Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer
Jeffrey A. Engelman, … , Lewis C. Cantley, Pasi A. Jänne
Jeffrey A. Engelman, … , Lewis C. Cantley, Pasi A. Jänne
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2695-2706. https://doi.org/10.1172/JCI28656.
View: Text | PDF
Research Article Oncology

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer

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, p110α 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.

Authors

Jeffrey A. Engelman, Toru Mukohara, Kreshnik Zejnullahu, Eugene Lifshits, Ana M. Borrás, Christopher-Michael Gale, George N. Naumov, Beow Y. Yeap, Emily Jarrell, Jason Sun, Sean Tracy, Xiaojun Zhao, John V. Heymach, Bruce E. Johnson, Lewis C. Cantley, Pasi A. Jänne

×

Figure 2

H3255 GR harbors a T790M mutation and is growth-inhibited by an irreversible EGFR inhibitor (CL-387,785) and EGFR-specific shRNA.

Options: View larger image (or click on image) Download as PowerPoint
H3255 GR harbors a T790M mutation and is growth-inhibited by an irrevers...
(A) Exon 20 was amplified by PCR analysis, subjected to SURVEYOR analysis, and analyzed using the WAVE system (see Methods). Shown are HPLC tracings; numbers represent fragment size. Note the presence of a 154-bp fragment in H3255 GR, suggesting a T790M mutation (asterisks), as observed in the H1975 cells (positive control). A549 (dashed line) is shown as a negative control. (B) Exon 20 was amplified from A549 (negative control), H1975 (positive control), H3255, and H3255 GR cells. Products were separated at a partially denaturing temperature using the WAVE system. The indicated fractions (boxed areas) were directly sequenced (see Methods). The T790M mutation was observed in H3255 GR and H1975 cells (asterisks). (C) H3255 and H3255 GR cells were treated with increasing concentrations of CL-387,785 or gefitinib and subjected to an MTS survival assay. (D) Western blot analysis of H3255 and H3255 GR cells treated with increasing concentrations of CL-387,785. Blots of H3255 and H3255 GR extracts for each antibody are the same exposure from a single gel and blot; irrelevant lanes between the H3255 and H3255 GR extracts were omitted. (E) Western blot for PARP with H3255 and H3255 GR cells following treatment with gefitinib or CL-387,785 for 72 hours. (F) Lentiviral constructs containing EGFR shRNAs were infected into H3255 and H3255 GR cells, followed by an MTS assay. Growth was normalized to shRNA construct A (control), which did not cause significant EGFR downregulation (Supplemental Figure 4).