Determination of Musashi1/CD44v6 signaling in mesenchymal glioma stem cells

描述（由申请人提供）：胶质母细胞瘤（GBM）仍然是最难治疗的癌症之一，目前的治疗方法，包括放疗和替莫唑胺化疗，只有姑息作用。几乎所有的患者都会出现肿瘤复发，并且复发的肿瘤对目前的细胞毒性治疗更有抵抗力。最近强有力的转录谱分析将GBM分为4种具有不同临床特征的亚型。特别是，间充质（MES） GBM似乎是最常见的亚型，预后最差。然而，调控MES GBM细胞生长的机制尚不清楚。在肿瘤的异质性细胞中，胶质瘤干细胞（GSCs）是一个，如果不是唯一的，关键的治疗靶点；然而，亚型特异性GSCs的特征却很差。我们的初步数据表明：a-间充质，而不是原质，GSCs表达CD44v6；b- CD44v6的siRNA抑制MES体外生长，但不抑制PN GSCs；间充质GSCs中的c- CD44v6（+）细胞表达神经干细胞相关基因MELK，而CD44v6（-）细胞不表达；CD44v6的d- siRNA降低MELK的表达；e- CD44位点显示多个Musashi 1 （Msi1）结合序列；f- shRNA介导的Msi1缺失减少CD44的表达，并对外显子v6的剪接产生负面影响。为了实现本研究的目标，我们建立并表征了患者源性GBM球培养物，并创建了再现原始肿瘤组织病理学的患者源性小鼠GBM肿瘤模型。通过这些临床前模型，我们将验证Msi1-CD44v6信号是治疗耐药间充质胶质母细胞瘤生长和存活所必需的假设。具体而言，在Aim 1中，我们将验证CD44v6通过与MELK的信号相互作用对MES GSCs的增殖在功能上至关重要的假设。在Aim 2中，我们将确定CD44转录本是否是GBM中rna结合蛋白Msi1的直接靶点，以及Msi1是否通过CD44v6-MELK轴在放射抗性中发挥作用。

DESCRIPTION (provided by applicant): Glioblastoma (GBM) remains among the most formidable cancers to treat, and current therapies, including radiation and temozolomide chemotherapy, have only palliative effects. Tumor recurrence occurs in virtually all patients, and relapsed tumors are more resistant to current cytotoxic therapies. Recent robust transcriptional profiling has classified GBM into 4 subtypes with distinct clinical features. In particular, mesenchymal (MES) GBM appears to be the most common subtype with the poorest prognosis. Nonetheless, the mechanisms that regulate growth of MES GBM cells have not been clarified. Among the heterogeneous cells in tumors, glioma stem cells (GSCs) are one of the, if not the only, critical therapeutic targets; however, subtype-specific GSCs are poorly characterized. Our preliminary data suggest the following: a- mesenchymal, but not proneural, GSCs express CD44v6; b- siRNA for CD44v6 reduces in vitro growth of MES, but not PN GSCs; c- CD44v6(+) cells but not CD44v6 (-) cells in mesenchymal GSCs express the neural stem cell-associated gene MELK; d- siRNA for CD44v6 reduces MELK expression; e- CD44 locus exhibits multiple Musashi 1 (Msi1)-binding sequences; f- shRNA- mediated Msi1 depletion diminishes CD44 expression and negatively affects splicing of exon v6. To achieve the goals of this study, we established and characterized patient-derived GBM sphere cultures and created patient-derived mouse GBM tumor models that recapitulate the histopathology of the original tumors. With these pre-clinical models, we will test the hypothesis that the Msi1-CD44v6 signaling is required for the growth and survival of therapy-resistant mesenchymal glioblastoma. Specifically, in Aim 1, we will test the hypothesis that CD44v6 is functionally essential for the proliferation of MES GSCs through signal interaction with MELK. In Aim 2, we will determine whether CD44 transcripts are a direct target of the RNA-binding protein Msi1 in GBM and if Msi1 via the axis CD44v6-MELK plays a role in radio-resistance.