Biologic and therapeutic impact of the AKT1E17K mutation in meningiomas

脑膜瘤是常见的颅内肿瘤，其中很大一部分患者除了手术和放疗外还可受益于药物治疗，尤其是侵袭性脑膜瘤亚型。然而，到目前为止，还没有任何治疗方案被证明是有效的。我们的研究小组最近在体外和体内证明mTORC 1抑制剂是一种有效的脑膜瘤治疗选择。遗传因素在脑膜瘤生物侵袭性和化学敏感性中的作用还不明确。这甚至适用于肿瘤抑制因子NF 2（梅林），尽管该基因的功能丧失频率很高。最近，AKT 1基因中的致癌突变E17 K（AKT 1 E17 K），导致该激酶的组成性激活，已被确定为一部分脑膜瘤中的体细胞突变，而没有NF 2损失，这表明一个独立的AKT 1驱动的肿瘤促进途径。AKT 1的这种组成性激活是非常有趣的，因为AKT 1与两种mTOR复合物双向相互作用，并且可以预期改变mTOR相关的生长调节和化学敏感性。在所提出的项目中，我们将分析a）AKT 1 E17 K对脑膜瘤细胞的mTOR复合物和mTOR依赖性特征的调节作用，即体外增殖、粘附、迁移、侵袭、集落形成和化学敏感性，B）在小鼠模型中AKT 1 E17 K对荷瘤裸鼠中脑膜瘤的致瘤特性和生长动力学的影响，和c）后一参数对AKT 1抑制剂、mTORC 1抑制剂或mTORC 1/2双重抑制剂的体内反应。体外研究将基于表达突变体和野生型AKT 1的同基因细胞系。体内实验将包括人肿瘤细胞的颅内异种移植物，以及遗传诱导的小鼠脑膜瘤模型。后者的特征在于AKT 1 E17 K或AKT 1 wt的脑膜表达。小鼠模型的产生需要产生两种小鼠品系，其在所有细胞中含有适当的AKT 1转基因，通过floxed转录终止信号（tpa）沉默。它们将与Cre驱动小鼠杂交，Cre驱动小鼠在脑膜特异性胰蛋白酶-D2-合酶（PGDS）启动子的控制下表达Cre。我们期望在小鼠脑膜中实现AKT 1 wt或AKT 1 E17 K的特异性表达，从而允许通过该癌基因对脑膜瘤的潜在肿瘤启动效应进行建模。

Meningiomas are frequent intracranial tumours, in which a significant proportion of patients may benefit from pharmacological treatment in addition to surgery and irradiation, especially in aggressive meningioma subtypes. However, so far no treatment scheme has been proven to be effective. Our group has recently demonstrated in vitro and in vivo that mTORC1 inhibitors represent an effective meningioma treatment option. The role of genetic factors for biologic aggressiveness and chemosensitivity of meningiomas is not well defined. This applies even to the tumour suppressor NF2 (merlin), despite the high frequency of functional losses of this gene. Recently, the oncogenic mutation E17K in the AKT1 gene (AKT1E17K), which leads to a constitutive activation of this kinase, has been identified as a somatic mutation in a fraction of meningiomas without NF2 loss, suggesting an independent AKT1-driven tumour-promoting pathway. This constitutive activation of AKT1 is highly interesting, because AKT1 interacts bi-directionally with two mTOR-complexes and can be expected to modify mTOR-associated growth regulation and chemosensitivity. In the proposed project, we will analyse a) the role of AKT1E17K for the regulation of mTOR complexes and mTOR-dependent features of meningioma cells, i.e. proliferation, adhesion, migration, invasion, colony formation, and chemosensitivity in vitro, b) in mouse models the impact of AKT1E17K for tumorigenic properties and growth kinetics of meningiomas in tumour-bearing nude mice, and c) the response of the latter parameters in vivo towards inhibitors of AKT1, of mTORC1, or dual inhibitors of mTORC1/2. The in vitro studies will be based upon syngenic cell lines expressing mutant and wild-type AKT1. In vivo experiments will include intracranial xenografts of human tumour cells, as well as a genetically induced mouse meningioma model. The latter will be characterized by meningeal expression of AKT1E17K or AKT1wt. The generation of the mouse model requires the generation of two mouse strains, which contain in all cells the appropriate AKT1 transgene, silenced by a floxed transcriptional stop signal (tpa). They will be crossed with Cre driver mice, which express Cre under the control of the meningeal-specific prostaglandin-D2-synthase (PGDS) promoter. We expect to achieve a specific expression of AKT1wt or AKT1E17K in mouse meninges, allowing modelling of a potential tumour-initiating effect for meningiomas by this oncogene.