SON-mediated RNA splicing in glioblastoma

项目总结 多形性胶质母细胞瘤(GBM)是最常见和致命的脑部恶性肿瘤，中位生存期仅为 确诊后一年。我们目前对GBM潜在基础的了解主要集中在以下几个方面 特定基因的反复突变。然而，非遗传因素促进了GBM的发育和 进展在很大程度上是未知的。由于我们对基底膜生物学缺乏了解，治疗选择有限。 化疗(替莫唑胺，TMZ)结合放射治疗。因此，新的治疗方法是 迫切需要治疗这种致命的肿瘤。新出现的证据表明，异常的RNA 剪接位点突变和/或剪接因子突变导致的剪接促进癌基因表达 多种类型的实体瘤和血液系统恶性肿瘤。在GBM中，一些RNA剪接因子，包括 多嘧啶结合蛋白1(Ptbp1)和hnrnpa2b1最近被发现是驱动 致癌剪接中的因素，表明RNA剪接是一种关键的，但尚未探索的机制， 调控广泛的致癌基因表达。我们大量的初步数据显示儿子， 一种具有dna和rna结合能力的大核斑点蛋白在GBM中高度上调。 患者样本中，Son上调与患者短生存期之间有很强的相关性。我们 发现SON促进了PTBP1的表达，从而激活了PTBP1介导的致癌剪接 程序。相反，SON抑制PTBP2的表达，PTBP2是神经外显子包涵体所需的剪接因子 和神经分化。我们进一步揭示了SON调控内含子的去除过程 PTBP1转录本中的构成剪接位置并调控盒外显子的包含/跳过 PTBP2转录本中的剪接位点。我们还证明了SON基因敲除显著抑制了GBM细胞 生长和神经干细胞基因表达以及SON缺失导致患者来源的胶质瘤干细胞 (GSCs)体外对TMZ敏感。根据我们的初步数据，我们假设Son是一个主要的RNA 位于剪接因子层次结构顶端的剪接调节器，它同时影响本质性和 替代的RNA剪接，从而打开致癌剪接程序并阻止神经剪接。 因此，SON有望成为治疗基底膜的新靶点。为了检验这一假设，我们建议 剖析SON在结构性和选择性RNA调控中作用的分子机制 在GBM中进行剪接(目标1)，并确定靶向SON在体内的治疗潜力(目标2)。 这项拟议研究的成功完成将极大地提高我们对异常基因的认识 在GBM中表达，并为未来开发SON抑制剂提供了基本的理论基础。

PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most common and lethal brain malignancy with a median survival of only one year after diagnosis. Our current knowledge of the underlying basis of GBM centers mostly on several recurrent mutations in specific genes. However, non-genetic factors contributing to GBM development and progression are largely unknown. Due to our poor understanding of GBM biology, treatment options are limited to chemotherapy (temozolomide, TMZ) combined with radiotherapy. Thus, new therapeutic approaches are desperately needed to treat this deadly tumor. Emerging evidence has demonstrated that aberrant RNA splicing due to splice site mutations and/or splicing factor mutations drives oncogenic gene expression in multiple types of solid tumors and hematologic malignancies. In GBM, a few RNA splicing factors, including polypyrimidine tract-binding protein 1 (PTBP1) and hnRNP A2B1 have been recently identified as driving factors in oncogenic splicing, indicating that RNA splicing is a critical, yet to be explored, mechanism that governs a broad range of oncogenic gene expression. Our extensive preliminary data demonstrated that SON, a large nuclear speckle protein possessing DNA- and RNA-binding abilities, is highly upregulated in GBM patient samples, and there is a strong correlation between SON upregulation and short patient survival. We found that SON facilitates expression of PTBP1, thereby activating the PTBP1-meditated oncogenic splicing program. In contrast, SON inhibits the expression of PTBP2, a splicing factor required for neural exon inclusion and neural differentiation. We further revealed that SON regulates the intron removal process at the constitutive splice site in the PTBP1 transcript and regulates cassette exon inclusion/skipping at the alternative splice site in the PTBP2 transcript. We also demonstrated that SON knockdown markedly inhibits GBM cell growth and neural stem cell gene expression, and SON depletion renders patient-derived glioma stem cells (GSCs) sensitive to TMZ in vitro. Based on our preliminary data, we hypothesize that SON is a master RNA splicing regulator positioned at the apex of the splicing factor hierarchy that affects both constitutive and alternative RNA splicing, consequently turning on the oncogenic splicing program and blocking neural splicing. Thus, SON could represent a promising novel therapeutic target for GBM. To test this hypothesis, we propose to dissect the molecular mechanisms of SON functions in the regulation of constitutive and alternative RNA splicing in GBM (Aim 1), and to determine the therapeutic potential of targeting SON in vivo (Aim 2). Successful completion of this proposed study will significantly advance our knowledge of abnormal gene expression in GBM and provide a fundamental rationale for future endeavors to develop SON inhibitors.