Lung cancers with acquired resistance to EGFR
inhibitors occasionally harbor BRAF gene mutations
but lack mutations in KRAS, NRAS, or MEK1
Kadoaki Ohashia, Lecia V. Sequistb,c, Maria E. Arcilad, Teresa Morane, Juliann Chmieleckif, Ya-Lun Lina, Yumei Pana,
Lu Wangd, Elisa de Stanchinag, Kazuhiko Shienh, Keisuke Aoei,j, Shinichi Toyookah, Katsuyuki Kiurak,
Lynnette Fernandez-Cuestal, Panos Fidiasb,c, James Chih-Hsin Yangm,n, Vincent A. Millero, Gregory J. Rielyo,
Mark G. Kriso, Jeffrey A. Engelmanb,c, Cindy L. Vnencak-Jonesp, Dora Dias-Santagataq,r, Marc Ladanyid, and William Paoa,1
aDivision of Hematology-Oncology, Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville,
TN 37232;bMassachusetts General Hospital Cancer Center, Boston, MA 02114; Departments ofcMedicine andrPathology, Harvard Medical School, Boston,
MA 02115;dDepartment of Pathology,gAnti-Tumor Assessment Core Facility, andoThoracic Oncology Service, Division of Solid Tumor Oncology, Department
of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10065;eCatalan Institute of Oncology, Universitat Autonoma de Barcelona and Hospital
Universitari Germans Trias i Pujol, 08916 Badalona, Barcelona, Spain;fWeill Cornell Graduate School of Medical Sciences, New York, NY 10065;hDepartment
of Thoracic Surgery, Okayama University Hospital, Okayama, Okayama 700-8558, Japan; Departments ofiMedical Oncology andjClinical Research, National
Hospital Organization Yamaguchi-Ube Medical Center, Ube, Yamaguchi 755-0241, Japan;kDepartment of Hematology, Oncology, and Respiratory Medicine,
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Okayama 700-8558, Japan;lMax Planck Institute for
Neurological Research with Klaus Joachim Zülch Laboratories of the Max Planck Society and the Medical Faculty, Cologne 50931, Germany;mGraduate
Institute of Oncology andnCancer Research Center, National Taiwan University, Taipei 100, Taiwan;pDepartment of Pathology, Microbiology and
Immunology, Vanderbilt University Medical Center, Nashville, TN, 37232; andqDepartment of Pathology, Massachusetts General Hospital, Boston, MA 02114
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved May 31, 2012 (received for review March 2, 2012)
modeled disease progression using EGFR-mutant human tumor cell
lines. Although five of six models displayed alterations already
found in humans, one harbored an unexpected secondary NRAS
Q61K mutation; resistant cells were sensitive to concurrent EGFR
and MEK inhibition but to neither alone. Prompted by this finding
and because RAS/RAF/MEK mutations are known mediators of ac-
quired resistance in other solid tumors (colon cancers, gastrointesti-
nal stromal tumors, and melanomas) responsive to targeted
therapies, we analyzed the frequency of secondary KRAS/NRAS/
BRAF/MEK1 gene mutations in the largest collection to date of lung
cancers with acquired resistance to EGFR TKIs. No recurrent NRAS,
KRAS, or MEK1 mutations were found in 212, 195, or 146 patient
samples, respectively, but 2 of 195 (1%) were found to have muta-
tions in BRAF (G469A and V600E). Ectopic expression of mutant
NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resis-
tance to EGFR TKIs that was overcome by addition of a MEK inhib-
itor. Collectively, these positive and negative results provide deeper
insight into mechanisms of acquired resistance to EGFR TKIs in lung
cancer and inform ongoing clinical trials designed to overcome re-
sistance. In the context of emerging knowledge about mechanisms
of acquired resistance to targeted therapies in various cancers, our
datahighlightthe notionthat,eventhough solid tumors share com-
mon signaling cascades, mediators of acquired resistance must be
elucidated for each disease separately in the context of treatment.
found in ∼10% of white and 30% of Asian non-small cell
lung cancers, respectively (1–4). EGFR-mutant tumors with exon
19 deletions and L858R substitutions are highly sensitive initially
to the EGFR tyrosine kinase inhibitors (TKIs), gefitinib or erlo-
tinib (1–4). Unfortunately, tumor cells eventually acquire resis-
tance, with progression of disease occurring in patients around
10–16 mo after starting treatment (5). Genetic mechanisms of
resistance found in tumor samples from patients with acquired
resistance include second-site EGFR mutations [>50% (6–9)],
amplification of the gene encoding an alternative kinase, MET
[(5–10% (9–12)], and mutations in the downstream signaling lipid
involve a threonine to methionine change at codon 790 of EGFR,
which alters binding of drug to the ATP-binding pocket (6–9).
