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

项目摘要 癌细胞需要由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靶点，因为阻断依赖性功能的药物可以诱导 凋亡和持久的肿瘤消退。遗传依赖性的发现和表征， 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诱变研究了多种推定的遗传依赖性。我们 已经发现，许多癌症药物靶点的经验证的诱变不能概括已发表的结果， 当这些基因被RNAi敲除时。此外，我们发现，目前多种“靶向抑制剂” 在临床试验中继续杀死在其报告的靶点中携带CRISPR诱导的无效突变的癌细胞， 证明了临床抑制剂中普遍的脱靶细胞杀伤。这些结果-加上 观察到97%进入肿瘤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性缺陷， 研究了在这项工作中，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，并验证推定的靶向活性。 临床抑制剂。特别是，我们将选择被报道为癌症依赖性的基因， 被小分子抑制剂靶向，我们将研究它们缺失或抑制的细胞后果 (Aim 1）。接下来，我们将使用携带CRISPR诱导的这些假定药物靶点敲除的细胞来研究 化学抑制剂，已被用来针对他们（目标2）。如果这些试剂继续杀死细胞， 完全缺乏它们报告的靶点，那么这将表明它们通过脱靶诱导细胞死亡。 机制然后，我们将部署自发和CRISPR定向诱变，以产生 突变，赋予这些小分子抑制剂的耐药性，从而帮助确定他们真正的细胞， 目标（目标3）。最后，通过分离耐药突变，我们发现一个错误的特征， 事实上，抗癌药物是第一个有效的和特异性的CDK 11B激酶抑制剂。使用此 知识，我们将寻求确定生物标志物，可以预测这种药物的治疗反应（目的4）。在 总的来说，这些实验将描绘一个强大的临床前管道的目标验证，揭示了遗传 构建癌症必需基因的基础，并允许多种临床研究的药物再利用研究。 通过发现它们的真正目标来抑制它们。

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.