Employing CRISPR inactivation screening and in vivo models towards improving treatments for SCLC

摘要 在小细胞肺癌(SCLC)中缺乏可药物的致癌基因突变，并且没有批准的靶向 对于小细胞肺癌的治疗，我们需要找到新的治疗方法。在这个提案中，我们应用了全基因组 CRISPR失活筛查，以确定广泛的小细胞肺癌必需基因，或关键的基因定义 子集。然后我们确定对已识别的HITS的药物抑制是否也会导致选择性地杀死 小细胞肺癌细胞。我们应用体内系统，包括患者来源的异种移植(PDX)和基因工程 用小鼠模型来测试在这些遗传筛选中确定的关键目标的药物抑制物。目标1将专注于 在我们已经使用CRISPR失活筛选确定的Rb/P53突变所必需的途径上 小细胞肺癌细胞。控制cullin活性和蛋白质泛素化的NEDD8/RBX1途径的成分是 对小细胞肺癌细胞至关重要。MLN4924对NEDD8激活酶的药理抑制作用 衍生异种移植(PDX)模型证实了SCLC细胞对这一途径的依赖。令人兴奋的是，我们确定了 在某些PDX型号中对MLN4924的异常响应示例。目标1：将体内模型应用于指导 NEDD8抑制对小细胞肺癌亚群最有可能的反应。在这里，我们探索抑制的可能性 NEDD8通路在小细胞肺癌中的表达。我们将联合抑制NAE和顺铂-依托泊苷化疗和 确定是否会产生持久响应。一些SCLC PDX模型对NAE表现出极高的敏感性 抑制；我们将确定超反应PDX模型的潜在机制和遗传特征 可以用来帮助预测特殊的应答者。我们将确定是否应该抑制NAE 针对所有小细胞肺癌患者，还是针对关键亚群，以及这种方法是否增强了对化疗的反应。 MLN4924已经在对其他癌症类型的临床试验中进行测试，这项工作可能很快就会进入临床 在小细胞肺癌的试验。目的2：应用CRISPR失活筛选寻找小细胞肺癌新的治疗靶点。 这一目标将扩大我们的CRISPR失活屏幕，以包括具有关键驱动程序突变的小鼠SCLC细胞系 除Rb/P53外，包括Pten、CREBBP和Myc1。我们的方法利用了这样一个事实，即虽然人类 小细胞肺癌具有极高的突变负担，导致小细胞肺癌与给定的 驱动程序突变，具有已定义驱动程序更改的小鼠SCLC更加同质，增加了信噪比 在识别漏洞方面。我们将确定SCLC基因定义的子集中的依赖关系，并将测试 在PDX和GEM模型中发现的可用药靶点的抑制剂，这些模型包含定义的基因改变。我们 利用现有的小鼠模型和衍生的细胞系，以及一组带有基因注释的PDX模型， 在体内和体外进行研究，以确定和评估靶向小细胞肺癌关键基因亚群的全新方法。

SUMMARY With a lack of druggable oncogenic mutations in small cell lung cancer (SCLC) and no approved targeted therapies, we need to identify new treatment approaches for SCLC. In this proposal, we apply whole genome CRISPR inactivation screens to identify genes that are essential for SCLC broadly, or for key genetically defined subsets. We then determine whether pharmacologic inhibition of identified hits also results in selective killing of SCLC cells. We apply in vivo systems including patient derived xenograft (PDX) and genetically engineered mouse models to test pharmacologic inhibitors of key targets identified in these genetic screens. Aim 1 will focus on a pathway that we already identified using CRISPR inactivation screens to be essential for Rb/p53-mutant SCLC cells. Components of a NEDD8/RBX1 pathway, controlling Cullin activity and protein ubiquitylation, were essential for SCLC cells. Pharmacologic inhibition of NEDD8 activating enzyme (NAE) using MLN4924 in patient derived xenograft (PDX) models confirmed reliance of SCLC cells on this pathway. Excitingly, we identified examples of exceptional responses to MLN4924 in some PDX models. Aim 1: Apply in vivo models to direct NEDD8 inhibition to subsets of SCLC most likely to respond. Here, we explore the potential for inhibiting the NEDD8 pathway in SCLC. We will combine NAE inhibition with cisplatin-etoposide chemotherapy and determine whether durable responses result. Some SCLC PDX models exhibited exquisite sensitivity to NAE inhibition; we will identify underlying mechanisms and genetic features of super-responding PDX models that could be used to help predict exceptional responders. We will determine whether NAE inhibition should be directed to all SCLC patients, or to key subsets, and whether this approach augments response to chemotherapy. MLN4924 is already being tested in clinical trials for other cancer types and this work could rapidly lead to clinical trials in SCLC. Aim 2: Apply CRISPR inactivation screens to identify new therapeutic targets for SCLC. This Aim will extend our CRISPR inactivation screens to include mouse SCLC cell lines with key driver mutations beyond Rb/p53, including Pten, Crebbp and Mycl. Our approach takes advantage of the fact that while human SCLC has an extremely high mutational burden, leading to extensive heterogeneity among SCLC with a given driver mutation, mouse SCLC with defined driver alterations are more homogenous, increasing signal to noise in identifying vulnerabilities. We will identify dependencies in genetically-defined subsets of SCLC and will test inhibitors of identified druggable targets in PDX and GEM models that harbor defined genetic alterations. We leverage available mouse models and derived cell lines, along with a bank of genetically annotated PDX models, studied in vivo and ex vivo, to identify and evaluate completely new ways to target key genetic subsets of SCLC.