Novel MEK1 mutation identified by mutational analysis of epidermal growth factor receptor signaling pathway genes in lung adenocarcinoma

Abstract

Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

Figure 1

Identification of a MEK1^K57N mutation in human lung adenocarcinoma. A, reverse sequencing chromatograms display presence of a G→T mutation at position 171 in exon 2 of MEK1 in two tumor samples. This mutation was absent from matched normal control samples. (Forward sequence chromatograms not shown due to space constraints.) B, protein sequence alignment of MEK1 from various species shows that K57 is a highly conserved residue. Numbers indicate amino acid positions. C, K57N is located in between the nuclear export signal (NES) and the kinase domain of MEK1. D, docking domain. Shown are mutations found in patients with cardio-facio-cutaneous (CFC) syndrome.

Figure 2

Functional characterization of the MEK1^K57N mutant. A, 293T cells were transiently transfected with expression plasmids encoding various cDNAs, and corresponding lysates from cells maintained in serum were subjected to immunoblotting with the indicated antibodies. Lysates from cells harboring MEK1 cDNAs displayed higher levels of total MEK1 protein compared with control-transfected cells, but only cells transfected with MEK1^K57N cDNAs displayed enhanced phospho-ERK expression, at levels comparable to those induced by mutants BRAF and KRAS. B, Ba/F3 cells were stably transfected with vectors encoding wild-type or MEK1^K57N cDNAs. The resulting cells were then cultured in the absence of IL-3. Numbers of live cells were counted daily for 4 d. Parental Ba/F3 cells grown in the absence or presence of IL-3 serve as controls. C, lysates from IL-3–independent K57N Ba/F3 cells display high levels of pERK and tMEK1 compared with parental Ba/F3 cells grown in the absence of IL-3. Immunoblotting was done with the indicated antibodies.

Figure 3

MEK1^K57N-induced activity is sensitive to MEK inhibition by AZD6244. A, 293T cells were transiently transfected with expression plasmids encoding various cDNAs, and corresponding lysates were subjected to immunoblotting with the indicated antibodies. Lysates from cells harboring mutant MEK1^K57N are sensitive to AZD6244 as shown by inhibition of pERK expression. MAPK signaling is also inhibited by drug treatment in the cells expressing BRAF^V600E and KRAS^G12V mutant constructs. B, Ba/F3 cells were stably transfected with vectors encoding wild-type or MEK1^K57N cDNAs. The resulting cells were then cultured for 48 h with AZD6244 in the absence or presence of IL-3. Parental Ba/F3 cells cultured in the presence of IL-3 were also treated with the drug. Growth inhibition assays were done using CellTiter Blue Reagent as described in Materials and Methods.