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。 肿瘤复发发生在几乎所有患者中，并且复发的肿瘤对当前的细胞毒性疗法更具抗性。 最近稳健的转录谱分析已将GBM分为具有不同临床特征的4种亚型。 特别是间充质（MES）GBM似乎是预后最差的最常见亚型。 然而，调节MES GBM细胞生长的机制尚未阐明。 在肿瘤中的异质细胞中，胶质瘤干细胞（GSC）是关键治疗靶点之一（如果不是唯一的话）;然而，亚型特异性GSC的特征很差。 我们的初步数据表明：a-间充质而非前神经GSC表达CD 44 v6;针对CD 44 v6的B- siRNA降低MES而非PN GSC的体外生长; c-间充质GSC中的CD 44 v6（+）细胞而非CD 44 v6（-）细胞表达神经干细胞相关基因MELK;针对CD 44 v6的d- siRNA降低MELK表达; e-CD 44位点显示多个Musashi 1（Msi 1）结合序列; f-shRNA介导的Msi 1缺失减少了CD 44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来源的GBM球体培养物，并创建了患者来源的小鼠GBM肿瘤模型，其重现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Msi 1-CD 44 v6信号传导是耐药性间充质胶质母细胞瘤生长和存活所需的假设。 具体而言，在目标1中，我们将检验CD 44 v6通过与MELK的信号相互作用对MES GSC的增殖功能至关重要的假设。 在目标2中，我们将确定CD 44转录物是否是GBM中RNA结合蛋白Msi 1的直接靶点，以及Msi 1是否通过轴CD 44 v6-MELK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.