL发病中起关键作用。MBD2蛋白是识别特定DNA甲基化信息的重要媒介，与传统的DNA甲基转移酶抑制剂相比，阻断MBD2的读取功能，是干预DNA甲基化调控的全新思路。本项目组前期研究也表明MBD2可能参与T-ALL的发病。基于此，我们提出：靶向甲基化阅读器MBD2，可能为治疗T-ALL提供新的切入点。本研究利用MBD2敲除小鼠，从造血重建及微环境上探索MBD2对T细胞分化发育的基因表达模式的作用；同时通过逆转录病毒构建T-ALL小鼠模型，揭示MBD2敲除对小鼠白血病发生发展的影响。并利用基因芯片和生物信息学，结合临床标本，发掘在T-ALL发病和T细胞分化中受DNA甲基化调控的关键基因或信号通路，为高效低毒地治疗T-ALL开辟新的方向。

背景：T-ALL是一种高度侵袭且易复发的白血病，目前尚缺乏高效低毒的靶向药物。新一代测序技术显示T-ALL患者具有独特的甲基化图谱，提示DNA甲基化可能在T-ALL发病中起关键作用。靶向DNA甲基化治疗已经在多种肿瘤中取得了突出的疗效。MBD2蛋白是识别特定DNA甲基化信息的重要媒介，与传统的DNA甲基转移酶抑制剂相比，阻断MBD2的读取功能，是干预DNA甲基化调控的全新思路。.结果：本研究利用MBD2敲除小鼠，探索MBD2对T细胞分化发育及急性T淋巴细胞性白血病中的作用。发现MBD2敲除后，小鼠HSC自我更新能力下降，但不影响HSC的数量和比例；外周血T和B淋巴细胞减少；小鼠T细胞在胸腺DN阶段的分化受阻，通过下调TCF7来抑制Wnt通路的活性。同时通过逆转录病毒构建T-ALL小鼠模型，利用基因芯片和生物信息学，揭示MBD2敲除后可能促使Wnt通路抑制分子的表达上调，导致核内β-catenin降低，最终引起Wnt通路活性下降，进而影响到了白血病细胞的增殖和周期，延缓白血病发病。同时，利用甲基化测序发现在T-ALL临床标本中，一系列的Wnt通路上游抑制分子处于高甲基化状态。并在174例成人T-ALL临床样本中，通过GSEA富集及Pearson相关性分析揭示Wnt通路与MBD2表达存在明显的正相关关系。此外，利用CRISPR/Cas9技术建立人T-ALL细胞系MBD2敲除模型，发现MBD2敲除后，Jurkat细胞恶性程度亦减低，Wnt下游公认的关键激活标志β-catenin蛋白的总蛋白和核蛋白表达亦随着MBD2敲除而下降，与小鼠结论相符合。.综上所述，靶向阻断MBD2，使T细胞发育在DN阶段出现了分化阻滞，且在T-ALL白血病中，敲除MBD2阻断了Wnt通路抑制分子的高甲基化状态的读取，使高甲基化的肿瘤抑制基因表达上升，并影响其下游靶分子的表达，从而延缓白血病的发生发展，为高效低毒地治疗T-ALL开辟新的方向。