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利用了NF2的新型鼠标模型（Postn-cre; NF2Flox/Flox小鼠），该模型紧密地概括了人类NF2疾病的重要方面，包括完全渗透的schwannaMAS型表型，以及导致schnwannoms schwannoms schwannompular schwannom schwannom schwannom schwannom schwannom schwannom的障碍。利用这种小鼠模型，我们采取了一种候选方法来识别NF2下游的哪些蛋白质是Schwannoma Genesis所需的关键信号中间体。先前的研究已将NF2鉴定为激酶PAK1的内源性抑制剂，并且在原代人造型样品中已经观察到了PAK1激酶活性的升高。重要的是，尚未使用自发的体内切旺纳马瘤模型来探索PAK1激酶活性与NF2缺陷型肿瘤发生的相关性。为了解决这个问题，我们已经与Postn-Cre进行了跨系统的PAK1敲除小鼠（PAK1 - / - ）。 NF2Flox/Flox小鼠生成邮政邮编； NF2Flox/Flox小鼠； pak1 - / - 鼠标。在初步研究中，我们观察到，PAK1的遗传消融可防止肿瘤的发育和前庭切割症相关的听力损失。 NF2Flox/Flox小鼠。在本提案的目标1中，我们将全面地表征邮政邮政的表型。 NF2Flox/Flox小鼠； PAK1 - / - 小鼠在听觉功能，肿瘤发育和生存方面。小鼠听力的功能评估将在很大程度上通过点击和音调引起的听觉脑干响应（ABR）测试确定。鉴于PAK1在复杂的致癌信号通路网络中作为关键节点的作用，尚不清楚哪些途径和底物在NF2缺乏的肿瘤中影响了多活跃PAK1的下游。在AIM 2中，我们建议阐明在NF2缺乏的肿瘤细胞中被PAK1激活的关键分子底物，以此作为进一步表征NF2缺陷细胞类型的基本病理生理学的一种策略，并确定了其他治疗靶标。来自其他实验室的多项研究以前已将NF2表征为MTORC信号传导和蛋白质翻译的新调节剂。 NF2调节MTORC激活的机制仍然未知。鉴于我们的初步数据表明，PAK1是NF2缺陷型造口瘤开发所必需的； NF2Flox/Flox小鼠，我们假设PAK激酶活性可能是MTORC多动症的介体，并且在NF2缺陷型细胞类型中失调了翻译。细胞系中的初步数据确实表明了这种情况，而PAK激酶活性的衰减减少了MTORC下游转化活性的生化标志物。 AIM 2中的研究旨在进一步剖析使用siRNA敲低，PAK突变体构建体的慢病毒过表达和PAK的药理抑制，进一步剖析了PAK和PAK和失调的MTORC活性之间的这种未开发的连接。这些结果将通过整个裂解物和免疫组织化学研究在体内验证。