Biologic and therapeutic impact of the AKT1E17K mutation in meningiomas

脑膜瘤是一种常见的颅内肿瘤，其中相当大比例的患者在手术和放疗的基础上可以从药物治疗中受益，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。我们小组最近在体外和体内证明了mTORC1抑制剂是脑膜瘤有效的治疗选择。遗传因素对脑膜瘤的生物侵袭性和化疗敏感性的作用尚不清楚。这甚至适用于肿瘤抑制基因NF2(Merlin)，尽管该基因功能丧失的频率很高。最近，AKT1基因的致癌突变E17K(AKT1E17K)导致该激酶的结构性激活，在一些没有NF2缺失的脑膜瘤中被发现是一种体细胞突变，提示AKT1驱动的一条独立的促肿瘤途径。AKT1的这种结构性激活是非常有趣的，因为AKT1与两个mTOR-复合体双向相互作用，并有望改变mTOR相关的生长调节和化疗敏感性。在拟议的项目中，我们将分析a)AKT1E17K在调节mTOR复合体和脑膜瘤细胞mTOR依赖的特征(即体外增殖、黏附、迁移、侵袭、克隆形成和化疗敏感性)中的作用，b)在小鼠模型中AKT1E17K对荷瘤裸鼠脑膜瘤的致瘤特性和生长动力学的影响，以及c)后一种参数在体内对AKT1、mTORC1或mTORC1/2的双重抑制物的反应。体内实验将包括人类肿瘤细胞的颅内异种移植，以及遗传诱导的小鼠脑膜瘤模型。后者的特征是脑膜表达AKT1E17K或AKT1wt。小鼠模型的产生需要产生两个小鼠品系，它们在所有细胞中都含有适当的AKT1转基因基因，并被一个漂浮的转录停止信号(TPA)沉默。它们将与CRE驱动小鼠杂交，后者在脑膜特异性前列腺素D2合成酶(PGDS)启动子的控制下表达Cre。我们希望实现AKT1wt或AKT1E17K在小鼠脑膜中的特定表达，从而能够通过这种癌基因对脑膜瘤的潜在致瘤作用进行建模。

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.