Project 3: A New Therapeutic Target for TERT Promoter Mutant Glioma

项目摘要/摘要 该项目的目标是开发GABP作为一种治疗靶点，以逆转携带ABP的肿瘤的不朽 突变的端粒酶逆转录酶(TERT)启动子。TERT启动子突变是第三常见的 人类癌症中的突变，影响80%以上的基底膜和少牙本质胶质瘤(OD)。由于缺乏TERT 在体细胞中转录，端粒随着每一次连续的细胞分裂而缩短，直到它们达到临界 触发衰老并限制细胞寿命的水平。TERT表达的重新激活克服了这些 屏障，使肿瘤细胞能够无限增殖。尽管控制突变的TERT启动子的蛋白质 重新激活和肿瘤细胞永生化可能是理想的治疗靶点，这两者的确切身份 分子仍然未知。我们发现，一种普遍表达的转录因子GABP， 与突变型TERT启动子唯一结合并在TERT启动子-突变型胶质瘤中驱动TERT重新激活 和其他癌症。GABP以异二聚体或异四聚体的形式与DNA结合，调节不同的功能 转录程序。在我们的初步数据中，我们确定了形成GABPβ1的特定的异位四聚体 异构体(GABPβ1L)，在正常细胞中是必不可少的，但对突变的TERT启动子激活可能是关键的 和肿瘤细胞永生化。如果突变的TERT启动子均匀存在于每个肿瘤中，并且如果 GABPβ1L调节导致肿瘤细胞死亡而保留正常细胞，GABP途径可能代表一种 突变的TERT启动子驱动的恶性肿瘤的新治疗选择。我们将用三个例子来检验这一假设 具体目标：在目标1中，我们将确定TERT启动子突变的克隆程度 诊断和复发。我们设计了一个强大的系统来收集和分析10个空间映射的克隆 来自每个基底膜和外径的样本，代表最大的肿瘤地理位置。在目标2中，我们将确定是否 在TERT启动子突变的中枢神经系统肿瘤中，需要GABP异四聚体来维持细胞永生。我们 将使用GABPB1L亚型的CRISPR-Cas9基因靶向来确定TERT的后果 表达、端粒长度、细胞活力和肿瘤形成。转录组效应与死亡机制 将确定GABPβ1L缺陷肿瘤细胞的脆弱性，以利用现有的治疗方法。 在目标3中，我们将确定增加TERT启动子突变肿瘤细胞死亡的治疗方法 GABPβ1L.在我们的初步数据中，GBM细胞未能通过GABP完全激活TERT表达 异源四聚体最终导致端粒功能障碍和DNA损伤。我们将进行一次集中的，探索性的 GABP1L缺陷细胞DNA损伤及损伤反应抑制物的筛选及鉴定 将增加细胞死亡和减少肿瘤形成的治疗方法。这些研究可以确定 GABPTERT1L亚型作为β启动子突变的中枢神经系统肿瘤的有价值的治疗靶点 可能还有很多其他人。同时，我们将推动药物发现和开发工作向小规模 与行业合作伙伴Telo Treeutics和GlaxoSmithKline共同开发不同GABP亚单位的分子抑制剂。

PROJECT SUMMARY/ABSTRACT The goal of this project is to develop GABP as a therapeutic target to reverse immortality of tumors harboring a mutant telomerase reverse transcriptase (TERT) promoter. TERT promoter mutation is the third most common mutation in human cancer, affecting over 80% of GBM and oligodengroglioma (OD). Due to a lack of TERT transcription in somatic cells, telomeres shorten with each successive cell division until they reach a critical level that triggers senescence and limits cell lifespan. Reactivation of TERT expression overcomes these barriers, enabling tumor cells to proliferate indefinitely. Although proteins controlling mutant TERT promoter reactivation and tumor cell immortalization may be ideal therapeutic targets, the exact identity of these molecules remained unknown. We discovered that a single ubiquitously expressed transcription factor, GABP, uniquely bound to the mutant TERT promoter and drove TERT reactivation in TERT-promoter-mutant glioma and other cancers. GABP binds DNA as a heterodimer or a heterotetramer which regulate functionally distinct transcriptional programs. In our preliminary data, we identify a specific heterotetramer forming GABPβ1 isoform (GABPβ1L) that is dispensable in normal cells but may be critical for mutant TERT promoter activation and tumor cell immortalization. If the mutant TERT promoter is uniformly present throughout each tumor, and if GABPβ1L modulation leads to tumor cell death while sparing normal cells, the GABP pathway may represent a new therapeutic option for mutant TERT promoter-driven malignancies. We will test this hypothesis with three specific aims: In Aim 1, we will determine the extent to which the TERT promoter mutation is clonal at diagnosis and recurrence. We devised a robust system to collect and analyze clonality in 10 spatially mapped samples from each GBM and OD, representing maximal tumor geography. In Aim 2, we will determine if the GABP heterotetramer is required to maintain cellular immortality in TERT promoter mutant CNS tumors. We will use CRISPR-Cas9 genetic targeting of the GABPB1L isoform to determine the consequences on TERT expression, telomere length, cell viability and tumor formation. The transcriptome effects and death mechanism of GABPβ1L deficient tumor cells will be determined to identify vulnerabilities to exploit with existing therapies. In Aim 3, we will identify therapies that will increase cell death in TERT promoter mutant tumors deficient in GABPβ1L. In our preliminary data, failure of GBM cells to fully activate TERT expression by a GABP heterotetramer culminates in telomere dysfunction and DNA damage. We will perform a focused, exploratory screen of DNA damaging and DNA damage response-inhibiting agents on GABPβ1L deficient cells to identify therapies that will increase cell death and decrease tumor formation. These studies could establish the GABPβ1L isoform as a valuable therapeutic target specifically for TERT promoter mutant CNS tumors, and potentially many others. In parallel, we will advance drug discovery and development efforts towards small molecule inhibitors of different GABP subunits with industry partners Telo Therapeutics and GlaxoSmithKline.