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

描述（由申请人提供）：一个非小细胞肺癌（NSCLC）亚组由表皮生长因子受体（EGFR）的激活突变驱动。突变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一过性反应，尽管这些抑制剂的临床疗效受到耐药性发展的限制。守门突变EGFR T790 M是临床上观察到的最普遍的耐药机制，占耐药病例的近60%。在这里，我们建议评估T790 M的易感性和生物学，并确定出现的耐药机制时，T790 M靶向一类新的多药选择性EGFR抑制剂。第一个目标是研究为什么一些患者和细胞系发展EGFR T790 M，而另一些患者和细胞系通过替代机制产生耐药性。目前尚不清楚EGFR T790 M是否在药物治疗后重新发生，或者是否在未经治疗的肿瘤中以非常低的水平存在，并且由于药物治疗赋予的选择性优势而变得可检测。为了测试这些可能性，将对已知通过T790 M可重复产生耐药性的未经治疗的细胞系和患者原代细胞进行亚克隆，并在药物治疗前后分析T790 M耐药性。表征细胞克隆亚群中的抗性发展将提供对细胞系内抗性机制库的深入了解，并有助于阐明为什么T790 M在某些细胞环境中是主导抗性机制。接下来，CRISPR-Cas9基因组编辑将用于将T790 M突变工程化到通过T790 M或替代耐药机制产生耐药性的未经治疗的细胞系中。这将允许在内源性和非内源性背景下研究T790 M突变，将T790 M发展与体外或离体产生耐药性期间出现的任何潜在乘客突变分开。还将探索阻碍T790 M发育或将EGFR T790 M回复为野生型密码子（T790）的后果。拟议研究的最终目的是表征对特异性靶向EGFR T790 M的MUR选择性抑制剂的耐药性的新机制，沿着活化突变EGFR。正在进行的临床试验中研究这些抑制剂的疗效。使用细胞系和患者样本对MUR选择性抑制剂的耐药性进行建模将有助于预测临床中出现的耐药机制。总之，拟议的实验将提供对EGFR T790 M的开发和治疗的深入了解。这些发现将对我们理解靶向治疗的耐药性产生直接影响，并可以指导临床决策，最终防止EGFR突变型NSCLC患者发生耐药性。

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.