Preclinical Testing of PI3K Inhibitors for Vestibular Schwannomas

摘要 Merlin（NF 2）肿瘤抑制基因突变导致良性肿瘤疾病，神经纤维瘤病型 2（NF 2）。这种疾病倾向于个体发展双侧前庭神经鞘瘤（VS）， 进行性听力损失，并可能导致危及生命的脑干压迫。因为手术切除 VS常引起耳聋、面瘫和失衡，有必要开发药物疗法来减缓或 防止VS生长并保护神经功能。我们致力于建立一种体外和体内药物 筛选平台，以确定新的化合物以及FDA批准的药物， 开发/重新用于VS疗法。为了实现这一目标，我们已经创造了小鼠和人类merlin缺陷 Schwann细胞系，并优化其在384孔高通量和高含量测定中的使用， 使用机器人平台筛选大型化合物/药物文库。这种方法鉴定了几种磷酸肌醇- 3激酶（PI3K）抑制剂，其与野生型相比选择性地降低小鼠merlin缺陷型的活力 具有纳摩尔IG50的雪旺细胞。这一初步发现在人类merlin缺陷型雪旺细胞中得到了证实。 用于多种PI3K、双重PI3K/mTOR和PI3K/HDAC抑制剂的线。由于PI3K在细胞内起着关键作用， 增殖，存活和侵袭，目前有15种不同的PI3K抑制剂在临床试验中用于各种 血癌和实体瘤。第一种PI3K抑制剂（idelalisib）于2014年被批准用于白血病。 在这个提议中，我们通过在体外进行PI3K通路抑制剂的系统筛选来推进我们的发现 和体内。该提议的目的是：1）描述PI3K通路抑制剂的文库在降低PI3K通路中的功效。 人merlin缺陷型雪旺细胞系和原代人VS细胞的存活力; 2）测试所述细胞的功效， 新型PI3K抑制剂在新型大鼠异种移植物中减缓移植物扩张并保护听力和平衡 模型，以及3）进行表型、激酶组和转录组分析以揭示分子特征， 细胞和移植物对抑制剂的适应性变化。我们希望获得必要的临床前数据， 支持PI3K抑制剂在NF 2相关VS患者中的潜在用途。

ABSTRACT Mutations in the merlin (NF2) tumor suppressor gene cause the benign tumor disorder, Neurofibromatosis type 2 (NF2). This disorder predisposes individuals to develop bilateral vestibular schwannomas (VS) that cause progressive hearing loss and can cause life-threatening brainstem compression. Because surgical removal of a VS often causes deafness, facial paralysis, and imbalance, there is a need to develop drug therapies to slow or prevent VS growth and preserve nerve function. We have worked to establish an in vitro and in vivo drug screening platform to identify novel compounds as well as FDA-approved drugs that can be developed/repurposed for VS therapies. Toward this goal, we have created mouse and human merlin-deficient Schwann cell lines and optimized their use in 384-well high-throughput and high-content assays in order to screen large compound/drug libraries using robotic platforms. This approach identified several phosphoinositide- 3 kinase (PI3K) inhibitors that selectively reduce viability of mouse merlin-deficient compared to wild-type Schwann cells with nanomolar IG50. This initial finding was confirmed in human merlin-deficient Schwann cell lines for multiple PI3K, dual PI3K/mTOR and PI3K/HDAC inhibitors. Because PI3K plays a critical role in cell proliferation, survival, and invasion, there are currently 15 different PI3K inhibitors in clinical trials for various blood cancers and solid tumors. The first in class PI3K inhibitor (idelalisib) was approved in 2014 for leukemia. In this proposal, we advance our findings by conducting a systematic screen of PI3K pathway inhibitors in vitro and in vivo. The aims of this proposal are to: 1) profile a library of PI3K pathway inhibitors for efficacy in reducing viability of human merlin-deficient Schwann cell lines and primary human VS cells; 2) test efficacy of the advanced PI3K inhibitors to slow graft expansion and preserve hearing and balance in a novel rat xenograft model, and 3) conduct phenotypic, kinome, and transcriptome analysis to reveal the molecular signatures and adaptive changes of the cells and grafts to the inhibitors. We expect to obtain the necessary pre-clinical data to support the potential use of PI3K inhibitors in patients with NF2-associated VS.