A few percent seemingly undergo histologic transformation, dis-
playing features of small cell lung cancer (12, 13) or epithelial–
mesenchymal transition (EMT) (12). Up to 40% of cases of ac-
quired resistance are mechanistically unexplained.
The RAS/RAF/MEK/MAPK signaling pathway downstream of
EGFR plays a significant role in tumorigenesis, including lung
cancers. Oncogenic recurrent driver mutations in KRAS, NRAS,
non-small cell lung cancers, respectively (14–18). Unlike PIK3CA
mutations, which occur concurrently with EGFR mutations in in-
dividual tumors, genetic alterations in KRAS, NRAS, BRAF, and
these genes have been associated with primary resistance to tar-
geted EGFR therapy, including EGFR TKIs in lung cancer (23)
and anti-EGFR monoclonal antibodies in colon cancer (24). Re-
cently, KRAS mutations also have been associated with acquired
resistance to the anti-EGFR antibody cetuximab in colorectal
cancers (25); NRAS and MEK1 mutations have been shown to
mediate acquired resistance to the mutant BRAF kinase inhibitor,
Author contributions: K.O. and W.P. designed research; K.O., M.E.A., J.C., Y.-L.L., Y.P.,
L.W., E.d.S., K.S., and L.F.-C. performed research; M.E.A., Y.-L.L., Y.P., C.L.V.-J., D.D.-S., and
M.L. contributed new reagents/analytic tools; L.V.S., T.M., K.A., S.T., K.K., L.F.-C., P.F., J.C.-H.
Y., V.A.M., G.J.R., M.G.K., and J.A.E. collected patient samples; K.O., L.V.S., M.E.A., T.M.,
Y.-L.L., Y.P., L.W., K.S., K.A., S.T., K.K., P.F., J.C.-H.Y., V.A.M., G.J.R., M.G.K., J.A.E., C.L.V.-J.,
D.D.-S., M.L., and W.P. analyzed data; and K.O. and W.P. wrote the paper.
Conflict of interest statement: L.V.S. received consulting fees from Clovis Oncology, Glax-
oSmithKline, and Celgene Corporation. J.C.-H.Y. received consulting fees from Boehringer
Ingelheim. V.A.M. is an employee at Foundation Medicine and has equity value in the
company. M.G.K. has received consulting fees from Boehringer Ingelheim and research
funding for other projects from Pfizer and Boehringer. J.A.E. has received consulting fees
and has stock option ownership in Agios Pharmaceuticals and has received research
funding for other projects from Novartis, GlaxoSmithKline, and AstraZeneca. D.D.-S. re-
ceived consulting fees from Bio-Reference Laboratories. W.P. has received consulting fees
from MolecularMD, AstraZeneca, Bristol-Myers Squibb, Symphony Evolution, and Clovis
Oncology and research funding for other projects from Enzon, Xcovery, AstraZeneca, and
Symphogen. W.P. and V.A.M. are part of a patent regarding EGFR T790M mutation
testing that was licensed by Memorial Sloan-Kettering Cancer Center to MolecularMD.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
See Author Summary on page 12282 (volume 109, number 31).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online July 6, 2012
vemurafenib, in melanomas (26, 27); and BRAF mutations have
been found in patients with acquired resistance to imatinib in KIT/
PDGFRα-mutant gastrointestinal stromal tumors (GISTs) (28).
Previous reports have shown that RAS mutations are not found in
(7, 12, 29). However, the sample sizes were too small (n = 6, 37,
and 14, respectively) to rule them out definitively as mediators of
resistance.Only one study looked forBRAF mutations, and MEK1
status was not assessed.
Here, we modeled acquired resistance in vitro and, surprisingly,
found that one cell-line model displayed a secondary NRAS mu-
tation. Prompted by the data above and by the additional finding
that a clinically relevant mouse lung tumor model of acquired re-
sistance also has identified secondary Kras mutations (30), we sys-
mutations in samples from patients with acquired resistance. The
findings provide deeper insight into mechanisms of acquired re-
sistance, inform ongoing clinical trials designed to overcome re-
sistance, and suggest which mutations should be screened for
routinely in samples from patients with acquired resistance.
