We analyzed genomic DNA from a total of 261 resected, clinically annotated NSCLC specimens (10
). We screened the coding sequences of 39 genes encoding mostly components of the EGFR signaling pathway for somatic mutations via high-throughput dideoxynucleotide sequencing of PCR-amplified gene products. Sequencing of 9MB of tumor sequence identified 239 putative nonsynonymous sequence variations that differed from reference sequences listed in the National Center for Biotechnology Information (RefSeq) database for each respective gene. We previously reported the examination of 22 sequence variations found in RAS family genes and 135 sequence variations localized to exons encoding the protein kinase domains (10
). In that study, we identified a total of 37 nonsynonymous somatic mutations, found collectively in EGFR, HER2, KRAS, BRAF, PIK3CA
, and FGFR4
Here, we examined the remaining putative genetic variants occurring in exons encoding domains outside of the kinase regions of their respective kinases. Of 82 putative nonkinase domain sequence variations, representing 69 distinct types of mutations, we confirmed the existence of 27 (18 distinct types) on sequencing a second independent PCR product from tumor DNA. We found 22 of the 27 (12 types) in corresponding matched normal tissue, suggesting that they were single nucleotide polymorphisms. Two variants were of unknown significance (ERBB4: P572L, FGFR4: C56S) because we were unable to obtain a PCR product from matched normal samples.
Of the remaining three confirmed somatic mutations, one involved an A→G change at nucleotide 542 in exon 4 of ERBB4, which substitutes serine for asparagine at position 181 (data not shown). This mutation was found in 1 of 171 lung tumor samples. The N181S substitution does not seem to grossly affect kinase activity; (a) according to the polymorphism phenotyping bioinformatics tools PolyPhen and SIFT, an N181S substitution is predicted to be “benign” and “tolerated,” respectively; and (b) lysates from 293T cells transiently transfected with cDNAs encoding wild-type or N181S ERBB4 do not display major differences by immunoblotting in phosphotyrosine reactivity of ERBB4 protein (data not shown).
The remaining two confirmed somatic mutations involved the same G→T transversion in exon 2 at nucleotide 171 of MEK1, found initially in 2 of 93 independent lung tumor samples (of which, 89, including the two with MEK1 changes, were of adenocarcinoma histology; ). This change substitutes asparagine for lysine at position 57. K57 is highly conserved among various species () and is located between the nuclear export signal (amino acids 33–44) and kinase domain (amino acids 68–271) of MEK1 (). Consistent with the G→T mutation, a type of transversion known to be smoking related, both samples harboring the MEK1K57N mutation were from former smokers. Neither patient had prior chemotherapy or exposure to asbestos or radiation. Both patients presented with stage IA disease and are alive with nearly 4 years of follow-up. Notably, neither of these two tumors harbored mutations in EGFR, HER2, KRAS, BRAF, PIK3CA, or FGFR4.
Figure 1 Identification of a MEK1K57N 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 (more ...)
To determine if a K57N substitution in MEK1 affected MAPK signaling, we assayed the ability of the mutant to induce ERK phosphorylation in 293T cells. Extracts from cells transiently transfected with plasmids encoding wild-type or mutant MEK1 cDNAs displayed higher levels of total MEK1 protein than did control-transfected cells, but only cells transfected with MEK1K57N cDNAs displayed enhanced levels of ERK phosphorylation (). We observed similar levels of induced ERK phosphorylation in lysates from cells transfected with cDNAs encoding two well-characterized mutants, BRAFV600E and KRASG12V, known to activate the ERK pathway ().
Figure 2 Functional characterization of the MEK1K57N 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 (more ...)
To characterize additional functional consequences of the K57N change, we generated stable polyclonal populations of Ba/F3 cells expressing wild-type or mutant MEK1. Ba/F3 cells are a murine pro-B cell line that is normally dependent on IL-3 for growth, but they can be rendered IL-3 independent by introduction of transforming tyrosine kinases such as BCR-ABL (11
). For some oncogenic proteins, very high levels of mutant kinase are required to derive growth factor independence (12
). In two independent assays, we derived Ba/F3 cells harboring cDNAs encoding MEK1K57N
that grew in the absence of IL-3. By contrast, we could not derive IL-3–independent Ba/F3 cells expressing the wild-type kinase (). The growth rate of IL-3–independent Ba/F3 cells expressing mutant MEKK57N
was similar to that of parental Ba/F3 cells cultured in the presence of IL-3 (). Immunoblotting of lysates from the cells harboring mutant MEK1
revealed very high levels of total MEK1 protein and ERK phosphorylation compared with parental Ba/F3 controls (). Thus, overexpression of mutant, but not wild-type, MEK1 is able to convert Ba/F3 cells to cytokine-independent growth.
We next assessed whether a small-molecule MEK inhibitor, AZD6244 (13
), could affect biological properties induced by the MEK1 mutant. AZD6244 treatment of 293T cells transiently transfected with plasmids encoding MEK1K57N
cDNAs readily inhibited the appearance of ERK phosphorylation (). Moreover, compared with parental Ba/F3 cells growing in IL-3, IL-3–independent MEK1K57N
-harboring Ba/F3 cells were more sensitive to the MEK inhibitor (). Because the administration of IL-3 to cultures of cytokine-independent MEK1K57N
-harboring Ba/F3 cells prevented AZD6244-induced growth inhibition, these data suggest that MEK inhibitors may effectively target cancer cells that depend on the activity of mutant MEK1 for survival.
Figure 3 MEK1K57N-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. (more ...)
Finally, we screened an additional 114 NSCLCs (including 33 squamous cell carcinomas) for the MEK1K57N
change or other mutations in exon 2 of MEK1
. No tumors harbored the MEK1K57N
mutation. We also examined an additional 85 NSCLC cell lines. Whereas no lines carried the K57N mutation, we did find that NCI-H1437 cells harbor a mutation encoding a Q56P MEK1 variant (data not shown). These cells were originally derived from a male smoker. They do not carry mutations in EGFR, KRAS, BRAF
, or PIK3CA
but do display an inferred copy number of up to 3 at the genomic region of MEK1
on chromosome 15.10
H1437 cells also display sensitivity in the nanomolar range to AZD6244 (data not shown).11
We did not detect any other MEK1
exon 2 mutations in 19 primary samples from patients with chronic myelomonocytic leukemia (with wild-type KRAS
) or in 54 additional tumor cell lines (14 melanomas, 13 colon carcinomas, 13 breast carcinomas, 6 neuroblastoma/neuroepithelial tumors, 4 prostate carcinomas, and 4 pancreas carcinomas).