Novel relationships of splicing factors in temozolomide-resistant glioblastoma

替莫唑胺耐药胶质母细胞瘤中剪接因子的新关系

• 批准号: 10085005
• 负责人: Deanna Marie Tiek
• 金额: $ 8.66万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10085005
• 关键词: Address; Agonist; Alkylating Agents; Alternative Splicing; Apoptotic; Arginine; Back; Biology; Blood - brain barrier anatomy; Brain; Brain Glioblastoma; Brain Neoplasms; Cell model; Cell physiology; Cells; DNA; DNA Damage; DNA Repair; Diagnosis; Doctor of Philosophy; Drug resistance; Equilibrium; Event; FDA approved; Genes; Genetic Transcription; Glioblastoma; Goals; In Vitro; Knowledge; Lesion; Malignant Neoplasms; Mentorship; Methods; Methyltransferase; MicroRNAs; Modeling; Molecular; Nuclear Orphan Receptor; Operative Surgical Procedures; Organ; Paper; Pathway interactions; Patients; Phase; Phosphorylation; Phosphotransferases; Play; Porifera; Postdoctoral Fellow; Process; Production; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA-Binding Protein EWS; RNA-Binding Proteins; Radiation therapy; Regulation; Research; Research Personnel; Research Project Grants; Research Technics; Resistance; Role; Serine; Signal Transduction; Testing; Therapeutic; Training; Transcript; Translating; Tubulin; Untranslated RNA; Work; Xenograft procedure; anticancer research; base; cell growth; cell motility; chemotherapy; circular RNA; combat; estrogen-related receptor; experience; gene product; improved; in silico; in vivo; inhibitor/antagonist; metabolic phenotype; neoplastic cell; new therapeutic target; novel; novel strategies; post-doctoral training; pre-doctoral; programs; receptor function; repaired; research study; response; success; temozolomide; therapeutic target; transcriptome; treatment strategy

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.

胶质母细胞瘤（GBM）是一种毁灭性的癌症，因为我们对其分子驱动因素的了解狭窄，并且 有限的理论策略。一个潜在的机械驱动程序是替代剪接。大脑包含 任何器官的剪接成绩单最多，许多剪接因子在正常之间上调 大脑和GBM。虽然化学治疗量受到身体血脑屏障（BBB）的限制，但DNA- 破坏性剂替莫唑胺（TMZ）能够跨入大脑。但是，大多数患者迅速成为 对TMZ和抗TMZ抗性GBM的抗性均匀致命。我的博士研究的最初目标是建立 新型TMZ耐药细胞模型，以识别可以针对GBM治疗的途径。我的 我两个新的TMZ-的细胞生长，运动性和代谢表型的全面表征 耐药的GBM模型构成了我初始的第一任撰稿人纸的基础。在我的论文研究中（AIM 1）， 我已经进行了两项完整的研究，以识别针对替代剪接的新方法 GBM中的活动。第一个（AIM 1.1）是靶向与雌激素相关的受体beta（ERRβ）。我 已经开始使用硅和体外方法定义该基因的促凋亡同工型errβ2是如何 处理。我发现丝氨酸/精氨酸（SR）富剪接因子SRSF6在ERRβ2的产生中起作用 抑制CDC样激酶（clks，clks，PhotophoryLate SR蛋白）与TG-003结合使用 ERRβ合成激动剂DY-131可能会在体外和颅内异种移植物中抑制耐TMZ的GBM细胞。 第二个（AIM 1.2）是对抗TMZ耐药GBM的剪接抑制和调节的广泛研究。我发现 TMZ降低了对TMZ敏感但不具有TMZ抗性模型中SR蛋白的磷酸化（P）。这是 伴随着PSR蛋白的错误定位，并增加了DNA损伤的基线水平。在TMZ- 抗性GBM细胞，RNA结合蛋白EWS也未定位并形成稳定的聚集体 由小管蛋白。我的工作假设是，由于TMZ耐药GBM的DNA损伤增加了 DNA损伤响应被重新编程，从而导致剪接因子（例如EWS和PSR蛋白） 从正常的细胞室中移动，并中毒以异常聚集。另外，这个新 剪接因子/DNA损伤修复轴可以用新型的剪接抑制剂热靶向。在 博士后培训期（AIM 2），我将解决我们对GBM转录组的理解的关键差距： 非编码RNA的作用，特别是非规范的后置或圆形RNA（CIRCRNA）。我建议 定义GBM的CIRCRNA景观，以确定调节功能并提出潜在 这些丰富而动态的剪接和转录调节剂的治疗应用。在一起，我的 博士后研究经验将使我准备平衡大型思想和专注 当我作为独立癌症研究人员建立自己的研究计划时，对机制的研究。