NRAS Q61K Mutation in an EGFR-Mutant Cell-Line Model of Acquired
Resistance to Erlotinib. To explore potential modes of drug re-
sistance, we used well-established protocols (31) to develop cell
models of acquired resistance to erlotinib. Including two lines
previously reported (31), we developed a total of six resistant lines
over 3–6 mo from five parental cells (PC-9, HCC827, HCC4006,
HCC4011, and 11-18) with drug-sensitive EGFR mutations and
detect mutations at an allele frequency of 5% (32), we found only
two resistant lines (PC-9R and HCC827R1) that harbored the
EGFR T790M second-site mutation (Table 1 and Fig. S1A). PC-
9R cells displayed increased amplification of EGFR compared
with parental cells (31), but HCC827R1 and R2 cells appeared to
have no change in EGFR gene status. Two lines (HCC4006R and
HCC4011R) appeared to lose copies of EGFR (Table 1). Two
lines (HCC827R2 and HCC4011R) harbored MET amplification
MET protein by immunoblotting studies (Fig. S1B). Consistent
with the loss of copies of EGFR, HCC4006R cells displayed loss
of dependence on EGFR protein expression for cell growth. In-
stead, they showed features of EMT, i.e., increased expression of
vimentin with loss of E-cadherin (Fig. S1 B and C). Similar results
have been reported previously by us and/or others (10, 31, 33–36).
No resistant cells displayed features of small cell lung cancer.
One EGFR T790M-negative line, 11-18R cells, had increased
levels of EGFR and MET protein but lacked EGFR or MET
and Figs. S1B, S2, and S3). Growth-inhibition studies after
knockdown of EGFR protein using EGFR-specific siRNAs
revealed that, unlike PC-9R cells (Fig. S1C), 11-18R cells were no
longer dependent solely on EGFR for cell growth (Fig. 1B). Con-
sistent with these findings, erlotinib treatment of 11-18R cells
inhibited phosphorylation of EGFR but not the downstream sig-
naling protein ERK (Fig. 1C). 11-18R cells also were resistant to
growth inhibition by the more potent second-generation EGFR
TKI afatinib (37), except at high doses (>1 μM) (Fig. S1D). These
with erlotinib (Fig. S1E).
To determine possible mechanisms of resistance in 11-18R cells,
we screened corresponding extracted cell DNA with a platform (a
combination of SNaPshot and PCR-based sizing assays) that can
detect more than 40 recurrent mutations in nine genes (AKT1,
BRAF, EGFR, HER2, KRAS, MEK1, NRAS, PIK3CA, and PTEN)
relevant to existing and emerging targeted therapies in lung cancer
(32). Surprisingly, we found that, compared with parental cells,
DNA from 11-18R cells harbored an acquired NRAS Q61K mu-
tation in addition to the primary EGFR L858R substitution (Fig.
1D, Table 1, and Table S1). None of the other cell lines harbored
any additional mutations. Consistent with these results, levels of
phospho-ERK decreased after knockdown of EGFR in 11-18 pa-
the same cell. Because cell-line models of acquired resistance to
date have harbored mechanisms of resistance found in human
tumors, we performed additional characterization of 11-18R cells.
Functional Role of NRAS Q61K in 11-18R Cells. We assessed whether
the acquired NRAS mutation plays a functional role in 11-18R
cells. First, we validated the activation status of NRAS in 11-18R
cells, using a Ras GTPase-specific pulldown assay. As expected,
the expression of NRAS-GTPase was much higher in 11-18R
cells than in 11-18 parental cells. Treatment with erlotinib had
no effect on NRAS activation (Fig. 2A).
erlotinib on 11-18R cell growth. The MEK serine-threonine kinase
is downstream of RAS; therefore, its inhibition might be expected
to affect cell viability in cells with activated RAS. The MEK in-
hibitorAZD6244 alonehad noeffect ineither parental orresistant
11-18 cells. Addition of AZD6244 to erlotinib had no additive ef-
fect in parental cells, but thecombinationsuppressed growthin the
Table 1.EGFR mutant cell-line models of acquired resistance to erlotinib
Cell line Primary EGFR mutationDrug selectionErlotinib IC50(μM)EGFR T790MEGFR ampMET ampOther
Erlotinib NRAS Q61K
Six resistant cell lines were established from five parental cell populations. PC-9R and HCC827R1 cells harbored EGFR T790M.
HCC827R2 and HCC4011R cells displayed MET amplification (amp) and increased levels of MET protein (Figs. S1A and S2). HCC4006R
cells showed features of EMT (Fig. S1A). 11-18R cells harbored an NRAS Q61K mutation. N/a, not applicable.
*PC-9R cells showed further EGFR amplification.
