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

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。

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.