title: 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, 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.
authors.affiliation: Massachusetts General Hospital Cancer Center, Boston, Massachusetts, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Engelman
authors.forename: Toru
authors.initials: T
authors.lastname: Mukohara
authors.forename: Kreshnik
authors.initials: K
authors.lastname: Zejnullahu
authors.forename: Eugene
authors.initials: E
authors.lastname: Lifshits
authors.forename: Ana M
authors.initials: AM
authors.lastname: Borrás
authors.forename: Christopher-Michael
authors.initials: CM
authors.lastname: Gale
authors.forename: George N
authors.initials: GN
authors.lastname: Naumov
authors.forename: Beow Y
authors.initials: BY
authors.lastname: Yeap
authors.forename: Emily
authors.initials: E
authors.lastname: Jarrell
authors.forename: Jason
authors.initials: J
authors.lastname: Sun
authors.forename: Sean
authors.initials: S
authors.lastname: Tracy
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhao
authors.forename: John V
authors.initials: JV
authors.lastname: Heymach
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Johnson
authors.forename: Lewis C
authors.initials: LC
authors.lastname: Cantley
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
mesh_terms: D000483:Alleles; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000077156:Gefitinib; D005784:Gene Amplification; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D051379:Mice; D020125:Mutation, Missense / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology / Q000627:therapeutic use; D034622:RNA Interference; D020893:Receptor, ErbB-3 / Q000378:metabolism; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
chemical_list: D047428:Protein Kinase Inhibitors; D011799:Quinazolines; C118029:CL 387785; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D020893:Receptor, ErbB-3; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D000077156:Gefitinib