†HCC827R1and HCC827R2 cells showed no further EGFR amplification.
| www.pnas.org/cgi/doi/10.1073/pnas.1203530109 Ohashi et al.
as in vivo with another MEK inhibitor, GSK1120212 (Fig. S4A and
Fig. 2C). By contrast, addition of AZD6244 to erlotinib had no
additive effect against PC-9 parental or resistant cells, the latter of
which harbor EGFR T790M (Fig. 2B). Consistent with these
findings, only the combination of erlotinib and AZD6244 strongly
inhibited levels of phospho-ERK in 11-18R cells (Fig. S4B). In-
terestingly, the MEK inhibitor alone was able to inhibit the growth
of H1299 cells, which harbor no drug-sensitive EGFR mutations
but do have an NRAS Q61K mutation (Fig. S4C). This discrepancy
suggests that signaling pathways in NRAS Q61K mutant cells are
different in the context of wild-type and mutant EGFR. A selective
PI3K inhibitor, GDC-0941, did not restore sensitivity of 11-18R
cells to erlotinib (Fig. S4D). These data suggest that the MEK
rather than the PI3K pathway is active downstream of mutant
NRAS in these cells.
Third, we examined the effect of NRAS protein knockdown
using siRNAs against NRAS. siRNA knockdown modestly inhibi-
ted the growth of 11-18R but not parental cells (Fig. 2D). When
combined with erlotinib, NRAS knockdown led to an even greater
growth reduction in resistant cells (Fig. 2D). Conversely, when
combined with AZD6244, EGFR knockdown led to similar levels
of growth inhibition (Fig. 2D). Consistent with these results, only
the combinations of erlotinib plus siRNA knockdown of NRAS
led to significant inhibition of phospho-ERK in resistant cells. Fi-
nally, to extend these results further, we performed growth-in-
hibition assays using siRNAs and kinase inhibitors as above with
two separate 11-18R single-cell clones (C1 and C4) and observed
essentially the same results (Fig. S4 F and G). Collectively, these
data indicate that in 11-18R cells NRAS Q61K functions down-
MEK signaling pathway.
RAS Signaling Pathway Gene Mutations in Tumors from Patients with
Acquired Resistance. Given the RAS-related findings in the murine
(30) and cell-line models of acquired resistance and the reported
data regarding KRAS/NRAS/BRAF/MEK1 mutations in other
cancers that grow after responding to targeted therapies (25–28),
we reexamined the frequency of mutations occurring in KRAS,
NRAS, BRAF, and MEK1 in 195, 212, 195, and 146 tumor samples,
Tumors were screened by a variety of methods including a SNap-
Shot/sizing platform (32, 38), a Sequenom mass spectrometry-
based assay (39), and/or direct sequencing. Although we did not
detect any NRAS, KRAS, or MEK1 mutations, we did identify two
BRAF mutations. One tumor harbored simultaneous EGFR
exon19 deletion, EGFR T790M, and BRAF V600E mutations, and
another tumor harbored EGFR exon 19 deletion and BRAF
to have tumors with an EGFR exon19 deletion that initially
responded radiographically to erlotinib monotherapy. Lack of
sufficient tissue precluded further analysis of the acquired-re-
sistance specimens. A pretreatment tumor sample was unavailable
for the first patient. This patient also had no history of melanoma,
a disease in which BRAF V600E mutations are common. In the
second case, the BRAF G469A mutation was confirmed to be ab-
sent before treatment. Collectively, these data demonstrate that
RAS pathway gene mutations are rare, but BRAF mutations can
occur in 1% of tumors with acquired resistance to EGFR TKIs.
Ectopic Expression of NRAS Q61K, BRAF V600E, or BRAF G469A Confers
Resistance to Cells Harboring Drug-Sensitive EGFR Mutations. To
confer acquired resistance to EGFR-TKIs, we introduced cDNAs
encoding NRAS Q61K or BRAF G469A in PC-9 cells and BRAF
V600E in both PC-9 and PC-9R cells and treated cell transfectants
with various inhibitors. We did not express mutant forms of KRAS
or MEK1, because we did not find correlative human data. Ectopic
afatinib (Fig.3 A and B and Fig.S6A and B). Consistent with these
data, EGFR-mutant cells with stable expression of either mutant
phospho-ERK activation was maintained in the presence of drug
(Fig. S6 C and D). In the stable transfectants, addition of MEK
inhibitors to erlotinib led to greater growth inhibition (Fig. 4 A and
C) and enhanced reduction ofphospho-ERK levels compared with
either drug alone (Fig. 4 B and D). In the mutated BRAF trans-
fectants, addition of the BRAF inhibitor vemurafenib to erlotinib
induced greater growth inhibition than either drug alone (Fig. S7).
Elucidating how patients with EGFR-mutant lung tumors develop
acquired resistance to EGFR TKIs has been an active area of in-
mutation, i.e., the T790M amino acid change, which results in al-
tered binding of drug to the ATP-binding pocket (6–9). Rarer
mechanisms (1–10%) include MET amplification, PIK3CA muta-
% Growth Inhibition
% Viable cells/
lipofectamine treated control
11 - 1811-18R
1µ µM 6h
the relative sensitivity of 11-18 and 11-18R cells to erlotinib. Data are expressed
as percentage of viability compared with vehicle by the cell titer blue assay.
