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Discovering the mechanisms of-action-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：
10390462
负责人：
Jason Sheltzer
金额：
$ 22.04万
依托单位：
YALE UNIVERSITY
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/10390462
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 drug repurposing experimental study fitness genetic architecture genetic technology in vivo inhibitor 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.

项目摘要癌细胞需要某些基因编码的蛋白质才能增殖。这些“基因依赖” 是治疗干预的有希望的目标，因为阻断依赖功能的药物可以诱导细胞凋亡和持久的肿瘤消退。基因依存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。我的实验室已经使用CRISPR/Cas9突变研究了多个假定的遗传相关性。我们已经发现，许多抗癌药物靶点的已验证突变未能概括已发表的结果当这些基因被RNAi打倒时。此外，我们发现，目前有多种“靶向抑制物” 在临床试验中，继续杀死在其报告的靶点中含有CRISPR诱导零突变的癌细胞，证明了临床抑制剂中普遍存在的非靶向细胞杀伤。这些结果-再加上观察到，进入肿瘤学临床试验的药物适应症对中有97%未能获得FDA的批准- 表明在如何识别癌症基因依赖性方面存在根本性缺陷学习。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，以表征潜在药物靶点功能丧失改变的后果和验证推定的靶点活性临床抑制剂。特别是，我们将选择那些被报告为癌症相关性的基因，以及以小分子抑制剂为靶点，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诱导的这些假定药物靶点的敲除的细胞来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那些完全没有报告的靶点，那么这就表明它们通过非靶点诱导细胞死亡机制。然后，我们将部署自发和CRISPR指导的突变，以便生成对这些小分子抑制剂产生抗药性的突变，从而帮助识别它们真正的细胞目标(目标3)。最后，通过分离耐药突变，我们发现一种错误的特征事实上，抗癌药物是第一个被描述的CDK11B激酶的有效和特异的抑制剂。使用这个在了解这些知识后，我们将寻求确定可以预测该药物治疗反应的生物标记物(目标4)。在……里面总之，这些实验将描绘出一个强大的临床前通道，用于靶标验证，揭示基因架构是癌症必需基因的基础，并允许对多种临床研究进行药物再利用研究通过发现它们的真实目标来抑制它们。