Engineered precancerous cells and tissues for discovery of lung cancer drivers

描述 小细胞肺癌(SCLC)仍然是一个影响男性和女性的重大临床问题 据估计，在美国每年导致30,000名患者死亡。这种癌症的高死亡率是 主要原因是难以及早发现，以及目前的遗传毒性化疗方法不足， 在过去的30年里基本保持不变。至关重要的是，新的预防和治疗 开发的方法是为了改善患者的预后。基因重复性突变的功能特征 小细胞肺癌基因组将有助于发现用于预防和靶向治疗的生物标记物，但这仍然是 由于缺乏强大的实验模型，这极具挑战性。转基因小鼠模型 人小细胞肺癌(SCLC)的GEMM已成为确定癌基因和肿瘤功能的重要工具 抑制基因和评估治疗方法。然而，利用GEMM需要付出艰苦的努力 和昂贵的基因杂交来测试即使是单个基因的功能。为了克服这些挑战，我们 我已经开发了一个体外肿瘤进展模型来确定CRISPR介导的致癌功能 突变基于它们转化癌前细胞的能力(前SC)，这些细胞是从基因上分离出来的 携带Rb和p53条件等位基因的小鼠小鼠小细胞肺癌模型，Rb和p53是最频繁突变的 人类小细胞肺癌的基因。一种候选突变转化的突变前SC与 控制前SC使我们能够将突变特异的基因和途径从大量的继发性或 适应性变化。我们还开发了一种基于体细胞工程的模型，在该模型中，腺病毒 CRISPR-Cre杂合载体，直接在肺内输送，使我们能够表征CRISPR介导的 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 新的体内方法实现了对体内候选突变的快速功能询问，而不需要 昂贵、耗时的基因杂交。使用这些互补的模型，我们将检验这一假设 小细胞肺癌中反复发生的突变足以导致Rb/P53突变的肿瘤进展 前体细胞。在目标1中，我们将使用比较分析来发现突变驱动的致癌途径 经过改造的癌前细胞。在目标2中，我们将使用CRISPR-CRE混合载体来表征新的 体内肿瘤发展中的突变。这项提案的成功完成将定义十几个最 小细胞肺癌的反复突变，并提供携带特定突变集合的小细胞肺癌临床前模型。这些 这些结果将是未来旨在阐明肿瘤发生机制的研究的开创性成果 以及针对这种癌症的预防和治疗策略的开发。此外，CRISPR-CRE 杂交工具很容易扩展到测试更多的突变，并很容易适用于其他癌症模型 接受腺病毒基因的传递，包括非小细胞肺癌的基因。

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.