Mutation of Threonine 766 in the Epidermal Growth Factor Receptor
Reveals a Hotspot for Resistance Formation against Selective Tyrosine
Kinase Inhibitors*
Stephanie
Blencke
,
Axel
Ullrich§, and
Henrik
Daub
¶
From
Axxima Pharmaceuticals AG, Max-Lebsche-Platz 32,
81377 München and the § Department of Molecular
Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A,
82152 Martinsried, Germany
Received for publication, October 31, 2002, and in revised form, February 5, 2003
 |
ABSTRACT |
Small molecule inhibitors of protein tyrosine
kinases such as STI571 represent a major new class of therapeutics for
target-selective treatment of human cancer. Clinical resistance
formation to the BCR-ABL inhibitor STI571 has been observed in patients
with advanced chronic myeloid leukemia and was frequently caused by a C
to T single nucleotide change in the Abl kinase domain, which
substituted Thr-315 with isoleucine and rendered BCR-ABL resistant to
STI571 inhibition. The corresponding mutation in the epidermal growth factor receptor (EGFR) tyrosine kinase replaced Thr-766 of the EGFR by
methionine and dramatically reduced the sensitivity of EGFR to
inhibition by selective 4-anilinoquinazoline inhibitors such as
PD153035. Inhibitor-resistant EGFR exhibited the same signaling
capacity as wild-type receptor in vivo and provides a
useful tool for analyzing EGFR-mediated signal transduction. Our data
identify Thr-766 of the EGFR as a structural determinant that bears the
potential to become a relevant feature in resistance formation during
cancer therapy with EGFR-specific 4-anilinoquinazoline inhibitors.
 |
INTRODUCTION |
Protein tyrosine kinases are key regulators of complex signaling
cascades that control a variety of physiological responses, such as
cell proliferation, differentiation, and survival (1, 2).
Deregulation of protein tyrosine kinase-mediated cellular signaling is critically involved in various human malignancies and has
therefore fueled the development of target-selective drugs for
anticancer therapy. Small molecule inhibitors of tyrosine kinase
activity represent one major new class of therapeutics, and several
target-specific compounds are currently undergoing clinical evaluation
(3, 4). The Abl tyrosine kinase inhibitor STI571 (GleevecTM) is the most advanced of these novel
cancer drugs and has received approval for the treatment of chronic
myeloid leukemia (CML)1 (5). In CML, the defined
Philadelphia chromosomal translocation generates the BCR-ABL fusion
protein, which exhibits constitutive Abl tyrosine kinase activity and
is causative for disease development. STI571 has demonstrated
remarkable effectiveness in early, chronic-phase CML patients, whereas
patients suffering from the advanced form of CML, also known as blast
crisis, initially responded to STI571 but then became resistant to drug
treatment (6). In a subset of these cases, resistance formation has
been attributed to a single C to T nucleotide mutation that replaced
Thr-315 of Abl by isoleucine and thereby rendered BCR-ABL insensitive
to STI571 treatment (7). Thr-315 is located at a hydrophobic cavity
near the nucleotide binding site of c-Abl and is critical for binding of the ATP-competitive inhibitor STI571 but not essential for positioning of ATP itself, explaining why the activity is preserved in
the mutant kinase (8). These findings establish that even targeted
intervention strategies can be prone to resistance formation and
further raise the important question of whether similar mechanisms might generally apply to tyrosine kinase inhibitors for cancer therapy.
Apart from STI571, the most advanced small molecule drugs for treatment
of malignancies belong to the 4-anilinoquinazoline class of compounds
and selectively target the epidermal growth factor receptor (EGFR)
tyrosine kinase, which has been implicated in the progression of
various tumors (3, 4, 9). Specific EGFR inhibitors such as the
quinazolines derivatives ZD1839 (Iressa®) and OSI-774
(Tarceva®) are already in late stages of clinical
development, but the structural determinants of the EGFR kinase domain
required for quinazoline binding and potentially involved in resistance
formation have not been analyzed yet. In this report, we
demonstrate that mutations equivalent to those found in BCR-ABL from
relapsed CML patients dramatically desensitize EGFR to inhibition by
4-anilinoquinazolines without affecting its kinase activity and signal
characteristics in vivo.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines, Reagents, and Plasmids--
CHO-K1 cells were from
ATCC. Immortalized embryonic EF1.1
/
fibroblasts derived from EGFR
knockout mice were a generous gift from Maria Sibilia and Erwin Wagner
(Vienna, Austria). Cell culture media and LipofectAMINE were purchased
from Invitrogen. Radiochemicals were from Amersham Biosciences.
