Functional Inhibition of Deubiquitylase HAUSP to Disrupt Glioblastoma Stem Cells

 描述(申请人提供)：胶质母细胞瘤(GBM)是一种高度致命的脑肿瘤，预后极差。由于治疗耐药，对GBMS的治疗仍然是姑息性的。GBM表现出明显的细胞异质性，分化层级的顶端有大量的肿瘤起始细胞(BTICs)或GBM干细胞(GSCs)。我们先前已经证明GSCs促进治疗抵抗、肿瘤血管生成和肿瘤侵袭，表明GSCs的治疗靶向可能有效地改善GBM的治疗。GSCs的干细胞样表型由包括SOX2在内的一组核心转录因子维持。SOX2的蛋白质稳定性受到翻译后修饰的严格调控。我们的初步研究表明，在胶质瘤细胞中，SOX2受泛素化(Ub)和去泛素化(DUb)的调节。SOX2被E3泛素连接酶HUWE1泛素化，然后被非干细胞胶质瘤细胞降解。在GSCs中，SOX2蛋白通过脱泛素化酶(Hausp)(疱疹病毒相关泛素特异性蛋白水解酶，又称USP7)介导的脱泛素化来稳定。与神经前体细胞相比，Hausp在神经干细胞中优先表达。通过shRNA靶向Hausp可以减少SOX2蛋白的表达，促进细胞分化，破坏GSC的维持，并有效地抑制GSC的生长。一种小分子抑制剂对Hausp的功能抑制也抑制了原位GBM移植瘤的生长，并使GSC对辐射或替莫唑胺(TMZ)增敏。基于这些初步数据，我们假设Hausp介导的去泛素化在GSCs的维持中拮抗HUWE1介导的泛素化以稳定SOX2，从而提出了一种靶向治疗策略来破坏GSCs并抑制GBM肿瘤的生长。我们提出了以下具体目标：1.确定Hausp抑制对抑制GBM肿瘤生长的治疗效果。2.明确Hausp介导的GSC维持的分子机制。3.评价Hausp抑制与当前GBM治疗的协同作用。该提案的目的是在一项临床前研究中评估Hausp抑制的治疗潜力。我们将确定靶向Hausp是否可以作为一种有效的治疗策略来改善GBM的治疗。本研究将为新型抗GSC治疗药物的开发奠定坚实的基础，从而有效提高GBM患者的生存率。

 DESCRIPTION (provided by applicant): Glioblastomas (GBMs) are highly lethal brain tumors with extremely poor prognosis. The treatment of GBMs remains palliative due to therapeutic resistance. GBM displays striking cellular heterogeneity with a population of tumor-initiating cell (BTICs) or GBM stem cells (GSCs) at the apex of differentiation hierarchy. We previously demonstrated that GSCs promote therapeutic resistance, tumor angiogenesis and cancer invasion, indicating that therapeutic targeting of GSCs may effectively improve GBM treatment. The stem cell-like phenotype of GSCs is maintained by a set of core transcription factors including SOX2. The protein stability of SOX2 is tightly regulated by post-translational modifications. Our preliminary study indicated that SOX2 is regulated by both ubiquitylation (Ub) and deubiquitylation (Dub) in glioma cells. SOX2 is ubiquitylated by the E3 ubiquitin ligase, Huwe1, and then targeted for degradation in non-stem glioma cells. In GSCs, SOX2 protein is stabilized through deubiquitylation mediated by the deubiquitylase, HAUSP (Herpesvirus-Associated Ubiquitin-Specific Protease, also known as USP7). HAUSP is preferentially expressed in GSCs relative to neural progenitors. Targeting HAUSP by shRNA reduced SOX2 protein, promoted cell differentiation, disrupted GSC maintenance and potently inhibited GSC tumor growth. Functional inhibition of HAUSP by a small molecule inhibitor also inhibited tumor growth in orthotopic GBM xenografts and sensitized GSCs to radiation or temozolomide (TMZ). Based on these preliminary data, we hypothesize that HAUSP-mediated deubiquitylation antagonizes Huwe1-mediated ubiquitylation to stabilize SOX2 in the maintenance of GSCs, presenting a therapeutic target strategy to disrupts GSCs and suppress GBM tumor growth. We propose the following specific aims: 1. Determine the therapeutic impact of HAUSP inhibition on suppressing GBM tumor growth. 2. Define the molecular mechanisms underlying HAUSP-mediated GSC maintenance. 3. Evaluate synergy of HAUSP inhibition and current GBM therapies. The goal of this proposal is to evaluate the therapeutic potential of HAUSP inhibition in a preclinical study. We will determine whether targeting HAUSP can serve as an effective therapeutic strategy to improve GBM treatment. The proposed studies will lay a solid foundation for the development of novel anti-GSC therapeutics to effectively improve the survival of GBM patients.