Data shown are mean ± SD of three independent experiments performed in
hextuplicate. (B) (i) EGFR knockdown experiments using siRNAs show that,
compared with parental cells,11-18R cells are no longer dependent upon EGFR
for survival. Data shown are mean ± SD of three independent experiments
performed in hextuplicate. Scr, Scramble. Two different siRNAs against EGFR
were used. (ii) Immunoblotting studies using the indicated antibodies show
that downstream signaling is inhibited in parental but not resistant cells after
knockdown of EGFR. (C) The effect of erlotinib on EGFR pathway signaling in
11-18/11-18R cells. (D) (i) SNaPshot assay reveals that, in addition to a baseline
EGFR L858R mutation, 11-18R cells have acquired an NRAS Q61K mutation but
not an EGFR T790M mutation. (ii) Direct sequencing confirms presence of the
NRAS 181C > A (Q61K) mutation in 11-18R cells.
Characterization of 11-18R cells. (A) Cell growth-inhibition assays show
Ohashi et al.PNAS
| Published online July 6, 2012
tion, and changes in tumor morphology (i.e., transformation to
small cell lung cancer or development of features of EMT) (9–13).
Up to 40% of cases are still unexplained.
Knowledge of these mechanisms coupled with prospective test-
ing in the relevant clinical samples has led to the creation of
rational strategies to overcome acquired resistance. For example,
using mouse lung tumor models of drug-sensitive and -resistant
EGFR-mutant alleles, we previously showed that dual inhibition of
EGFR with the second-generation EGFR TKI afatinib plus the
anti-EGFR antibody cetuximab could eradicate T790M-driven
tumors (40). A trial based on these data has now shown a 40%
partial response rate in patients with acquired resistance (41).
Here, we performed in vitro modeling to determine additional
mechanisms of resistance. Surprisingly, we found that one human
EGFR-mutant tumor cell line acquired a new mutation in NRAS.
Because these models have recapitulated findings in humans (no-
tably T790M mutations and MET amplification), and because
a variety ofRASsignaling pathwaygenes havebeenassociated with
acquired resistance to other targeted therapies in other solid
tumors, we systematically screened for recurrent mutations in
KRAS/NRAS/BRAF/MEK1 in nearly 200 tumor samples from
patients with acquired resistance to EGFR TKIs. Although no
KRAS, NRAS, or MEK1 mutations were detected, we did find one
case with concurrent EGFR exon19 deletion and EGFR T790M
and BRAF V600E mutations and another case with EGFR exon19
deletion and the BRAF G469A mutation (2/195, 1.0%). Although
our cell-line models indicated that the NRAS Q61K and EGFR
L858R mutations were in the same cell, we could not determine if
the BRAF and EGFR mutations were in the same or different tu-
mor cells in the patient samples. In case they were present in the
same cells, we studied further the biological and therapeutic con-
sequences of acquired NRAS and BRAF mutations in EGFR-mu-
tant lung tumor cells and showed that these tumor cells were
resistant to erlotinib alone but were sensitive to combination
treatment with EGFR and MEK inhibition. Transfectants with
mutant BRAF were sensitive to the combination of EGFR and
BRAF inhibition as well. Collectively, these data suggest that RAS
pathway gene mutations are rare but do occur, with BRAF gene
mutations found in 1% of these patients.
In previous smaller studies, with only 6, 37, and 14 patients, re-
in lung cancers from patients with acquired resistance to EGFR
TKIs (7, 12, 29). The sample sizes were too small to be definitively
conclusive. Only 37 tumors have been examined for BRAF muta-
tions (7, 12, 29), and MEK1 status has not been assessed. By con-
cancers, including in tumors from patients with acquired resistance
(12, 19, 20). Why RAS signaling pathway gene mutations are in-
frequent in lung cancers as opposed to other types of solid tumors
treated with targeted therapies is unclear. One possibility is that
introduction of mutant RAS or BRAF into EGFR mutant lung cells
is toxic in most instances, whereas other cell types are more per-
Erlotinib 1 µ µM 6h
Drug Concentration [nM]
% Growth Inhibition
+ Erlotinib 1µ µM + Erlotinib 1µ µM
% Viable cells/
lipofectamine treated control
+ Erlotinib 1 µ µM
11 - 1811-18R
+ AZD6244 1µ µM
Tumor Volume (mm3)
7 11 14 18 21 25 29
E + GSK
11 - 1811 - 18R
parental cells. Erlotinib has no effect on activated NRAS activity in 11-18R cells. (B) Cell growth-inhibition assays show the relative sensitivity of 11-18/11-18R
and PC-9/PC-9R cells to erlotinib, the MEK inhibitor AZD6244, or the combination of erlotinib with AZD6244. Data shown are mean ± SD of three independent
experiments performed in hextuplicate. (C) Athymic nude mice with 11-18R tumors were administered vehicle, erlotinib, MEK inhibitor, GSK1120212, or
erlotinib plus GSK1120212. Tumor volume was determined at the indicated times after the onset of treatment. n = 5 mice per group. Error bars indicate SE.
