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.

Public Health Relevance

Because all EGFR-mutant lung adenocarcinomas eventually develop resistance to targeted therapies such as erlotinib or afatinib/cetuximab; understanding mechanisms of acquired resistance may improve clinical outcomes. This proposed research uses patient-derived data to address mutation in NF2 as a previously unrecognized potential mechanism of acquired resistance to targeted therapy in lung cancer. 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
1F30CA180353-01
Application #
8593718
Study Section
Special Emphasis Panel (ZRG1-F09B-P (20))
Program Officer
Damico, Mark W
Project Start
2013-12-01
Project End
2017-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
Fiscal Year
2013
Total Cost
$47,232
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Pirazzoli, Valentina; Nebhan, Caroline; Song, Xiaoling et al. (2014) Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1. Cell Rep 7:999-1008
Cross, Darren A E; Ashton, Susan E; Ghiorghiu, Serban et al. (2014) AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov 4:1046-61