Research Article: BIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR Mutations

Date Published: October 30, 2007

Publisher: Public Library of Science

Author(s): Daniel B Costa, Balázs Halmos, Amit Kumar, Susan T Schumer, Mark S Huberman, Titus J Boggon, Daniel G Tenen, Susumu Kobayashi, Ingo K Mellinghoff

Abstract: BackgroundEpidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.Methods and FindingsTo study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.ConclusionsOur results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.

Partial Text: Sequencing of the epidermal growth factor receptor (EGFR) gene in a large number of tumor samples has identified somatic activating mutations in the tyrosine-kinase pocket of EGFR [1,2]. These mutations were first described in non-small cell lung cancer (NSCLC) patients treated with specific EGFR tyrosine kinase inhibitors (TKIs)—gefitinib and erlotinib—who had radiographic and clinical responses to such agents [3–5]. Two recent transgenic mouse models, in which the overexpression of EGFR mutations was targeted in type II pneumocytes, demonstrated that these mutations led to the development of adenocarcinomas and that the tumors responded both to suppression of the EGFR driving signal and EGFR TKIs [6,7]. As data accumulate, it seems clear that EGFR-mutant “oncogene-addicted” cancers represent a distinct form of NSCLC that can be targeted through novel approaches [8]. The tumor cells are dependent on, or addicted to, the EGFR mutated oncogene for both maintenance of the malignant phenotype and cell survival. At the time of this writing, phase II trials in which patients with advanced NSCLC are included on the basis of presence of the two most common EGFR mutations (either exon 19 deletions or the exon 21 arginine-for-leucine substitution at amino acid 858, or L858R) and are given gefitinib as first-line treatment show radiographic response rates that exceed 75% [9–11]. Mature results of such trials will likely confirm the improved time to progression and survival seen in retrospective studies of patients treated with TKIs in which EGFR mutations had been identified [12–15].

We identified BIM as a key apoptotic effector of EGFR TKIs in sensitive cells with the activating L858R or exon 19 deletion EGFR mutations. The common T790M secondary mutation and the novel L747S, in conjunction with an activating mutation, attenuated the up-regulation of BIM and apoptosis.



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