Targeting the mutant promoter of Telomerase Reverse Transcriptase (TERT)

项目总结 端粒酶逆转录酶(TERT)是端粒酶维持端粒长度的限速酶 也具有端粒不依赖的功能，如调节细胞生长。我们发现了TERT启动子突变 (Muttert)发生在约70%的膀胱癌(BC)中，最常见的是-146个碱基和−124个碱基，这会产生 由共同的上游信号机制识别的相同的11个碱基的序列。这些突变推动了 TERT过度表达，维持BC生长，与BC患者预后不良有关。这些数据领先 美国将开发一组创新的分析方法，专门设计通过化学图书馆筛选来识别 方法，减少Muttert中TERT表达但不减少wtTERT表达的小分子。发现了这样的 化合物是实现我们开发对BC细胞具有选择性毒性的药物的总体目标的第一步， 同时对需要TERT进行自我更新的正常干细胞的毒性最小。初步数据： 使用CRISPR，我们构建了在突变体或wt TERT下游表达HiBiT或EGFP报告基因的BC细胞 推动者。我们通过证明siRNA介导的GABPA的枯竭来验证这些检测，GABPA是一种 突变启动子的表达，通过HiBiT或EGFP监测选择性地降低Muttert。我们部署了我们的 Muttert-HiBiT在高通量筛选(HTS)中试筛选605种激酶抑制剂，发现23种 降低Muttert-HiBiT的命中率。其中包括已知的TERT表达抑制物，如 Aurora Kinase A，以及Muttert选择性的抑制剂，所有这些都针对mTOR。此数据领先于 我们支持这样的假设，即由突变体驱动的特异性抑制TERT表达的小分子 启动子可以通过表型筛选来鉴定。有三个具体目标验证了这一假设。在AIM 1中，HTS 将使用Muttert-HiBiT报告试验对350K化学文库进行筛选以确定 降低TERT表达的化合物在16小时内将突变启动子赶走。然后将确认命中情况， 并对柜台进行筛选，以删除那些有不必要活动的人。在目标2中，优先命中将被购买， 在初步试验中再次确认，他们对影响突变体的选择性进行了正面和正面的TERT评估 在BC细胞和等位基因特异的qPCR中部署EGFP报告程序。一种迭代的按目录模拟(ABC) 该方法将用于建立HIT支架的新生构效关系，并提高其效力 和选择性。在目标3中，每个脚手架中满足一套严格的效力和选择性标准的最佳命中 将被选为探针，并在多种检测中进行表征，以绘制其活性对下游影响的图 TERT。这些检测使用了一组带有和没有TERT启动子突变的BC和非BC细胞株来 通过对我们的TERT的评估，确定探针对：1)下游TERT相关基因转录的影响 表达标记(TES)；2)端粒长度定量；3)端粒酶依赖和独立的TERT 功能包括细胞生长。未来方向：我们为针对癌症特异性的新药奠定了基础， Muttert驱动，BC或其他TERT驱动的恶性肿瘤患者的端粒酶活性。

PROJECT SUMMARY TElomerase Reverse Transcriptase (TERT) is rate limiting in maintenance of telomere length by telomerase and also has telomere independent functions such as regulating cell growth. We showed TERT promoter mutations (mutTERT) occur in ~70% of Bladder Cancers (BC), most commonly at -146bp and −124bp which generate an identical 11 bp sequence that is recognized by a common upstream signaling mechanism. These mutations drive TERT overexpression, maintain BC growth and are associated with poor BC patient prognosis. These data led us to develop a panel of innovative assays specifically designed to identify, through a chemical library screening approach, small molecules that reduce TERT expression from mutTERT but not wtTERT. Discovery of such compounds is a first step in attaining our overall goal of developing a drug that is selectively toxic for BC cells, while having minimal toxicity on normal stem cells, which require TERT for self-renewal. Preliminary Data: Using CRISPR, we constructed BC cells expressing HiBiT or EGFP reporters downstream of mutant or wt TERT promoters. We validated these assays by showing that siRNA-mediated depletion of GABPA, a regulator of mutant promoter expression, selectively reduced mutTERT as monitored by HiBiT or EGFP. We deployed our mutTERT-HiBiT assay in an High-Throughput Screen (HTS) pilot screen of 605 kinase inhibitors and found 23 hits that reduced mutTERT-HiBiT. Among these were inhibitors to known drivers of TERT expression such as Aurora Kinase A, as well as inhibitors that were mutTERT selective, all of which targeted mTOR. This data leads us to the Hypothesis that small molecules that specifically suppress TERT expression driven by a mutant promoter can be identified by a phenotypic screen. Three Specific Aims test this hypothesis. In Aim 1, an HTS screen of a 350K chemical library will be conducted using a mutTERT-HiBiT reporter assay to identify compounds that decrease TERT expression driven off the mutant promoter at 16hrs. Hits will then be confirmed, and counter screened to remove those with unwanted activities. In Aim 2, priority hits will be purchased, reconfirmed in the primary assay and their selectivity to effect mutant over wt TERT evaluated head to head deploying EGFP reporter assays in BC cells and by allele-specific qPCR. An iterative analog-by-catalog (ABC) approach will be used to establish nascent structure-activity relationship of hit scaffolds and improve potency and selectivity. In Aim 3, the best hit from each scaffold that meet a set of rigorous potency and selectivity criteria will be selected as a probe and characterized in multiple assays to map their activity on downstream effects of TERT. These assays use a panel of BC and non-BC cell lines with and w/o TERT promoter mutations to determine the probes’ effects on: 1) downstream TERT-related gene transcription via evaluation of our TERT Expression Signature (TES); 2) telomere length quantitation; 3) Telomerase-dependent and independent TERT functions including cell growth. Future Directions: We set the stage for novel drugs targeting cancer specific, mutTERT driven, telomerase activity in patients with BC or other TERT-driven malignancies.