Article

Yun, C. H. et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl Acad. Sci. USA 105, 2070-2075

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2008; 105(6):2070-5. DOI: 10.1073/pnas.0709662105
Source: PubMed

ABSTRACT

Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

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    • "EGFR determines acquired resistance by increasing the affinity of EGFR to ATP[16,17]. "
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    ABSTRACT: Introduction: KRAS oncogene mutations (MUTKRAS) drive resistance to EGFR inhibition by providing alternative signaling as demonstrated in colo-rectal cancer. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations (MUTEGFR). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS, or restoration of EGFR activity due to additional MUTEGFR, i.e., the c.2369C > T (p.T790MEGFR). Aim: The aim of this study was to investigate the appearance of MUTKRAS during EGFR-TKI treatment and their contribution to drug resistance. Methods: This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 MUTKRAS and p.T790MEGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI. Results: p.T790MEGFR was detected in 11 (33.3%) patients, MUTKRAS at codon 12 in 3 (9.1%) while both p.T790MEGFR and MUTKRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type (WTKRAS) and negative for p.T790MEGFR. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790MEGFR and 37.5% for MUTKRAS. The analysis of time to progression (TTP) and overall survival (OS) in WTKRAS vs. MUTKRAS were not statistically different, even if there was a better survival with WTKRAS vs. MUTKRAS, i.e., TTP 14.4 vs. 11.4 months (p = 0.97) and OS 40.2 vs. 35.0 months (p = 0.56), respectively. Conclusions: MUTKRAS could be an additional mechanism of escape from EGFR-TKI inhibition and cftDNA is a feasible approach to monitor the molecular development of drug resistance.
    Full-text · Article · Jan 2016 · Oncotarget
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    • "Previous results suggested ligand-independent activity of EGFR-L858R/T790M expressed in Ba/F3 cells was dimerization-independent based on resistance to Cetuximab (Cho et al., 2013). Whether the increased affinity for ATP of EGFR-L858R/T790M (Yun et al., 2008) accelerates receptor phosphorylation and effectively circumvents the requirement for stable dimer formation remains unknown. phosphorylation of EGFR-L858R (Figure 5, A and B), indicating that kinase domain dimerization alone, while perhaps less stable in the absence of ectodomain engagement, can still produce some level of ligand-independent signaling. "

    Full-text · Article · Nov 2015
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    • "Previous results suggested ligandindependent activity EGFR-L858R/T790M expressed in Ba/F3 cells was dimerizationindependent based on resistance to Cetuximab (Cho et al., 2013). The increased affinity for ATP of EGFR-L858R/T790M (Yun et al., 2008) may accelerate receptor phosphorylation and effectively circumvent the requirement for stable dimer formation, however this remains unknown. "
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    ABSTRACT: Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non-small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g. ΔL747-P753insS) which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color super-resolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live cell FRET measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization. © 2015 by The American Society for Cell Biology.
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