靶向mTORC2对MLL急性髓系白血病恶性造血维持的影响及分子机制研究

Acute myeloid leukemia (AML) is a clonal hematopoietic neoplasm characterized by the genetic and molecular diversity. Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the relapse of leukemia. Recent studies had found that the activation of PI3K/PTEN/AKT/mTOR pathway is one of the most common molecular events of acute leukemia onset. Here, we demonstrated that knockdown of Rictor in parts of MLL-rearranged AML cell lines resulted in reduction in colony formation in vitro. We also did functional analyses of Rictor in zebrafish embryonic hematopoiesis, and observed that Rictor knock-down had minor effects on embryonic hematopoiesis. Meanwhile, a higher level of Rictor in MLL-AML cells especially in LICs was revealed compared to healthy BMMNC control. Furthermore, using Rictor conditional deleted mice, and murine MLL-AF9 acute myeloid leukemia, we indicated that Rictor deletion was not contributed to MLL-AF9–induced transformation in vitro , but was required for maintenance of AML hematopoiesis. In this study, we will use both an established murine Rictor conditional deletion model and murine MLL-AF9 acute myeloid leukemia combined with serial transplantation model and limiting dilution analysis in vivo to define whether deletion of Rictor could affect maintaining of MLL-AML hematopoiesis. Meanwhile, Our research will demonstrate an unexpected molecular mechanism of targeting mTORC2 on maintenance of MLL-AML hematopoiesis by microarray and bioinformatics analysis which will provide a new strategy for targeting treatment of MLL-AML.

急性髓系白血病是一类在细胞遗传学及分子生物学上有很大异质性的血液系统恶性克隆性疾病，具有自我更新能力的白血病干细胞是该病容易复发的根源。研究发现，PI3K/PTEN/AKT/mTOR 通路活化，是急性白血病最常见的分子事件之一。在前期研究中，申请人及所在研究组发现 Rictor 分子对部分 MLL急性髓系白血病细胞系体外集落形成能力有明显影响，并观察到 Rictor 封闭不影响斑马鱼胚胎造血的现象，此外检测到 Rictor 高表达于MLL- AML标本尤其是 LSC 群体的现象，并进一步得出 Rictor 敲除不影响 MLL-AML 体外转化却可能影响 MLL-AML恶性造血维持的初步结论。基于前期研究基础，借助 Rictor 基因条件敲除小鼠和 MLL-AF9 逆转录病毒诱导的小鼠 AML 平台，运用连续移植模型和有限稀释分析，探讨Rictor 敲除对MLL-AML 恶性造血维持的影响。

Rictor是维持MLL白血病中白血病干细胞（LSCs）干性所必须的，Rictor敲除影响MLL白血病小鼠LSCs的静息状态，从而导致LSCs耗竭。Rictor敲除导致了MLL LSCs的mTORC1通路反应性活化。mTORC1调控细胞代谢，活化的mTORC1信号促使静息状态的LSCs纷纷进入细胞周期，走向耗竭，并最终成功阻止了小鼠白血病连续移植模型的发病。同时，我们还发现Rictor缺失也引起FoxO3a的活化，反应性活化FoxO3a抑制了Rictor缺失细胞mTORC1活性的过度增高，进而对白血病细胞产生保护作用。同时敲除Rictor并降低FoxO3a的表达能够快速促进MLL LSCs耗竭及阻止发病进程。我们的研究揭示了Rictor-FoxO3a轴在维持MLL白血病LSCs静息状态及干性中的潜在调控作用。