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: 8885487
• 负责人: Jeffrey R Gehlhausen
• 金额: $ 2.73万
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8885487
• 关键词: Ablation; Acoustic Neuroma; Address; Affect; Auditory; Benign Schwannoma; Biochemical Markers; Biochemical Pathway; Cell Line; Cloning; Complex; Cranial Nerves; Data; Development; Disease; Distal; Evoked Potentials, Auditory, Brain Stem; Excision; Functional disorder; Genes; Genetic; Genetically Engineered Mouse; Germ-Line Mutation; 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; meningioma; mortality; mouse model; mutant; neoplastic cell; novel; overexpression; prevent; public health relevance; 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NF2小鼠模型（Postn-Cre； Nf2flox/flox小鼠），该模型紧密概括了人类NF2疾病的重要方面，包括完全渗透的神经鞘瘤表型，以及由前庭神经鞘瘤发展导致的听力和前庭损伤。利用该小鼠模型，我们采用了一种候选方法来确定NF2下游的哪些蛋白质是神经鞘瘤发生所需的关键信号中间体。先前的研究已经确定NF2是PAK1激酶的内源性抑制剂，并且在原发性人神经鞘瘤样本中观察到PAK1激酶活性水平升高。重要的是，PAK1激酶活性与nf2缺陷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探讨。为了解决这个问题，我们将PAK1敲除小鼠（PAK1 -/-）与Postn-Cre交叉；Nf2flox/flox小鼠产生post - cre；Nf2flox /液氧老鼠;Pak1 - / -小鼠。在初步研究中，我们观察到Pak1的基因消融可以阻止肿瘤的发展和前庭神经鞘瘤相关的听力损失；Nf2flox /液氧老鼠。在本提案的目标1中，我们将全面表征post - cre的表型；Nf2flox /液氧老鼠;Pak1-/-对小鼠听觉功能、肿瘤发展和生存的影响。听觉的功能评估主要是通过点击和音调诱发的听觉脑干反应（ABR）测试来确定的。鉴于PAK1在复杂的致癌信号通路网络中是一个关键节点，目前尚不清楚在nf2缺失的肿瘤中，哪些途径和底物受到PAK1过度活跃下游的影响。在Aim 2中，我们建议阐明NF2缺陷肿瘤细胞中由PAK1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。此前，其他实验室的多项研究表明NF2是mTORC信号传导和蛋白质翻译的一种新型调节剂。NF2调控mTORC激活的机制尚不清楚。鉴于我们的初步数据表明，在n- cre后，nf2缺乏的神经鞘瘤的发展需要PAK1；在Nf2flox/flox小鼠中，我们假设PAK激酶活性可能是nf2缺陷细胞类型中mTORC过度活跃和翻译失调的中介。细胞系的初步数据表明确实如此，PAK激酶活性的衰减降低了mTORC下游翻译活性的生化标志物。Aim 2的研究旨在通过siRNA敲除、PAK突变体的慢病毒过表达和PAKs的药理学抑制，进一步剖析PAK与体外mTORC活性失调控之间的这种未被探索的联系。这些结果将在体内通过全裂解物和免疫组织化学研究进行验证。