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

项目总结/摘要 该项目的目标是将GABP开发为治疗靶点，以逆转具有免疫缺陷的肿瘤的永生性。 突变型端粒酶逆转录酶（TERT）启动子。TERT启动子突变是第三常见的 突变，影响超过80%的GBM和寡腺胶质瘤（OD）。由于缺乏TERT 在体细胞中，端粒随着每次连续的细胞分裂而缩短，直到它们达到一个关键的水平。 引发衰老并限制细胞寿命的水平。TERT表达的再活化克服了这些问题。 屏障，使肿瘤细胞能够无限增殖。尽管控制突变型TERT启动子的蛋白质 激活和肿瘤细胞永生化可能是理想的治疗靶点， 分子仍然未知。我们发现，一个普遍表达的转录因子GABP， 独特地与突变的TERT启动子结合，并在TERT启动子突变的胶质瘤中驱动TERT再活化 和其他癌症。GABP以异源二聚体或异源四聚体的形式结合DNA，其调节功能上不同的 转录程序。在我们的初步数据中，我们鉴定了形成GABPβ1的特异性异源四聚体， 一种在正常细胞中表达但可能对突变型TERT启动子激活至关重要的同种型（GABPβ 1 L 和肿瘤细胞永生化。如果突变的TERT启动子均匀地存在于每个肿瘤中， GABPβ 1 L调节导致肿瘤细胞死亡而不影响正常细胞，GABP通路可能代表了一种新的肿瘤细胞死亡机制。 突变型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个假设。 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个空间映射的克隆性 来自每个GBM和OD的样本，代表最大肿瘤地理分布。在目标2中，我们将确定 GABP异源四聚体是维持TERT启动子突变型CNS肿瘤细胞永生所必需的。我们 将使用CRISPR-Cas9基因靶向GABPB 1 L亚型，以确定对TERT的影响。 表达、端粒长度、细胞活力和肿瘤形成。转录组效应和死亡机制 将确定GABPβ 1 L缺陷肿瘤细胞的数量，以确定现有疗法的弱点。 在目标3中，我们将鉴定出增加TERT启动子突变型肿瘤细胞死亡的疗法， GABPβ1L。在我们的初步数据中，GBM细胞不能通过GABP完全激活TERT表达， 异源四聚体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 筛选GABPβ 1 L缺陷细胞的DNA损伤和DNA损伤反应抑制剂，以鉴定 增加细胞死亡和减少肿瘤形成的疗法。这些研究可以建立 GABPβ 1 L同种型是一种有价值的治疗靶点，可特异性治疗TERT启动子突变型CNS肿瘤， 可能还有很多人。与此同时，我们将推进药物发现和开发工作， 与行业合作伙伴Telo Therapeutics和GlaxoSmithKline合作开发不同GABP亚基的分子抑制剂。

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.