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

项目总结/摘要 项目2的目标是了解独特的因子如何募集到突变的TERT启动子（TERTp） 与野生型（WT）启动子天然的因子一起激活TERT，以实现肿瘤细胞 仙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， 肿瘤细胞变得不朽。我们发现，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出版物中， GBM中GABP的减少会导致TERT的减少，并且在一个细胞中逐渐和几乎完全丧失活力。 TERTp突变依赖性方式。GABP通常不存在于TERTp中，但其他方面知之甚少 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-RNF 2，一种似乎驱动突变TERTp激活的泛素连接酶;以及肿瘤 抑制型CIC，抑制WT和突变型TERTp，但OD反复突变， GBM。在这里，我们将测试GABP激活突变TERTp和肿瘤永生的假设， 涉及突变等位基因特异性因子和天然因子的关键贡献。在目标1中，我们将定义 RNF 2在促进TERT表达和永生中的作用。我们将研究 RNF 2在GBM和OD培养物中的募集和突变TERTp的调节，确定敲低是否 RNF 2在体外改变端粒酶活性、端粒长度和肿瘤细胞活力，在体内改变肿瘤发生， 以及GABP和RNF 2的联合抑制是否加速了这些过程。潜在耐药机制 尽管GABP降低，但肿瘤仍在生长。在目标2中，我们将确定WT和突变体 CIC调节TERT表达和永生。WT CIC抑制几种ETS因子的转录， 我们先前证明了正常激活WT和突变体TERT启动子。我们将测试作战信息中心 在GBM和OD培养物中抑制TERT表达，然后测试是否复发性功能丧失 OD中发现的突变上调了TERT，并且ETS因子介导了该效应。我们将击倒 CIC调节的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 仙在目标3中，我们建议使用体内免疫系统更广泛地发现新的永生调节剂。 CRISPRi筛选。基因抑制将在具有和不具有GABP的TERTp突变GBM细胞中建模。 编辑以鉴定调节体内细胞永生的因子， 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 在两种临床上不同的胶质瘤亚型-OD和GBM-中对比TERT调节机制，我们将 确定TERTp调控机制是共享的，或亚型特异性的，并可能与 不同的患者结局。肿瘤细胞特异性调节因子可能带来新的治疗机会。

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.