*P < 0.05 (Student’s t test) for the combination of erlotinib plus GSK1120212 versus either erlotinib or GSK1120212 alone. (D) siRNA-mediated knockdown of
NRAS combined with erlotinib or siRNA-mediated knockdown of EGFR combined with AZD6244 inhibits growth of 11-18R cells. Scr, Scramble. Two different
siRNAs against NRAS were used. Data shown are mean ± SD of three independent experiments performed in hextuplicate. *, **P < 0.05 (Student’s t test) for
the combination of erlotinib plus siNRAS knockdown versus erlotinib or siNRAS alone in 11-18R cells. ***P < 0.05 (Student’s t test) for the combination of
siEGFR plus AZD6244 knockdown versus AZD6244 alone in 11-18R cells. (E) Immunoblotting with the indicated antibodies demonstrates that siRNA-mediated
knockdown of NRAS combined with erlotinib inhibits ERK activation in 11-18R cells. These samples were run on the same gel but were noncontiguous.
Functional role of NRAS Q61K in 11-18R cells. (A) RAS GTPase-specific pulldown assay shows increased activated NRAS in 11-18R cells compared with
| www.pnas.org/cgi/doi/10.1073/pnas.1203530109Ohashi et al.
the pathway, such as amplification (e.g., involving KRAS) or loss of
downstream regulatory genes (e.g., NF1) either by genetic or epi-
genetic mechanisms, occur specifically in lung cancer but not in
In the 11-18R cell-line model of acquired resistance, we found
mutation acts in the ERK pathway downstream of mutant EGFR.
Consistent with these data, a recent report showed that chronic
exposure of cells harboring EGFR T790M to an EGFR T790M-
selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002,
led to the development of ERK2 amplification. Combined treat-
knockdown suppressed cell growth (42). These results demon-
strate that activation of the RAS signaling pathway can mediate
resistance; such activation may become more frequent in patients
as better ways to inhibit EGFR T790M are developed. Thus, it will
be interesting to determine if patients who develop acquired re-
sistance to afatinib/cetuximab or EGFR T790M-specific inhibitors
harbor activation of the RAS signaling pathway via either gene
mutation or amplification.
In summary, preclinical modeling coupled with emerging data
about acquired resistance to targeted therapies in solid tumors led
us to reexamine, in thelargest collection of tumor samples to date,
the contribution of mutations in RAS signaling pathway genes as
mediators of acquired resistance to EGFR TKIs in lung cancer.
Such samples occasionally harbored BRAF mutations but lacked
recurrent mutations in KRAS, NRAS, or MEK1. Although the
percent of cases with BRAF mutation is small (1%), the positive
findings coupled with the negative results provide deeper insight
into mechanisms of acquired resistance to EGFR TKIs in lung
cancer, inform ongoing clinical trials designed to overcome re-
sistance, and narrow the list of genes that should be routinely
screened for in samples from patients with acquired resistance. As
more data emerge on mechanisms of resistance to targeted ther-
apies in various cancers, the findings reported here further dem-
onstrate that, even though colorectal cancers, melanomas, GISTs,
and lung cancers share common signaling cascades, each disease
must be examined independently to determine disease-specific
mediators of acquired resistance.
Materials and Methods
Cell Culture. EGFR-mutant PC-9 (EGFR exon19del E746–A750), HCC827 (EGFR
exon19del E746–A750), HCC4006 (EGFR exon19del L747–E749), HCC4011
(EGFR L858R), 11-18 (EGFRL858R), and H1299 (NRAS Q61K) cells were cultured
in RPMI 1640 medium (Mediatech) supplemented with 10% heat-inactivated
FBS (Atlanta Biologicals) and penicillin-streptomycin solution (final concen-
tration 100 U/mL penicillin, 100 μg/mL streptomycin) (Mediatech). Cells were
grown in a humidified incubator with 5% CO2at 37 °C. To create EGFR TKI-
resistant lines, parental cells were cultured with increasing concentrations of
TKIs starting with the IC30. Doses were increased in a stepwise pattern when
Resistant cells that grew in 5 μM erlotinib were derived after 3–6 mo
of culturing with drug. 11-18R cells were maintained initially as polyclonal
populations under constant TKI selection. DNA identity testing on both the
parental and resistant cells confirmed that the cells were derived from the
same origin. Clonal resistant cells were isolated by limiting dilution.
