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

描述（由申请方提供）：在编码表皮生长因子受体（EGFR）的基因中携带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（TKI）（如吉非替尼或厄洛替尼）有反应，但最终发展为获得性耐药（AR）。在疾病进展时，超过一半的患者的肿瘤具有第二位点EGFR突变T790 M。我们的实验室在临床前模型中显示，第二代EGFR-TKI阿法替尼与抗EGFR抗体西妥昔单抗联合使用可能克服T790 M介导的耐药性。随后在AR患者中进行的吉非替尼/厄洛替尼Ib期临床试验显示了前所未有的30%缓解率。我从三名携带T790 M的肿瘤患者身上获得了肿瘤样本，这些患者的肿瘤在放射学上有反应，然后在药物组合中取得了进展。全基因组测序的生物信息学分析在一个样品中鉴定了两个意外的NF 2突变：剪接位点突变c.811- 2A>T和截短突变c.592C>T（p.R198*）。在患者的治疗前样本中没有检测到突变，这表明NF 2的突变是在治疗期间获得的。NF 2基因编码Merlin，一种参与细胞粘附的细胞骨架蛋白。在其推定的功能中，Merlin已被证明在EGFR信号传导中发挥作用。在融合环境中，Merlin在膜上结合EGFR，以抑制受体信号传导并防止内化和再循环。Merlin的缺失可导致增强的EGFR信号传导以及下游PI 3 K和MAPK信号传导途径的激活。Merlin在细胞粘附和生长因子受体应答中的作用使其成为介导对双重EGFR抑制的抗性的有趣候选物。我在EGFR突变型肺癌细胞系中的初步研究表明，瞬时转染一组针对NF 2的短干扰RNA（siRNA）可降低细胞对EGFR TKI厄洛替尼的敏感性。用单个siRNA获得了类似的结果。基于我的初步数据，我假设在EGFR突变型肺肿瘤中，NF 2的缺失介导了肿瘤对EGFR抑制的耐药性。为了验证这一假设，我提出了三个具体的目标：1）确定一组EGFR突变细胞系中NF 2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）检查药物敏感和耐药EGFR突变细胞中NF 2缺失对增殖/致癌信号通路的机制后果;和3）确定相关患者样品中NF 2突变的频率。了解NF 2突变的功能和信号传导后果可能会提供对EGFR信号传导的见解，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.