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

NF2 在肺癌靶向 EGFR 抑制获得性耐药中的作用

• 批准号: 8795093
• 负责人: Caroline Nebhan
• 金额: $ 4.81万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795093
• 关键词: Address; Antibodies; Binding; Bioinformatics; Biological Assay; Biological Process; Cancer cell line; Cell Adhesion; Cell Line; Cell Survival; Cells; Cetuximab; Clinical; Clinical Trials; Complementary DNA; Complex; Cytoskeletal Proteins; Data; Development; Disease Progression; Drug Combinations; Drug resistance; Drug-sensitive; Environment; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Erlotinib; Frequencies; Gefitinib; Generations; Genes; Growth Factor Receptors; Image; Immunoblot Analysis; Individual; Lead; Lung Adenocarcinoma; Lung Neoplasms; MAP Kinase Gene; MAPK Signaling Pathway Pathway; Malignant neoplasm of lung; Mediating; Membrane; Mutation; NF2 gene; Nature; Neurofibromin 2; Oncogenic; Outcome; PI3K/AKT; Patients; Phase; Physicians; Play; Pre-Clinical Model; Receptor Inhibition; Receptor Signaling; Recycling; Research; Resistance; Resistance development; Role; Sampling; Scientist; Signal Pathway; Signal Transduction; Site; Soft Agar Assay; Splice-Site Mutation; System; Testing; Transfection; Translational Research; Tyrosine Kinase Inhibitor; base; cancer cell; cell growth; genome sequencing; human FRAP1 protein; human disease; improved; insight; knock-down; mutant; overexpression; prevent; public health relevance; resistance mechanism; response; targeted treatment; tumor

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.

描述（由申请人提供）：表皮生长因子受体（EGFR）编码基因活化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，但最终发展为获得性耐药（AR）。在疾病进展过程中，超过一半的患者肿瘤携带第二位点EGFR突变T790M。我们的实验室在临床前模型中表明，第二代EGFR- tki、阿法替尼和抗EGFR抗体西妥昔单抗联合使用可能克服t790m介导的耐药性。随后在AR患者中进行的一项Ib期临床试验显示，吉非替尼/厄洛替尼的缓解率达到了前所未有的30%。我已经获得了三名患者的肿瘤样本，他们的t790m肿瘤在放射学上有反应，然后进行了药物联合治疗。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：剪接位点突变c.811- 2A>T和截断突变c.592C>T （p.R198*）。在患者治疗前的样本中未检测到这两种突变，这表明NF2突变是在治疗期间获得的。NF2基因编码Merlin，这是一种参与细胞粘附的细胞骨架蛋白。在其推测的功能中，Merlin已被证明在EGFR信号传导中发挥作用。在融合环境中，Merlin在膜上与EGFR结合，抑制受体信号传导，阻止内化和再循环。Merlin基因的缺失会导致EGFR信号通路的增强以及下游PI3K和MAPK信号通路的激活。Merlin在细胞粘附和生长因子受体反应中的作用使其成为介导双EGFR抑制的耐受性的有趣候选者。我对EGFR突变肺癌细胞系的初步研究表明，短暂转染一组短干扰rna （sirna）对抗NF2会降低细胞对EGFR TKI埃洛替尼的敏感性。单个sirna也得到了类似的结果。根据我的初步数据，我假设NF2的缺失介导了EGFR突变肺肿瘤对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)研究NF2缺失对药物敏感和耐药EGFR突变细胞增殖/致癌信号通路的机制影响；3)确定相关患者样本中NF2突变的频率。了解NF2突变的功能和信号转导后果可能有助于了解EGFR信号转导，并揭示预防或克服患者获得性耐药的方法。