EGFR-mutant lung cancer and acquired resistance to EGFR TKIs
RAS signaling pathway gene mutations in tumor samples from 212 patients with
Institution EGFR T790MNRAS KRAS BRAFMEK1
Vanderbilt-Ingram Cancer Center†
Massachusetts General Hospital‡
0/146107/195 (54.9%)2/195 (1.0%)
n/d, no data.
*Sixty-eight MSKCC patient samples were assessed by a mass spectrometry-based (Sequenom) assay (ref. 39), and
35 samples were assessed using a SNaPshot-based assay (ref. 32).
†Eight samples were analyzed at Vanderbilt-Ingram Cancer Center by the SNaPshot assay.
‡Eighty-four samples were analyzed at Massachusetts General Hospital by SNapshot assay (ref. 38).
§Seventeen samples were analyzed for NRAS mutations by direct sequencing at other institutions (nine patients
at Okayama University, Japan, five patients at National Taiwan University Hospital, and three patients at the
Max Planck Institute, Cologne, Germany).
% Growth Inhibition
- + - + -+
- + - + -+
% Growth Inhibition
mediates resistance to EGFR TKIs. (A and B) PC-9 cells were transiently trans-
fected with expression plasmidsencoding NRAS wild type or NRAS Q61K (A) or
of erlotinib for 6 h. Corresponding cell lysates were subjected to immuno-
blotting with the indicated antibodies. Lysates from cells harboring NRAS wild
type or NRAS Q61K displayed higher levels of total NRAS than seen in control-
transfected cells, but only cells transfected with NRAS Q61K displayed en-
hanced phospho-ERK expression in the presence of erlotinib. Similarly, lysates
from cells harboring BRAF wild type or BRAF V600E displayed higher levels of
BRAF V600E displayed enhanced phospho-ERK expression in the presence of
erlotinib. (C and D). PC-9 cells were stably transfected with control plasmids or
expression plasmids encoding NRAS Q61K (C) or BRAF V600E (D). Ectopic ex-
pression of NRAS Q61K (C) or BRAF V600E (D) mediated resistance of PC-9 cells
to erlotinib. C13, clone 13; C21, clone 21; C5, clone 5; C6, clone 6. Data shown
are mean ± SD of three independent experiments performed in hextuplicate.
Ectopic expression of NRAS Q61K or BRAF V600E in EGFR mutant cells
Ohashi et al.PNAS
| Published online July 6, 2012
Growth Inhibition Assay. For cell growth-inhibition experiments, cells were
day were exposed to drugs alone or in combination. Cell Titer Blue Reagent
on a Spectramax spectrophotometer (Molecular Devices) according to the
manufacturer’s instructions. All experimental points were set up in hextupli-
and afatinibwere synthesized by theMemorial Sloan-Kettering Cancer Center
(MSKCC) Organic Synthesis Core. AZD6244, GSK1120212, vemurafenib, SGX-
523, and GDC-0941 were purchased from Selleck Chemicals.
Antibodies and Immunoblotting. The following antibodies were obtained
from Cell Signaling Technology: phospho-EGFR, EGFR, MET, phospho-ERK,
ERK, phospho-AKT, AKT, HRP-conjugated anti-mouse, and HRP-conjugated
anti-rabbit. NRAS and BRAF antibody were purchased from Santa Cruz. For
immunoblotting, cells were harvested, washed in PBS, and lysed in 50 mmol/L
Tris·HCl (pH 8.0), 150 mmol/L sodium chloride, 5 mmol/L magnesium chlo-
ride, 1% Triton X-100. 0.5% sodium deoxycholate, 0.1% SDS, 40 mmol/L
sodium fluoride, 1 mmol/L sodium orthovanadate, and complete protease
inhibitors (Roche Diagnostics). Lysates were subjected to SDS/PAGE followed
by blotting with the indicated antibodies and detection by Western Light-
ning ECL reagent (Perkin-Elmer).
RAS Activation Assay. 11-18 and 11-18R cells were serum starved overnight
and supplemented with 1 μM of erlotinib for 6 h. RAS activity was measured
using the Ras-binding domain of Raf-1 to pull down active Ras according to
the manufacturer’s protocol (Cell BioLabs). Following separation by SDS
PAGE, proteins were transferred to membranes which were probed with an
Patient Samples and Data. Tumor specimens were obtained with patients’
consent under Institutional Review Board (IRB)-approved protocols in each
institution. Samples were frozen and stored at −80 °C in institutional tumor
banks or in formalin-fixed, paraffin-embedded blocks. Mutational profiling
results were performed within Clinical Laboratory Improvement Amend-
ments-certified laboratories on multiple samples as part of routine standard
of care and/or on IRB-approved protocols.
