Genetic Disruption of PAK prevents NF2-deficient schwannomas and hearing loss

描述(申请人提供)：本申请中提出的研究利用了一种新的NF2小鼠模型(Postn-CRE；NF2flx/Flox小鼠)，该模型紧密概括了人类NF2疾病的重要方面，包括完全穿透性神经鞘瘤表型，以及由前庭神经鞘瘤发展引起的听力和前庭损害。利用这个小鼠模型，我们采用了一种候选方法来确定NF2下游的哪些蛋白是神经鞘瘤发生所需的关键信号中间体。以前的研究已经确认NF2是一种内源性的蛋白激酶PAK1的抑制因子，并且在原发的人神经鞘瘤样本中观察到PAK1活性的升高。重要的是，PAK1激酶活性与NF2缺乏的肿瘤发生的相关性还没有利用自发的体内神经鞘瘤模型来探索。为了解决这个问题，我们让系统内的PAK1基因敲除小鼠(Pak1-/-)与Postn-CRE；Nf2flx/Flox小鼠产生Postn-Cre；Nf2Flox/Flox小鼠；Pak1-/-小鼠。在初步研究中，我们观察到，在Postn-CRE；Nf2flx/Flox小鼠中观察到，Pak1的基因消融可以防止肿瘤发展和前庭神经鞘瘤相关的听力损失。在这项提案的目标1中，我们将从听觉功能、肿瘤发展和生存方面综合表征Postn-CRE、Nf2flx/flx小鼠和pak1-/-小鼠的表型。对小鼠听力的功能评估将在很大程度上通过点击和音调诱发的听觉脑干反应(ABR)测试来确定。鉴于PAK1的S作为致癌信号通路复杂网络中的关键节点，目前尚不清楚在NF2基因缺陷的肿瘤中，哪些通路和底物受到高活性PAK1下游的影响。在目标2中，我们建议阐明PAK1在NF2缺陷肿瘤细胞中激活的关键分子底物，作为进一步表征NF2缺陷细胞类型的基本病理生理学和确定其他治疗靶点的策略。来自其他实验室的多项研究先前已将NF2描述为mTORC信号和蛋白质翻译的新调节因子。NF2调节mTORC激活的机制尚不清楚。鉴于我们的初步数据表明，在Postn-CRE；NF2FLOX/FLOX小鼠中，PAK1是NF2缺陷性神经鞘瘤发生所必需的，我们假设PAKK活性可能是NF2缺陷性细胞类型中mTORC过度活动和非调控翻译的中介。在细胞系中的初步数据表明，S确实是这种情况，PAK激酶活性的减弱降低了mTORC下游翻译活性的生化标记物。Aim 2中的研究旨在通过siRNA敲除、PAK突变结构的慢病毒过表达和PAK的药物抑制来进一步剖析PAK和体外放松调控的mTORC活性之间的这种未知联系。然后，这些结果将通过整个裂解液和免疫组织化学研究在体内得到验证。

DESCRIPTION (provided by applicant): The studies proposed in this application make use of a novel mouse model of NF2 (Postn-Cre; Nf2flox/flox mice) that closely recapitulates important aspects of human NF2 disease, including a fully-penetrant schwannoma phenotype, and hearing and vestibular impairment resulting from the development of vestibular schwannomas. Utilizing this mouse model, we have taken a candidate approach to identify which proteins downstream of NF2 are key signaling intermediates required for schwannoma genesis. Previous studies have identified NF2 as an endogenous inhibitor of the kinase PAK1, and elevated levels of PAK1 kinase activity have been observed in primary human schwannoma samples. Importantly, the relevance of PAK1 kinase activity to NF2-deficient tumorigenesis has not been explored utilizing a spontaneous in-vivo schwannoma model. To address this question, we have intercrossed systemic PAK1 knockout mice (Pak1-/-) with Postn-Cre; Nf2flox/flox mice to generate Postn-Cre; Nf2flox/flox mice; Pak1-/- mice. In preliminary studies, we have observed that genetic ablation of Pak1 prevents tumor development and vestibular schwannoma-related hearing loss observed in Postn-Cre; Nf2flox/flox mice. In Aim 1 of this proposal, we will comprehensively characterize the phenotype of Postn-Cre; Nf2flox/flox mice; Pak1-/- mice in terms of auditory function, tumor development, and survival. Functional assessment of hearing in mice will largely be determined through click and tone-evoked auditory brainstem response (ABR) testing. Given PAK1's role as a crucial node in a complex network of oncogenic signaling pathways, it remains unclear which pathways and substrates are affected downstream of hyperactive PAK1 in NF2-deficient tumors. In Aim 2, we propose to elucidate the key molecular substrates that are activated by PAK1 in NF2-deficient tumor cells as a strategy to further characterize the basic pathophysiology of NF2- deficient cell types and identify additional therapeutic targets. Multiple studies from other labs have previously characterized NF2 as a novel regulator of mTORC signaling and protein translation. The mechanism by which NF2 regulates mTORC activation remains unknown. Given our preliminary data suggesting PAK1 is required for NF2-deficient schwannoma development in Postn-Cre; Nf2flox/flox mice, we hypothesized that PAK kinase activity could be the mediator of mTORC hyperactivity and deregulated translation in NF2-deficient cell types. Preliminary data in cell lines indicates this s indeed the case, with attenuation of PAK kinase activity decreasing biochemical markers of translational activity downstream of mTORC. The studies in Aim 2 are designed to further dissect this unexplored connection between PAK and deregulated mTORC activity in vitro using siRNA knockdown, lentiviral overexpression of PAK mutant constructs, and pharmacologic inhibition of PAKs. These results will then be validated in vivo through whole lysate and immunohistochemical studies.