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：
10668942
负责人：
Jason Sheltzer
金额：
$ 23.02万
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/10668942
关键词：
Ablation Alleles Antineoplastic Agents Biological Assay Biological Markers CRISPR/Cas technology Cell Cycle Cell Cycle Progression Cell Death Induction 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 Induction of Apoptosis Knock-out Knowledge Laboratories Malignant - descriptor Malignant Neoplasms Mitotic Mitotic Cell Cycle Modeling Mutagenesis Mutation Oncology Patients Pharmaceutical Preparations 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 biomarker identification cancer cell cancer genetics cancer therapy cell killing cellular targeting clinical efficacy drug isolation drug repurposing experimental study fitness genetic architecture genetic technology in vivo inhibitor knock-down loss of function novel novel anticancer drug novel therapeutics null mutation patient population pharmacologic 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“遗传依赖性” 被承诺进行治疗干预的目标，因为阻止依赖功能的药物可以诱导细胞凋亡和耐用的肿瘤回归。遗传依赖性的发现和表征可以抑制它们的药物是临床前癌研究的关键目标。我的实验室使用CRISPR/CAS9诱变研究了多种推定的遗传依赖性。我们已经发现，经过验证的许多癌症药物靶标的诱变未能概括获得的已发表的结果当这些基因被RNAi击倒时。此外，我们发现目前有多个“靶向抑制剂” 在临床试验中，在其报告的靶标中杀死了带有CRISPR诱导的无效突变的癌细胞，证明临床抑制剂之间普遍存在的靶向细胞杀死。这些结果 - 与观察到，有97％的药物配对进入肿瘤学临床试验未能获得FDA批准 - 暗示存在如何确定癌症遗传依赖性的根本缺点和 Studiod。在这项工作中，我们将开发一个健壮的临床前目标验证管道，以表征潜在药物靶标的功能丧失改变并验证推定的目标活动的后果临床抑制剂。特别是，我们将选择据报道是癌症依赖性的基因由小分子抑制剂靶向，我们将研究其缺失或抑制作用的细胞后果（目标1）。接下来，我们将使用带有CRISPR诱导的这些假定药物靶标的敲除的细胞进行调查用于靶向它们的化学抑制剂（AIM 2）。如果这些试剂继续杀死细胞完全缺乏报告的目标，这将表明它们通过脱离目标影响细胞死亡机制。然后，我们将部署赞助商和CRISPR指导的诱变，以生成会议抗这些小分子抑制剂的突变，从而有助于识别其真正的细胞目标（目标3）。最后，通过隔离药物抗性突变，我们发现一个错误的表征实际上，抗癌药物是要描述的CDK11b激酶的第一个潜力和特异性抑制剂。使用此知识，我们将寻求确定可以预测这种药物治疗反应的生物标志物（AIM 4）。总计，这些实验将描述一个可靠的临床前管道，以验证目标验证基于癌症基因的结构，并允许对多个临床的药物进行重新修复研究抑制剂通过发现其真正的目标。