Discovering the mechanisms-of-action of mistargeted anti-cancer agents

发现错误靶向抗癌药物的作用机制

基本信息

批准号：
9886861
负责人：
Jason Sheltzer
金额：
$ 26.93万
依托单位：
COLD SPRING HARBOR LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9886861
关键词：
Ablation Alleles Antineoplastic Agents Apoptosis Biological Assay Biological Markers CRISPR/Cas technology Cell Cycle Cell Cycle Progression Cell Death Cell Line Cell Proliferation Cells Chemicals Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Combined Modality Therapy Coupled Cyclin-Dependent Kinase Inhibitor Dependence Dose Dose-Limiting Drug Design Drug Targeting Drug resistance Essential Genes Evaluation Exhibits Failure Genes Genetic Genetic Screening Genetic Techniques Goals Growth Human In Vitro Knock-out Knowledge Laboratories Light Malignant - descriptor Malignant Neoplasms Mitotic Mitotic Cell Cycle Modeling Mutagenesis Mutation Oncology Patients Pharmaceutical Preparations Pharmacology Phenotype Phosphotransferases Prediction of Response to Therapy Proliferating Proteins Publishing RNA Interference Reagent Regimen Reporting Research Resistance Role Series Specificity Techniques Testing Therapeutic Therapeutic Intervention Tissues Toxic effect Validation Work anticancer research cancer cell cancer genetics cancer therapy cell killing cellular targeting clinical efficacy experimental study fitness genetic architecture genetic technology in vivo inhibitor/antagonist knock-down loss of function non-drug novel novel anticancer drug novel therapeutics null mutation patient population pre-clinical research clinical testing resistance mutation response side effect small molecule small molecule inhibitor targeted treatment tumor tumorigenesis

项目摘要

Project Summary Cancer cells require the proteins encoded by certain genes in order to proliferate. These “genetic dependencies” are promising targets for therapeutic intervention, as drugs that block the function of a dependency can induce apoptosis and durable tumor regression. The discovery and characterization of genetic dependencies and the drugs that can inhibit them are key goals of preclinical cancer research. My laboratory has investigated multiple putative genetic dependencies using CRISPR/Cas9 mutagenesis. We have found that verified mutagenesis of many cancer drug targets fails to recapitulate published results obtained when these genes were knocked down with RNAi. Moreover, we find that multiple “targeted inhibitors” currently in clinical trials continue to kill cancer cells harboring CRISPR-induced null mutations in their reported targets, demonstrating pervasive off-target cell killing among clinical inhibitors. These results – coupled with the observation that 97% of drug-indication pairs that enter clinical trials in oncology fail to receive FDA approval - suggest the existence of fundamental shortcomings in how cancer genetic dependencies are identified and studied. In this work, we will develop a robust, preclinical target validation pipeline to characterize both the consequences of loss-of-function alterations in potential drug targets and to validate on-target activity of putative clinical inhibitors. In particular, we will select genes that are reported to be cancer dependencies and that are targeted by small-molecule inhibitors, and we will study the cellular consequences of their deletion or inhibition (Aim 1). Next, we will use cells harboring CRISPR-induced knockouts of these putative drug targets to investigate the chemical inhibitors that had been used to target them (Aim 2). If these reagents continue to kill cells that totally lack their reported targets, then this would indicate that they induce cell death through an off-target mechanism. Then, we will deploy both spontaneous- and CRISPR-directed mutagenesis in order to generate mutations that confer resistance to these small-molecule inhibitors, thereby helping to identify their true cellular targets (Aim 3). Finally, by isolating drug-resistance mutations, we have discovered that one mischaracterized anti-cancer drug is in fact the first potent and specific inhibitor of the CDK11B kinase to be described. Using this knowledge, we will seek to identify biomarkers that can predict therapeutic responses to this drug (Aim 4). In total, these experiments will delineate a robust preclinical pipeline for target validation, shed light on the genetic architecture that underlies cancer-essential genes, and allow drug re-purposing studies of multiple clinical inhibitors by uncovering their true targets.

项目概要癌细胞需要某些基因编码的蛋白质才能增殖。是治疗干预的有希望的目标，因为阻断依赖性功能的药物可以诱导细胞凋亡和持久的肿瘤消退。遗传依赖性的发现和表征以及能够抑制它们的药物是临床前癌症研究的关键目标。我的实验室使用 CRISPR/Cas9 诱变研究了多种假定的遗传依赖性。发现许多癌症药物靶标的经过验证的诱变无法概括已发表的结果当这些基因被RNAi敲除时，我们发现目前存在多种“靶向抑制剂”。在临床试验中继续杀死在其报告的靶标中含有 CRISPR 诱导的无效突变的癌细胞，这些结果表明临床抑制剂普遍存在脱靶细胞杀伤作用。观察发现，97% 进入肿瘤学临床试验的药物-适应症对未能获得 FDA 批准 - 表明在如何识别癌症遗传依赖性方面存在根本缺陷在这项工作中，我们将开发一个强大的临床前目标验证管道来表征两者。潜在药物靶点功能丧失改变的后果并验证假定的靶点活性特别是，我们将选择据报道与癌症相关的基因。小分子抑制剂靶向，我们将研究它们的缺失或抑制的细胞后果（目标 1）接下来，我们将使用含有 CRISPR 诱导敲除这些假定药物靶标的细胞来进行研究。用于靶向它们的化学抑制剂（目标 2）如果这些试剂继续杀死细胞。完全缺乏他们报告的目标，那么这表明它们通过脱靶诱导细胞死亡然后，我们将部署自发诱变和 CRISPR 定向诱变以产生。赋予对这些小分子抑制剂抗性的突变，从而有助于识别它们真正的细胞最后，通过分离耐药突变，我们发现一个被错误表征的突变。事实上，抗癌药物是第一个被描述的有效且特异性的 CDK11B 激酶抑制剂。知识，我们将寻求识别可以预测对该药物的治疗反应的生物标志物（目标 4）。总的来说，这些实验将描绘出用于目标验证的强大临床前管道，揭示遗传癌症必需基因的结构，并允许对多个临床进行药物再利用研究通过发现其真正目标来抑制抑制剂。