Targeting the mutant promoter of Telomerase Reverse Transcriptase (TERT)

靶向端粒酶逆转录酶 (TERT) 的突变启动子

• 批准号: 10677899
• 负责人: Michael Jackson
• 金额: $ 77.22万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677899
• 关键词: Alleles; Biological Assay; Bladder; Cancer Biology; Cancer cell line; Catalogs; Cell Death; Chemicals; Chimeric Proteins; Cisplatin; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependence; Disease; Dose; Drug Targeting; Effectiveness; Ensure; Evaluation; FRAP1 gene; Future; Gene Expression Profile; Genetic Transcription; Goals; Growth; Head; Heterozygote; Human; Immunologic Surveillance; Immunotherapy; In Vitro; Length; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Maps; Marrow; Mediating; Monitor; Morphologic artifacts; Mutation; Patients; Performance; Pharmaceutical Preparations; Phenotype; Promoter Regions; Proteins; RNA-Directed DNA Polymerase; Reporter; Reproducibility; Role; Signal Transduction; Small Interfering RNA; Specificity; Structure-Activity Relationship; Telomerase; Telomere Length Maintenance; Testing; Therapeutic; Toxic effect; analog; aurora kinase A; cancer cell; cancer therapy; cell growth; cheminformatics; chemotherapy; clinical practice; cytotoxicity; design; experimental study; high throughput screening; immune checkpoint blockade; improved; inhibitor; innovation; kinase inhibitor; mRNA Expression; mTOR inhibition; mutant; new therapeutic target; novel; novel therapeutics; overexpression; patient prognosis; promoter; protein degradation; reconstitution; response; scaffold; screening; self-renewal; small molecule; small molecule libraries; stem cells; telomere; therapeutic target; tool; tumor; tumor growth

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.

项目摘要 端粒酶逆转录酶（TERT）在通过端粒酶维持端粒长度方面是限速的， 还具有调节细胞生长等端粒非依赖性功能。我们发现TERT启动子突变 （mutTERT）发生在约70%的膀胱癌（BC）中，最常见的是在-146bp和-124bp处， 由共同上游信号传导机制识别的相同11 bp序列。这些突变驱动 TERT过表达维持BC生长，并与BC患者预后不良相关。这些数据导致 我们开发了一组创新的检测方法，专门用于通过化学库筛选 方法，减少来自mutTERT而不是wtTERT的TERT表达的小分子。通知发现 化合物是实现我们开发对BC细胞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具有最小的毒性。初步数据： 使用CRISPR，我们构建了在突变体或野生型TERT下游表达HiBiT或EGFP报告基因的BC细胞， 发起人。我们通过显示siRNA介导的GABPA（GABPA的一种调节因子）的耗竭来验证这些测定。 突变启动子表达，选择性地减少mutTERT，如通过HiBiT或EGFP监测的。我们部署了 在605种激酶抑制剂的高通量筛选（HTS）中试筛选中进行了mutTERT-HiBiT测定，发现23 减少mutTERT-HiBiT的命中率。其中包括已知的TERT表达驱动因子的抑制剂， 极光激酶A，以及mutTERT选择性抑制剂，所有这些都靶向mTOR。这些数据导致 我们的假设是，小分子特异性抑制突变驱动的TERT表达， 启动子可以通过表型筛选来鉴定。三个具体目标验证了这一假设。在目标1中， 将使用mutTERT-HiBiT报告基因测定进行350 K化学文库的筛选，以鉴定 降低TERT表达的化合物在16小时时从突变型启动子上被驱动。点击将被确认， 并进行反筛选，以清除那些有不必要活动的人。在目标2中，优先命中将被购买， 在初步测定中再次证实，并且头对头评价了它们对效应突变体超过wt TERT的选择性 在BC细胞中部署EGFP报告基因测定和通过等位基因特异性qPCR。迭代的按目录类比法（ABC） 方法将用于建立新生的结构-活性关系的命中支架和提高效力 和选择性。在目标3中，每个支架的最佳命中符合一组严格的效力和选择性标准 将被选为探针，并在多个测定中表征，以绘制它们对 叔这些测定使用一组具有和不具有TERT启动子突变的BC和非BC细胞系， 确定探针对以下方面的影响：1）通过评估我们的TERT， 表达标签（TES）; 2）端粒长度定量; 3）端粒酶依赖性和非依赖性TERT 功能包括细胞生长。未来方向：我们为靶向癌症特异性的新药奠定了基础， 在患有BC或其他TERT驱动的恶性肿瘤的患者中，mutTERT驱动的端粒酶活性。