Sequencing of NRAS and Systematic Mutation Screening. Genomic DNA was
extracted from patient samples (>70% tumor cells) and cell lines using
standard procedures. NRAS exons 2 and 3 were amplified from genomic
DNA and were sequenced directly. NRAS mutations, along with other
mutations, also were screened using a SNapShot-based (32, 38) or mass
spectrometry-based (Sequenom) assays (39).
siRNA Experiments. EGFR, NRAS, and negative control oligos (Dharmacon)
were used at a concentration of 10 nM and transfected with Lipofectamine
RNAimax according to the manufacturer’s protocol (Invitrogen).
Expression Constructs and Transfections. A cDNA for NRAS was purchased
from Origene and subcloned into a Flag-N-CMV6 entry vector (Origene). A
cDNA for BRAF was kindly provided by David Solit (MSKCC) and was subcl-
oned into a pcDNA3.1 vector (Invitrogen). The NRAS Q61K and BRAF V600E
and BRAF G469A mutations were introduced into the cDNAs using site-di-
rected mutagenesis (Agilent) with mutant-specific primers according to the
manufacturer’s instructions. The cDNAs were fully resequenced to ensure
that no additional mutations were introduced. Plasmid transfections into PC-
9 or PC-9R cells were performed with Lipofectamine 2000 (Invitrogen) fol-
lowing the manufacturer’s protocol. Selection of cells was started 48 h later
in 96-well plates with appropriate antibiotics.
Xenograft Studies. Nude mice (nu/nu; Harlan Laboratories) were used for in
vivo studies andwere cared forin accordance with guidelinesapproved by the
MSKCC Institutional Animal Care and Use Committee and Research Animal
11-18R cells together with Matrigel. Once tumors reached an average volume
of 150 mm3, mice were randomized and dosed via oral gavage with either
erlotinib (MSKCC Organic Synthesis Core), GSK1120212 (Selleck Chemicals), or
the combination at the indicated doses. A uniform volume for administration
(200 μL) was used for each group. Mice were observed daily throughout the
treatment period for signs of morbidity/mortality. Tumors were measured
twice weekly using calipers, and volume was calculated using the formula
length × width2× 0.52. Body weight also was assessed twice weekly. The
experiment was terminated after 4 wk of treatment.
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health/National Cancer Institute (NCI) Grants R01-CA121210, P01-CA129243,
and U54-CA143798. W.P. received additional support from Vanderbilt’s Spe-
cialized Program of Research Excellence in Lung Cancer Grant CA90949 and
from Vanderbilt-Ingram Cancer Center Core Grant P30-CA68485. L.V.S. re-
ceived additional support from NCI Grant 1R21-156000 and from Uniting
Against Lung Cancer: New England and the Marjorie E. Korff Fund.
% Growth Inhibition
PC-9/NRASQ61K C13PC-9/NRASQ61K C21
Drug Concentration [nM] Erlotinib x1 GSK1120212 x1/5
Erlotinib 1µ µM 6h
C13 C21 C13 C21 C13 C21 C13 C21
+ -- + + -
C5 C6 C5 C6 C5 C6 C5 C6
PC-9/BRAFV600E C5PC-9/BRAFV600E C6
Drug Concentration [nM] Erlotinib x1 GSK1120212 x1/5
+ + + +
+ + -
- + +
- + + + +
% Growth Inhibition
Erlotinib 1 M 6h
µ µ µ
BRAF V600E stable clones to erlotinib. C13, clone 13; C21, clone 21; C5, clone
5; C6, clone 6. (A and C) The combination of erlotinib and GSK1120212 leads
to greater inhibition of cell growth in PC-9 cells stably expressing NRAS Q61K
(PC-9/NRAS Q61K cells) (A) or BRAF V600E (PC-9/BRAF V600E cells) (C) than
seen either drug alone. Data shown are mean ± SD of three independent
experiments performed in hextuplicate. (B and D) In stable transfectants the
combination of erlotinib plus GSK1120212 leads to a greater reduction in
phospho-ERK levels than either drug alone. PC-9/NRAS Q61K (B) or PC-9/
BRAF V600E (D) cells were cultured in the absence or presence of erlotinib
or/and GSK1120212 for 6 h; corresponding cell lysates were subjected to
immunoblotting with the indicated antibodies.
MEK inhibition restores the sensitivity of PC-9/NRAS Q61K or PC-9/
| www.pnas.org/cgi/doi/10.1073/pnas.1203530109Ohashi et al.
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