PD153035, AG1478, and human recombinant EGF were from Calbiochem. All
other reagents were obtained from Sigma.
Antibodies purchased were mouse monoclonal anti-HA antibody (Roche
Molecular Biochemicals), polyclonal anti-EGFR antibody (Santa Cruz
Biotechnology), mouse monoclonal anti-SHC antibody (BD Transduction
Laboratories), rabbit polyclonal anti-Gab1 antibody (Upstate),
mouse monoclonal anti-phospho-ERK1/2 antibody (Cell Signaling
Technology), rabbit polyclonal anti-ERK2 antibody (Santa Cruz
Biotechnology) and rabbit polyclonal anti-c-Fos antibody (Santa
Cruz Biotechnology). Rabbit polyclonal anti-SHC and mAb108.1 mouse monoclonal anti-EGFR antibodies have been described previously (10).
Human EGFR cDNA was either cloned in the expression vector pRK5 or
in the retroviral expression vector pLXSN, which allows moderate
protein expression upon transient plasmid transfection (11, 12).
pcDNA3-HA-ERK2 has been described previously (10). Mutants of EGFR
were generated using a mutagenesis kit according to the manufacturer's
instructions (Stratagene).
Cell Culture and Transfections--
COS-7 and EF1.1
/
cells
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum. CHO-K1 cells were maintained in
nutrient-mixture Ham's F12 medium containing 10% fetal bovine serum.
Retroviral infections of EF 1.1
/
cells were performed as described
(13). Polyclonal cell lines stably expressing wild-type or mutant EGFR
were established by selection in G418-containing medium. For plasmid
transfection experiments in 6-well dishes, COS-7 (CHO-K1) cells were
seeded at 2.0 × 105 (3.0 × 105) per
well 20 h before transfection. Cells were incubated for 4 h
in 1.0 ml of serum-free medium containing 9 µl (6 µl) of
LipofectAMINE (Invitrogen) and 1.5 µg (2 µg) plasmid of DNA per
well. The transfection mixtures were then either supplemented with 1 ml
of medium containing 20% fetal bovine serum (COS-7) or replaced by
fresh medium containing 10% fetal bovine serum (CHO-K1); 20 h
later, cells were either lysed or serum-starved for a further 20 h
in serum-free medium prior to stimulation and lysis.
Cell Lysis, Immunoprecipitation, and
Immunoblotting--
Serum-starved cells were treated with inhibitors
and growth factors as indicated prior to cell lysis in buffer
containing 50 mM HEPES, pH 7.5, 150 mM NaCl,
0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM sodium pyrophosphate plus additives (10 mM
sodium fluoride, 1 mM orthovanadate, 10 µg/ml aprotinin,
10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride).
Lysates were precleared by centrifugation and then immunoprecipitated
with the respective antibodies and protein G-Sepharose for 3 h at
4 °C. After three washes with lysis buffer without additives, bound
proteins were eluted with SDS sample buffer, boiled for 3 min, and then
resolved by SDS gel electrophoresis. After SDS-PAGE, proteins were
transferred to nitrocellulose membrane and immunoblotted with the
indicated antibodies. For analysis of c-Fos induction,
SDS-containing lysis buffer to solubilize nuclear proteins was used
(14).
EGFR in Vitro Kinase Assay--
CHO-K1 cells were transiently
transfected in 10-cm dishes with 12 µg of either pRK5-EGFR or
pRK5-EGFR-T766M expression plasmid and 36 µl of LipofectAMINE. On the
following day, dishes were stimulated with 100 ng/ml EGF for 5 min and
then lysed with 950 µl of lysis buffer. After preclearing by
centrifugation, aliquots of 140 µl of lysate were immunoprecipitated
with mAb108.1 antibody. Beads were washed twice with 300 µl of lysis
buffer without additives and twice with 200 µl of kinase buffer
containing 20 mM Tris-HCl, pH 7.5, 3 mM
MgCl2, 3 mM MnCl2, 100 µM orthovanadate, and 0.1 mM dithiothreitol.
Precipitated EGFRs were then preincubated on ice for 15 min in kinase
buffer supplemented with EGFR inhibitor as indicated. Kinase reactions
were then started by the addition of 10 µM ATP, 2 µCi
of [
-33P]ATP and 3 µg of glucose-6-phosphate
dehydrogenase (G6PDH) (Sigma) and performed for 5 min at room
temperature (15). Reactions were stopped by the addition of 3× SDS
sample buffer. After gel electrophoresis, phosphorylated G6PDH was
detected by autoradiography and quantified by phosphorimaging.
