EGFR T790M-mediated drug resistance in non-small cell lung cancer

描述(申请人提供)：非小细胞肺癌(NSCLC)的一个子集是由表皮生长因子受体(EGFR)的激活突变驱动的。突变的EGFR驱动的肺癌对EGFR激酶抑制剂吉非替尼和厄洛替尼的靶向治疗有短暂的反应，尽管这些抑制剂的临床疗效受到耐药性发展的限制。门卫突变EGFR T790M是临床上观察到的最常见的耐药机制，占耐药病例的近60%。在这里，我们建议评估T790M的易感性和生物学，并确定当T790M以一种新的突变选择性EGFR抑制剂为靶点时出现的耐药机制。第一个目标是研究为什么一些患者和细胞系产生EGFR T790M，而另一些人则通过替代机制产生耐药性。目前尚不清楚EGFR T790M是对药物治疗的反应而重新出现，还是在治疗初期的肿瘤中存在于非常低的水平，并由于药物治疗带来的选择性优势而变得可检测。为了测试这些可能性，已知可通过T790M复制产生耐药性的治疗初治细胞系和患者原代细胞将被亚克隆，并在药物治疗前后分析T790M耐药性。研究克隆细胞亚群中耐药发展的特征将有助于深入了解细胞系中的耐药机制，并有助于阐明为什么T790M是某些细胞环境中的主要耐药机制。接下来，CRISPR-Cas9基因组编辑将被用于将T790M突变改造成通过T790M或替代耐药机制变得耐药的治疗初始细胞系。这将允许在内源性和非内源性环境中研究T790M突变，将T790M的发展与在体外或体外产生耐药性期间出现的任何潜在的乘客突变分开。还将探讨阻碍T790M发育或将EGFR T790M恢复为野生型密码子(T790)的后果。这项拟议研究的最终目的是表征针对EGFR T790M的突变选择性抑制剂的新抗性机制，以及激活突变的EGFR。正在进行的临床试验中正在研究这些抑制剂的疗效。使用细胞系和患者样本对突变选择性抑制剂的耐药性进行建模将有助于预测将在临床上出现的耐药性机制。综上所述，拟议的实验将为EGFR T790M的开发和治疗提供深入的见解。这些发现将直接影响我们对靶向治疗耐药的理解，并可能指导临床决策，并最终防止EGFR突变的非小细胞肺癌患者的耐药性发展。

DESCRIPTION (provided by applicant): A subset of non-small cell lung cancer (NSCLC) is driven by activating mutations of the epidermal growth factor receptor (EGFR). Mutant EGFR-driven lung cancers respond transiently to targeted therapy with the EGFR kinase inhibitors gefitinib and erlotinib, although the clinical efficacy of these inhibitors is limited by the development of drug resistance. The gatekeeper mutation EGFR T790M is the most prevalent resistance mechanism observed in the clinic, accounting for nearly 60% of drug resistance cases. Here, we propose to assess T790M predisposition and biology, and identify resistance mechanisms that emerge when T790M is targeted with a new class of mutant-selective EGFR inhibitors. The first objective is to study why some patients and cell lines develop EGFR T790M, while others become drug resistant through alternative mechanisms. It is not currently known whether EGFR T790M develops de novo in response to drug treatment, or if it is present at very low levels in treatment-naive tumors and becomes detectable due to a selective advantage conferred by drug treatment. To test these possibilities, treatment-naïve cell lines and patient primary cells known to reproducibly become resistant through T790M will be subcloned and analyzed for T790M resistance before and after drug treatment. Characterizing resistance development in clonal subpopulations of cells will provide insight into the repertoire of resistanc mechanisms within a cell line, and help elucidate why T790M is the dominant resistance mechanism in some cellular contexts. Next, CRISPR-Cas9 genome editing will be used to engineer the T790M mutation into treatment-naive cell lines that become resistant through T790M or alternative resistance mechanisms. This will permit study of the T790M mutation in both endogenous and nonendogenous contexts, separating T790M development from any potential passenger mutations that arise during in vitro or ex vivo generation of drug resistance. The consequences of hindering T790M development or reverting EGFR T790M to the wild type codon (T790) will also be explored. The final aim of the proposed research is to characterize novel mechanisms of resistance to mutant-selective inhibitors that specifically target EGFR T790M, along with activating mutant EGFR. The efficacy of these inhibitors is being investigated in ongoing clinical trials. Modeling resistance to mutant-selective inhibitors using cell lines and patient samples will help predict resistance mechanisms that will emerge in the clinic. Taken together, the proposed experiments will provide insight into EGFR T790M development and treatment. These findings will have immediate implications for our understanding of resistance to targeted therapies, and could guide clinical decision-making, and ultimately prevent the development of drug resistance in patients with EGFR-mutant NSCLC.