The role of NF2 in Acquired Resistance to Targeted EGFR Inhibition in Lung Cancer

描述(申请人提供)：含有编码表皮生长因子受体(EGFR)基因的激活突变的转移性肺肿瘤最初对第一代EGFR酪氨酸激酶抑制剂(TKIs)如吉非替尼或厄洛替尼有反应，但最终产生获得性耐药(AR)。随着疾病的发展，超过一半的患者的肿瘤含有第二位点EGFR突变T790M。我们的实验室在临床前模型中显示，第二代EGFR-TKI，afatinib，加上抗EGFR抗体，西妥昔单抗，可以克服T790M介导的耐药性。随后在吉非替尼/厄洛替尼的变应性鼻炎患者中进行的Ib期临床试验显示出前所未有的30%的应答率。我已经从三名携带T790M的肿瘤患者身上获得了肿瘤样本，这些患者的肿瘤放射学反应良好，然后在药物组合方面取得了进展。全基因组测序的生物信息学分析在一个样本中发现了两个意想不到的NF2突变：剪接点突变C.811-2A和截断突变C.592C&gt；T(p.R198*)。在患者治疗前的样本中没有检测到这两种突变，这表明NF2的突变是在治疗过程中获得的。NF2基因编码Merlin，一种参与细胞黏附的细胞骨架蛋白。在其可能的功能中，Merlin已被证明在EGFR信号转导中发挥作用。在融合环境中，Merlin在膜上结合EGFR，以抑制受体信号转导，防止内化和再循环。Merlin的缺失可以导致EGFR信号的增强，以及下游PI3K和MAPK信号通路的激活。Merlin在细胞黏附和生长因子受体反应中的作用使其成为调节双重EGFR抑制的耐受性的有趣候选者。我在一个EGFR突变的肺癌细胞系中的初步研究表明，瞬时转染针对NF2的短干扰RNA(SiRNAs)池降低了细胞对EGFR TKI erlotinib的敏感性。对单个siRNA也得到了类似的结果。根据我的初步数据，我假设在EGFR突变的肺癌中，NF2的缺失介导了肿瘤对EGFR抑制的抵抗。为了验证这一假设，我提出了三个具体的目标：1)在一组EGFR突变细胞系中，确定NF2缺失和过表达对EGFR突变肺癌细胞对EGFR抑制的敏感性的影响；2)检测NF2缺失对药物敏感和耐药的EGFR突变细胞中增殖/致癌信号通路的机制影响；以及3)确定相关患者样本中NF2突变的频率。了解NF2突变的功能和信号后果可能有助于深入了解EGFR信号转导，并揭示预防或克服患者获得性耐药的方法。

DESCRIPTION (provided by applicant): Metastatic lung tumors that harbor activating mutations in the gene encoding the epidermal growth factor receptor (EGFR) initially respond to first-generation EGFR-tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib, but eventually develop acquired resistance (AR). Upon disease progression, more than half of patients' tumors harbor a second-site EGFR mutation, T790M. Our lab showed in preclinical models that the combination of a second-generation EGFR-TKI, afatinib, plus the anti- EGFR antibody, cetuximab, may overcome T790M-mediated resistance. A subsequent phase Ib clinical trial in patients with AR to gefitinib/erlotinib showed an unprecedented 30% response rate. I have obtained tumor samples from three patients whose T790M-harboring tumors responded radiographically, then progressed on the drug combination. Bioinformatic analysis of whole genome sequencing identified in one sample two unexpected NF2 mutations: a splice site mutation c.811- 2A>T and a truncating mutation c.592C>T (p.R198*). Neither mutation was detected in the patient's pre-treatment sample, suggesting that mutations in NF2 were acquired during treatment. The NF2 gene encodes Merlin, a cytoskeletal protein involved in cellular adhesion. Among its putative functions, Merlin has been shown to play a role in EGFR signaling. In confluent environments, Merlin binds EGFR at the membrane to inhibit receptor signaling and prevent internalization and recycling. Loss of Merlin can lead to enhanced EGFR signaling as well as activation of downstream PI3K and MAPK signaling pathways. The role of Merlin in both cellular adhesion and growth factor receptor response make it an intriguing candidate to mediate resistance to dual EGFR inhibition. My preliminary studies in an EGFR-mutant lung cancer cell line show that transient transfection with a pool of short-interfering RNAs (siRNAs) against NF2 decreases cell sensitivity to EGFR TKI erlotinib. Similar results were obtained with individual siRNAs. Based upon my preliminary data, I hypothesize that loss of NF2 mediates tumor resistance to EGFR inhibition in EGFR-mutant lung tumors. To test this hypothesis, I propose three specific aims: 1) determine the effect of NF2 loss and overexpression on the sensitivity of EGFR-mutant lung cancer cells to EGFR inhibition in a panel of EGFR mutant cell lines; 2) examine the mechanistic consequences of NF2 loss on proliferative/oncogenic signaling pathways in drug-sensitive and -resistant EGFR mutant cells; and 3) determine the frequency of NF2 mutation in relevant patient samples. Understanding the functional and signaling consequences of NF2 mutation may provide insights into EGFR signaling and reveal ways to prevent or overcome acquired resistance in patients.