Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer
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Published on: April 11, 2016
Jeffrey A Engelman1, Toru Mukohara, Kreshnik Zejnullahu
1Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA.
Abstract:
EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.
Insights
A specific EGFR mutation (T790M) can cause resistance to lung cancer drugs like gefitinib, even when diluted among other gene copies. This resistance can be overcome by targeting PI3K signaling pathways.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Epidermal growth factor receptor (EGFR) mutations and amplifications are common in lung adenocarcinomas, driving sensitivity to EGFR inhibitors like gefitinib.
- Acquired resistance to EGFR inhibitors is a significant clinical challenge, with the T790M mutation being a known, but not fully understood, contributor.
Purpose of the Study:
- To investigate the role of the T790M mutation in acquired resistance to gefitinib in EGFR-mutated and amplified lung cancer.
- To explore mechanisms of resistance beyond direct sequencing detection and the involvement of PI3K/Akt signaling.
Main Methods:
- Development of a gefitinib-resistant cell line model from a sensitive EGFR-mutated/amplified lung carcinoma cell line (H3255) through prolonged drug exposure.
- Utilized a highly sensitive HPLC-based technique to detect the T790M mutation, which was missed by direct sequencing.
- Exogenously introduced EGFR T790M and PIK3CA (p110alpha E545K) mutations into sensitive cells to assess their impact on gefitinib sensitivity and signaling.
Main Results:
- A gefitinib-resistant cell line acquired a T790M mutation in a subpopulation of amplified alleles, undetectable by standard sequencing.
- Exogenous expression of EGFR T790M conferred gefitinib resistance and sustained ErbB-3/PI3K/Akt signaling in EGFR-mutated/amplified cells.
- Activation of PI3K signaling via PIK3CA mutation (p110alpha E545K) was sufficient to block gefitinib-induced apoptosis.
Conclusions:
- Allelic dilution of resistance mutations like T790M can evade detection by direct sequencing in cancers with amplified oncogenes.
- Restoration of PI3K pathway activation, through mechanisms such as T790M mutation or other alterations, is a key driver of acquired resistance to gefitinib.
