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

基本信息

批准号：
10334507
负责人：
Deanna Marie Tiek
金额：
$ 9.4万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
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/10334507
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 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

项目摘要

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理解狭隘，而且一种潜在的机制驱动因素是大脑中包含的选择性剪接。任何器官的大多数选择性剪接转录物，并且许多剪接因子在正常之间上调虽然化疗方案受到物理血脑屏障 (BBB) 的限制，但 DNA- 损伤剂替莫唑胺 (TMZ) 能够进入大脑，但大多数患者很快就会发生这种情况。对 TMZ 耐药和对 TMZ 耐药的 GBM 都是致命的我博士研究的最初目标是确定。新型 TMZ 耐药细胞模型，以确定 GBM 治疗的目标途径。我的两个新 TMZ 的细胞生长、运动和代谢表型的综合表征抗性 GBM 模型构成了我最初的第一作者论文的基础（目标 1）。我进行了两项补充研究，确定了针对选择性剪接的新方法 GBM 中的第一个事件（目标 1.1）是针对选择性剪接的雌激素相关受体 β (ERRβ I)。已经开始使用计算机和体外方法来定义该基因的促凋亡亚型 ERRβ2 是如何发挥作用的我发现富含丝氨酸/精氨酸 (SR) 的剪接因子 SRSF6 在 ERRβ2 的产生和发挥作用。与 TG-003 结合使用可抑制 Cdc 样激酶（CLK，磷酸化 SR 蛋白） 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）是对 TMZ 耐药 GBM 剪接抑制和调节的更广泛研究，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。伴随着 pSR 蛋白的错误定位，以及 TMZ- 中 DNA 损伤基线水平的增加。耐药 GBM 细胞中，RNA 结合蛋白 EWS 也会错误定位并形成稳定的聚集体我的工作假设是，由于 TMZ 耐药 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）从正常的细胞区室中转移并准备好进行异常聚集。剪接因子/DNA损伤修复轴可以通过新型剪接抑制剂进行治疗。在博士后培训期间（目标 2），我将解决我们对 GBM 转录组理解中的一个关键差距：我建议非编码 RNA，特别是非规范反向剪接或环状 RNA (circRNA) 的作用。定义 GBM 的 circRNA 景观，确定调控功能并提出潜在的这些丰富且动态的剪接和转录治疗调节剂的应用在一起，我的。博士前和博士后研究经验将使我做好平衡大局想法和重点的准备当我作为一名独立的癌症研究者建立自己的研究计划时，我开始研究机制。