Novel relationships of splicing factors in temozolomide-resistant glioblastoma

胶质母细胞瘤（GBM）是一种毁灭性的癌症，由于我们对其分子驱动因素的狭隘理解， 有限的治疗策略。一个潜在的机械驱动因素是选择性剪接。大脑中含有 任何器官的大多数选择性剪接转录物，许多剪接因子在正常 脑和GBM。虽然化学治疗的选择受到物理血脑屏障（BBB）的限制，但DNA- 破坏剂替莫唑胺（TMZ）能够进入大脑。然而，大多数患者很快就会 对TMZ和TMZ耐药GBM具有一致的致命性。我博士研究的最初目标是建立 新的TMZ耐药细胞模型，以确定可以靶向GBM治疗的途径。我 我的两种新TMZ的细胞生长、运动和代谢表型的综合表征- 抗GBM模型构成了我最初的第一作者论文的基础。在我的论文研究期间（目标1）， 我进行了两项互补的研究，确定了针对选择性剪接的新方法。 GBM的事件。第一个（目标1.1）是靶向选择性剪接雌激素相关受体β（ERRβ）。我 我已经开始用计算机和体外方法来确定该基因的促凋亡亚型ERRβ2是如何被 已处理。我发现富含丝氨酸/精氨酸（SR）的剪接因子SRSF 6在ERRβ2的产生中起作用， 用TG-003与抗肿瘤药物组合抑制Cdc样激酶（使SR蛋白磷酸化的CLK）， ERRβ合成激动剂DY-131在体外和颅内异种移植物中有效抑制TMZ耐药GBM细胞。 第二个（目标1.2）是一个更广泛的研究剪接抑制和调控TMZ耐药GBM。我发现 TMZ降低TMZ敏感模型中SR蛋白的磷酸化（p），但不降低TMZ耐药模型中SR蛋白的磷酸化（p）。这是 伴随着pSR蛋白的错误定位，以及DNA损伤的基线水平增加。在TMZ- 在耐药GBM细胞中，RNA结合蛋白EWS也错误定位并形成稳定的聚集体 tubulin。我的工作假设是，由于TMZ耐药GBM的DNA损伤增加， DNA损伤反应被重新编程，导致剪接因子（如EWS和pSR蛋白） 从它们正常的细胞区室中被置换出来，并准备进行异常聚集。此外，这一新的 剪接因子/DNA损伤修复轴可以用新的剪接抑制剂治疗靶向。期间 在博士后培训期间（目标2），我将解决我们对GBM转录组理解的一个关键差距： 非编码RNA的作用，特别是非典型的反向剪接或环状RNA（circRNA）。我提议 定义GBM的circRNA景观，确定调控功能，并提出潜在的 这些丰富和动态的剪接和转录调节剂的治疗应用。一起我的 博士前和博士后的研究经验将使我准备好平衡大局的想法和重点 当我作为一个独立的癌症研究者建立自己的研究计划时，我对机制的研究。

Glioblastoma (GBM) is a devastating cancer, due to both our narrow understanding of its molecular drivers and limited therapeutic strategies. One potential mechanistic driver is alternative splicing. The brain contains the most alternatively spliced transcripts of any organ, and many splicing factors are upregulated between normal brain and GBM. While chemotherapeutic options are limited by the physical blood brain barrier (BBB), the DNA- damaging agent temozolomide (TMZ) is able to cross into the brain. However, most patients rapidly become resistant to TMZ and TMZ-resistant GBM is uniformly fatal. An initial goal of my PhD research was to establish novel TMZ-resistant cellular models in order to identify pathways that could be targeted for GBM treatment. My comprehensive characterization of the cell growth, motility, and metabolic phenotypes of my two new TMZ- resistant GBM models forms the basis for my initial first-author paper. During my dissertation research (Aim 1), I have conducted two complementary studies that identify novel approaches to targeting alternative splicing events in GBM. The first (Aim 1.1) is to target the alternatively spliced estrogen-related receptor beta (ERRβ). I have started to define with in silico and in vitro methods how the pro-apoptotic isoform of this gene, ERRβ2, is processed. I found that the serine/arginine (SR) rich splicing factor SRSF6 plays a role in ERRβ2 production and that inhibition of Cdc-like kinases (CLKs, which phosphorylate SR proteins) with TG-003 in combination with the ERRβ synthetic agonist DY-131 potently inhibits TMZ-resistant GBM cells in vitro and in intracranial xenografts. The second (Aim 1.2) is a broader study of splicing inhibition and regulation in TMZ-resistant GBM. I found that TMZ decreases the phosphorylation (p) of SR proteins in TMZ-sensitive, but not TMZ-resistant models. This is accompanied by mis-localization of pSR proteins, and increased baseline levels of DNA damage. In TMZ- resistant GBM cells, the RNA binding protein EWS also mis-localizes and forms aggregates that are stabilized by tubulin. My working hypothesis is that because of the increased DNA damage in TMZ-resistant GBM, the DNA damage response becomes reprogrammed which causes splicing factors (like EWS and pSR proteins) to be displaced from their normal cellular compartments and poised for aberrant aggregation. Also, that this new splicing factor/DNA damage repair axis can be therapeutically targeted with novel splicing inhibitors. During the postdoctoral training period (Aim 2), I will address a key gap in our understanding of the GBM transcriptome: the role of non-coding RNAs, specifically the noncanonical back-spliced or circular RNAs (circRNAs). I propose to define the circRNA landscape of GBM, to determine the regulatory functions and to propose potential therapeutic applications of these abundant and dynamic regulators of splicing and transcription. Together, my pre- and postdoctoral research experiences will have prepared me to balance both big picture ideas and focused studies of mechanism when I establish my own research program as an independent cancer researcher.