Engineered precancerous cells and tissues for discovery of lung cancer drivers

描述 小细胞肺癌（SCLC）仍然是影响男性和女性的重要临床问题， 每年在美国造成约3万名患者死亡。这种癌症的高死亡率是 主要是由于早期检测的困难和目前遗传毒性化疗的不足， 在过去的30年里基本保持不变。新的预防和治疗方法至关重要， 开发了改善患者结果的方法。基因突变的功能表征 SCLC基因组将有助于发现用于预防和靶向治疗的生物标志物，但这仍然是 由于缺乏可靠的实验模型，这是非常具有挑战性的。遗传工程小鼠模型 人小细胞肺癌的GEMM已成为研究癌基因功能和肿瘤发生的重要工具 抑制基因和评估治疗性治疗。然而，GEMM的利用需要费力的 和昂贵的基因杂交来测试哪怕是一个基因的功能。为了克服这些挑战，我们 已经开发了一种体外肿瘤进展模型来定义CRISPR介导的肿瘤细胞的致癌功能。 基于其转化癌前细胞（preSC）的能力， 携带Rb和p53的条件等位基因的SCLC的工程小鼠模型，Rb和p53是最常突变的 人类SCLC中的基因。通过候选突变转化的突变体preSC的比较分析， 控制preSC使我们能够区分突变特异性基因和途径，从众多的次级或 适应性变化。我们还开发了一种基于体细胞工程的模型， 直接在肺气道中递送的CRISPR-Cre混合载体使我们能够表征CRISPR介导的 在体内肿瘤发展过程中的突变与Cre介导的Rb和p53失活有关。这 新的体内方法能够在体内快速功能性询问候选突变，而不需要 昂贵耗时的基因杂交使用这些互补模型，我们将测试假设 小细胞肺癌中的复发性突变足以导致Rb/p53突变体的致瘤进展， 前体细胞在aim 1中，我们将通过比较分析 工程化的癌前细胞在目标2中，我们将使用CRISPR-Cre杂合载体来表征新的 在体内肿瘤发展中的突变。成功完成这一提案将定义一打最 SCLC中的复发突变，并提供携带特定突变集的SCLC的临床前模型。这些 结果将是突破性的，为未来的研究，旨在阐明机制的肿瘤发展， 以及发展这种癌症的预防和治疗策略。此外，CRISPR-Cre 混合工具易于扩展以测试更多的突变，并易于应用于其他癌症模型， 接受腺病毒基因递送，包括那些非小细胞肺癌。

DESCRIPTION Small cell lung cancer (SCLC) remains a significant clinical problem that affects both men and women and causes an estimated 30,000 patient deaths every year in the US. The high mortality rate of this cancer is mainly due to the difficulty of early detection and the inadequacy of current genotoxic chemotherapies that have remained largely unchanged for the past 30 years. It is essential that new preventive and therapeutic approaches are developed to improve patient outcomes. Functional characterization of recurrent mutations in the SCLC genome will facilitate discovery of biomarkers for prevention and targeted therapy, but this remains extremely challenging due to the paucity of robust experimental models. Genetically engineered mouse models (GEMM) of human SCLC have been important tools for determining functions of oncogenes and tumor suppressor genes and evaluating therapeutic treatments. However, utilization of the GEMMs requires laborious and expensive genetic crosses to test the function of even a single gene. To overcome these challenges, we have developed an in vitro tumor progression model to define oncogenic function of CRISPR-mediated mutations based on their ability to transform precancerous cells (preSC) which were isolated from a genetically engineered mouse model of SCLC that carries conditional alleles of Rb and p53, the most frequently mutated genes in human SCLC. Comparative profiling of mutant preSC transformed by a candidate mutation with control preSC allows us to distinguish mutation-specific genes and pathways from a multitude of secondary or adaptive changes. We have also developed a somatic engineering-based model in which an adenoviral CRISPR-Cre hybrid vector, delivered directly in the lung airways, allows us to characterize CRISPR-mediated mutation during in vivo tumor development in conjunction with Cre-mediated inactivation of Rb and p53. This new in vivo approach enables rapid functional interrogation of candidate mutations in vivo without need for expensive, time-consuming genetic crosses. Using these complementary models, we will test the hypothesis that recurrent mutations in SCLC are sufficient to cause the tumorigenic progression of Rb/p53-mutant precursor cells. In aim 1, we will discover mutation-driven oncogenic pathways using comparative analysis of engineered precancerous cells. In aim 2, we will use the CRISPR-Cre hybrid vector to characterize novel mutations in tumor development in vivo. Successful completion of this proposal will define a dozen of the most recurrent mutations in SCLC and provide preclinical models of SCLC carrying specific sets of mutations. These outcomes will be groundbreaking for future studies aimed at mechanistic elucidation of the tumor development as well as development of preventive and therapeutic strategies for this cancer. Moreover, the CRISPR-Cre hybrid tools are easily extendable to test more mutations and are readily applicable to other cancer models that are receptive to adenoviral gene delivery, including those of non-small cell lung cancers.