Determination of Musashi1/CD44v6 signaling in mesenchymal glioma stem cells

描述(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，包括放射和替莫唑胺化疗，只有缓解作用。肿瘤复发几乎在所有患者中都会发生，而复发的肿瘤对目前的细胞毒治疗更具抵抗力。最近，强健的转录图谱已经将GBM分为4个亚型，具有明显的临床特征。尤其是间充质(MES)GBM似乎是最常见、预后最差的亚型。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)即使不是唯一的，也是关键的治疗靶点之一；然而，亚型特异性GSCs的特征很差。我们的初步数据表明：A-间充质GSC表达CD44v6，但不表达CD44v6；CD44v6的b-siRNA抑制MES的体外生长，但不抑制PN GSCs的生长；间质GSC中的c-CD44v6(+)细胞但不表达CD44v6(-)细胞表达神经干细胞相关基因Melk；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缺失降低CD44的表达，并对外显子V6的剪接产生负面影响。为了实现这项研究的目标，我们建立并表征了患者来源的GBM球体培养，并创建了患者来源的小鼠GBM肿瘤模型，概括了原始肿瘤的组织病理学。通过这些临床前模型，我们将检验这一假设，即Msi1-CD44v6信号对于耐药间充质胶质母细胞瘤的生长和生存是必需的。具体地说，在目标1中，我们将测试CD44v6通过与Melk的信号相互作用而对MES GSCs的增殖起重要作用的假设。在目标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轴在辐射抗性中发挥作用。

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.