Project 2: The GABP-TERT axis in immortality of Oligodendroglioma and Glioblastoma

项目摘要/摘要 项目2的目标是了解因子如何唯一地招募到突变的TERT启动子(TERTp) 与野生型(WT)启动子固有的因子一起激活TERT以获得肿瘤细胞 永生。TERTp突变发生在几乎所有的胶质母细胞瘤(GBM)和少突胶质细胞瘤(OD)中，使 肿瘤细胞变得永生。我们发现这些突变允许GA结合蛋白(GABP) 在许多癌症类型中异常激活突变的TERTp。在我们的初步数据和新的出版物中， GBM中GABP的减少会导致TERT降低，并逐渐和几乎完全丧失生存能力 TERTp突变依赖方式。GABP通常不会出现在TERTp上，但对其他方面知之甚少 关于这个新发现的肿瘤细胞永生的中心节点。我们发现了两个新的候选人 分子-RNF2，一种似乎驱动突变TERTp激活的泛素连接酶；以及肿瘤 抑制因子CIC抑制WT和突变体TERTp，但在OD中反复突变，在 GBM。在这里，我们将检验这样一种假设，即突变TERTp的激活和GABP对肿瘤不朽的影响 涉及突变的等位基因特有因子和本地因子的关键贡献。在目标1中，我们将定义 RNF2突变体特异性招募在促进TERT表达和永生化中的作用。我们将研究 RNF2在GBM和OD培养上的招募和对突变TERTp的调节，决定了敲除 RNF2在体外改变端粒酶活性、端粒长度、肿瘤细胞活力和体内肿瘤形成， 如果联合抑制GABP和RNF2，则会加速这些过程。潜在的抗性机制 尽管GABP降低但仍在生长的肿瘤将得到解决。在目标2中，我们将确定WT和突变体 CIC调控TERT的表达和永生化。WT CIC抑制转录的几个ETS因子 我们以前证明了正常激活WT和突变的TERT启动子。我们将测试中投公司 在GBM和OD培养中抑制TERT的表达，然后测试是否反复功能丧失 在OD中发现的突变上调TERT，以及哪些ETS因素介导了这一影响。我们将推倒 CIC调节ETS因子并确定它们是否改变端粒酶活性、端粒长度和肿瘤细胞 永生。在目标3中，我们建议更广泛地发现新的永生调节因子，使用体内 CRISPRi屏幕。基因抑制将在有和没有GABP的TERTp突变GBM细胞中模拟。 编辑以确定调节体内细胞不朽的因素，无论是独立于或 与GABP协同作用。我们在胶质瘤中的结果将建立在TERT-GABP相互作用的中心节点上 这是我们发现的，可能与更广泛的TERTp突变肿瘤类型有关。通过 我们将对比两种临床上不同的胶质瘤亚型-OD和GBM-TERT的调节机制 确定共享的或特定于子类型并可能链接到 不同的病人结局。肿瘤细胞特异性调节因子可能提供新的治疗机会。

PROJECT SUMMARY/ABSTRACT The goal of Project 2 is to understand how factors uniquely recruited to the mutant TERT promoter (TERTp) together with factors that are native to the wildtype (WT) promoter activate TERT to achieve tumor cell immortality. TERTp mutation occurs in nearly all glioblastoma (GBM) and oligodendroglioma (OD), enabling tumor cells to become immortal. We showed that the mutations allow the GA-binding protein (GABP) to aberrantly activate the mutant TERTp across many cancer types. In our preliminary data and new publication, reduction of GABP in GBM causes decreased TERT and a gradual and nearly complete loss of viability in a TERTp mutation-dependent manner. GABP is not normally present at the TERTp, however little else is known about this newly discovered central node in tumor cell immortality. We have discovered two novel candidate molecules - RNF2, an ubiquitin ligase that appears to drive activation of the mutant TERTp; and the tumor suppressor CIC that represses WT and mutant TERTp, but is recurrently mutated in OD and downregulated in GBM. Here, we will test the hypothesis that activation of the mutant TERTp and tumor immortality by GABP involve critical contributions from mutant allele-specific factors and native factors. In Aim 1, we will define the role of mutant-specific recruitment of RNF2 in promoting TERT expression and immortality. We will examine RNF2 recruitment to, and regulation of mutant TERTp across GBM and OD cultures, determine if knockdown of RNF2 alters telomerase activity, telomere length and tumor cell viability in vitro and tumorigenesis in vivo, and if combined inhibition of GABP and RNF2 accelerates these processes. Potential resistance mechanisms in tumors that grow despite reduced GABP will be addressed. In Aim 2, we will determine how WT and mutant CIC regulates TERT expression and immortality. WT CIC suppresses transcription several ETS factors which we previously demonstrated normally activate the WT and mutant TERT promoter. We will test CIC suppression of TERT expression across GBM and OD cultures, then test whether the recurrent loss of function mutations found in OD upregulate TERT, and which ETS factors mediate the effect. We will knockdown the CIC-regulated ETS factors and determine if they alter telomerase activity, telomere length and tumor cell immortality. In Aim 3, we propose to more broadly discover novel regulators of immortality using an in vivo CRISPRi screen. The gene inhibition will be modeled in TERTp mutant GBM cells with and without GABP- editing in order to identify factors that regulate cellular immortality in vivo either independently of or synergistically with GABP. Our results in glioma will build upon the central node of TERT-GABP interaction which we discovered, and may be relevant to the wider spectrum of TERTp mutant tumor types. By contrasting mechanisms of TERT regulation in two clinically distinct glioma subtypes -OD and GBM- we will identify TERTp regulatory mechanisms that are shared, or subtype-specific and potentially linked to the different patient outcomes. The tumor cell specific regulators may present new therapeutic opportunities.