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）：编码表皮生长因子受体（EGFR）的基因中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）（TKIS）作出反应，例如gefitinib或erlotinib，但最终会产生产生的抵抗（AR）。疾病进展后，超过一半的患者肿瘤带有第二位EGFR突变，T790M。我们的实验室在临床前模型中表明，第二代EGFR-TKI，Afatinib以及抗EGFR抗体Cetuximab的组合可能会克服T790M介导的抗性。随后对Gefitinib/erlotinib的AR患者进行的IB临床试验显示了前所未有的30％缓解率。我已经从三名T790M-Harboring肿瘤在射线照相上反应的患者那里获得了肿瘤样本，然后在药物组合上进行了进展。在一个样品中鉴定出的整个基因组测序的生物信息学分析两个意外的NF2突变：剪接位点突变C.811-2A> T和截断的突变C.592C> T（P.R198*）。在患者的治疗样本中均未检测到突变，这表明在治疗期间获得了NF2中的突变。 NF2基因编码Merlin，这是一种与细胞粘附有关的细胞骨架蛋白。在其推定的功能中，Merlin已被证明在EGFR信号传导中起作用。在汇合环境中，梅林在膜上结合EGFR，以抑制受体信号传导并防止内在化和回收利用。 Merlin的丧失会导致EGFR信号增强，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对双EGFR抑制的耐药性的有趣候选者。我在EGFR突变的肺癌细胞系中的初步研究表明，对NF2的短裂RNA（siRNA）的瞬时转染降低了细胞对EGFR TKI Erlotinib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NF2的丧失介导了EGFR突变肺肿瘤中EGFR抑制的抑制。为了检验这一假设，我提出了三个具体目的：1）确定NF2丢失和过表达对EGFR突变肺癌细胞对EGFR突变细胞系中EGFR抑制的敏感性的影响； 2）检查NF2损失对药物敏感和耐药性EGFR突变细胞中增殖/致癌信号通路的机理后果； 3）确定相关患者样品中NF2突变的频率。了解NF2突变的功能和信号传导后果可能会提供有关EGFR信号传导的见解，并揭示预防或克服患者获得的耐药性的方法。