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

PAK 的基因破坏可预防 NF2 缺陷型神经鞘瘤和听力损失

• 批准号: 9088395
• 负责人: Jeffrey R Gehlhausen
• 金额: $ 4.47万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9088395
• 关键词: Ablation; Acoustic Neuroma; Address; Affect; Auditory; Auditory Brainstem Responses; Benign Schwannoma; Biochemical Markers; Biochemical Pathway; Cell Line; Cloning; Complex; Cranial Nerves; Data; Development; Disease; Distal; Excision; Functional disorder; Genes; Genetic; Genetically Engineered Mouse; Germ-Line Mutation; Health; Hearing; Hereditary Disease; Human; Hyperactive behavior; Impairment; In Vitro; Knockout Mice; Life; Link; Longevity; Mediating; Mediator of activation protein; Medical; Modeling; Molecular; Morbidity - disease rate; Mus; Nerve; Neurilemmoma; Neurofibromatosis 2; Oncogenic; Operative Surgical Procedures; PAK-1 kinase; Pathway interactions; Patients; Peripheral Nerves; Phenotype; Phosphotransferases; Premature Mortality; Proteins; Role; Sampling; Schwann Cells; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Spinal Ganglia; Spinal nerve structure; Surgical complication; System; Testing; Therapeutic; Translations; Tumor Suppressor Genes; Validation; Xenograft procedure; attenuation; cell type; design; hearing impairment; in vitro activity; in vivo; kinase inhibitor; knock-down; meningioma; mortality; mouse model; mutant; neoplastic cell; novel; overexpression; prevent; therapeutic target; tumor; tumorigenesis

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.

描述（由申请人提供）：本申请中提出的研究使用了一种新型NF 2小鼠模型（Postn-Cre; Nf 2flox/flox小鼠），该模型密切概括了人类NF 2疾病的重要方面，包括完全渗透性神经鞘瘤表型以及前庭神经鞘瘤发展导致的听力和前庭损害。利用这种小鼠模型，我们已经采取了一种候选的方法来确定哪些蛋白质的NF 2下游的神经鞘瘤发生所需的关键信号中间体。以前的研究已经确定NF 2作为激酶PAK 1的内源性抑制剂，并且在原发性人类神经鞘瘤样品中观察到PAK 1激酶活性水平升高。重要的是，PAK 1激酶活性与NF 2缺乏肿瘤发生的相关性尚未利用自发性体内神经鞘瘤模型进行研究。为了解决这个问题，我们将系统性PAK 1敲除小鼠（Pak 1-/-）与Postn-Cre; Nf 2flox/flox小鼠杂交，以产生Postn-Cre; Nf 2flox/flox小鼠; Pak 1-/-小鼠。在初步研究中，我们已经观察到Pak 1的基因消融预防了Postn-Cre; Nf 2flox/flox小鼠中观察到的肿瘤发展和前庭神经鞘瘤相关的听力损失。在本提案的目标1中，我们将在听觉功能、肿瘤发展和存活方面全面表征Postn-Cre; Nf 2flox/flox小鼠; Pak 1-/-小鼠的表型。小鼠听力的功能评估将主要通过点击和音调诱发的听觉脑干反应（ABR）测试来确定。鉴于PAK 1作为致癌信号通路复杂网络中的关键节点的作用，目前尚不清楚在NF 2缺陷型肿瘤中，哪些通路和底物受到过度活跃的PAK 1下游的影响。在目标2中，我们建议阐明在NF 2缺陷型肿瘤细胞中由PAK 1激活的关键分子底物，作为进一步表征NF 2缺陷型细胞类型的基本病理生理学并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NF 2表征为mTORC信号传导和蛋白质翻译的新型调节剂。NF 2调节mTORC激活的机制仍然未知。鉴于我们的初步数据表明PAK 1是Postn-Cre; Nf 2flox/flox小鼠中NF 2缺陷型神经鞘瘤发展所必需的，我们假设PAK激酶活性可能是NF 2缺陷型细胞中mTORC过度活跃和翻译失调的介导者。细胞系中的初步数据表明情况确实如此，PAK激酶活性的减弱降低了mTORC下游翻译活性的生化标志物。目的2中的研究旨在使用siRNA敲低、PAK突变体构建体的慢病毒过表达和PAK的药理学抑制进一步剖析PAK与体外失调mTORC活性之间的未探索联系。然后通过全裂解物和免疫组织化学研究在体内验证这些结果。