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，这是由于我们对其分子驱动因素和 有限的治疗策略。一个潜在的机械驱动因素是另一种剪接。大脑包含了 大多数选择性剪接的任何器官的转录本和许多剪接因子在正常 脑和GBM。虽然化疗选择受到物理血脑屏障(BBB)的限制，但DNA- 损害剂替莫唑胺(TMZ)能够进入大脑。然而，大多数患者很快就会变成 新的TMZ耐药细胞模型，以确定可作为GBM治疗靶点的途径。我的 我的两个新的TMZ-的细胞生长、运动和代谢表型的综合表征 阻力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)， 我已经进行了两项互补性研究，确定了针对选择性剪接的新方法 以GBM表示的事件。第一个(目标1.1)是靶向选择性剪接的雌激素相关受体β(ERRβ)。我 已经开始用电子计算机和体外方法确定该基因的促凋亡亚型errβ2是如何 已处理。我发现富含丝氨酸/精氨酸(SR)的剪接因子SRSF6在ERRβ2的产生和 用TG-003联合Tg-003抑制CDC样激酶(使SR蛋白磷酸化的CLKs) ERRβ合成激动剂DY-131在体外和颅内异种移植中都能有效地抑制耐药的基底膜细胞。 第二个(目标1.2)是对TMZ抗性GBM中剪接抑制和调控的更广泛研究。我发现 在TMZ敏感的模型中，TMZ降低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。这是 伴随着PSR蛋白的错误定位，以及DNA损伤基线水平的增加。在TMZ- 在耐药的GBM细胞中，RNA结合蛋白EWS也错误定位并形成稳定的聚集体 通过微管蛋白。我的工作假设是，由于抗TMZ的GBM中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) 另外，这一新的 剪接因子/DNA损伤修复轴可以用新型剪接抑制剂作为治疗靶点。在.期间 博士后培训期(目标2)，我将解决我们对GBM转录组理解中的一个关键差距： 非编码RNA的作用，特别是非规范的后剪接或环状RNA(CircRNA)。我建议 定义GBM的CircRNA格局，确定调节功能并提出潜在的 这些丰富和动态的剪接和转录调节因子在治疗中的应用。一起，我的 博士后和博士后的研究经验将使我准备好平衡大局想法和专注于 当我作为一名独立的癌症研究人员建立自己的研究计划时，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.