No detectable kinase activity was found in anti-EGFR immunoprecipitates
from control-transfected cells expressing no EGFR.
[3H]Thymidine Incorporation--
EF1.1
/
cell
lines expressing wild-type or mutant EGFR were seeded in 12-well dishes
and serum-starved at 50% confluency. After 24 h, cells were
treated with PD153035 and EGF as indicated and then cultivated for
18 h. For the last 4 h, 0.5 µCi of
methyl-[3H]thymidine were added per well. Cells were
washed twice with phosphate-buffered saline followed by 1 h of
precipitation with 10% (w/v) trichloroacetic acid on ice.
Precipitates were solubilized with 0.5 ml of 0.2 N NaOH/1% SDS and
then neutralized with 0.5 ml of 0.2 N HCl. Incorporated radioactivity
was quantified by scintillation counting.
 |
RESULTS |
Kinase-active EGFR Mutants Resistant to Quinazoline
Inhibitors--
Mutation of Thr-315 in Abl to isoleucine by a C to T
single nucleotide change (ACC to ATC) rendered
BCR-ABL kinase activity resistant to STI571 in advanced CML patients
(7). The molecular basis for these results was provided by the Abl
tyrosine kinase crystal structure, which identified Thr-315 in Abl as a
key determinant for STI571 binding (8). Alignment of the amino acid
residues surrounding Thr-315 in Abl with the EGFR sequence reveals a
conserved threonine residue in the equivalent position 766 of the EGFR
(Fig. 1A). We reasoned that
Thr-766 in EGFR might also be implicated in drug resistance formation
and therefore introduced the corresponding C to T single nucleotide
change into the EGFR sequence. In the case of EGFR, this clinically
relevant mutation replaces Thr-766 by methionine (ACG to
ATG). The resulting EGFR-T766M mutant and wild-type EGFR
were transiently expressed in CHO-K1 cells and isolated by
immunoprecipitation. Both wild-type and mutant receptors were then
assayed for kinase activity in vitro utilizing G6PDH as a
substrate (15). Wild-type EGFR was potently inhibited by the
quinazoline derivative PD153035 with an IC50 value of about 1 nM (Fig. 1B). PD153035 is highly related in
structure to the therapeutic agents ZD1839 and OSI-774 and was used as
a representative 4-anilinoquinazoline inhibitor of the EGFR in this
study (16). Mutation of Thr-766 to methionine resulted in 100-fold less
potent EGFR kinase inhibition by PD153035 (Fig. 1B). With 10 µM ATP present in the kinase reactions, the catalytic
activity of the mutant kinase was about 50% when compared with
wild-type EGFR (data not shown).

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Fig. 1.
EGFR mutants resistant to the
4-anilinoquinazoline inhibitor PD153035. A, residues
surrounding Thr-315 in Abl aligned with the corresponding EGFR
sequence. Thr-315 in Abl and the equivalent Thr-766 in EGFR are
highlighted in gray. B, in vitro
kinase assay. Wild-type EGFR and EGFR-T766M mutant were preincubated
with the indicated concentrations of PD153035 and then subjected to
in vitro kinase assays using G6PDH as a substrate. Kinase
activities in the absence of inhibitor were set to 100%; activities
measured with different PD153035 concentrations are expressed relative
to this value. C, CHO-K1 cells were transiently transfected
with either empty vector or pLXSN expression plasmids encoding EGFR,
EGFR-T766I, or EGFR-T766M. Following serum starvation for 24 h,
cells were preincubated with the indicated concentrations of PD153035
or an equal volume of Me2SO for 15 min prior to stimulation
with 10 ng/ml EGF for 5 min. After cell lysis, EGFR was
immunoprecipitated with mAb108.1. Following gel electrophoresis,
tyrosine-phosphorylated EGFR was detected by immunoblotting with
monoclonal anti-phosphotyrosine antibody ( PY, upper
panels). In parallel, the amount of EGFR in immunoprecipitates was
analyzed using polyclonal anti-EGFR antibody (lower panels).
D, serum-starved CHO-K1 cells transiently expressing either
wild-type EGFR or EGFR-T766V were preincubated with the indicated
concentrations of PD153035 for 15 min prior to stimulation with 10 ng/ml EGF. After cell lysis, EGFR was immunoprecipitated and
analyzed as described in panel C.
|
|
For further analysis of inhibitor sensitivity, we studied EGFR mutants
in intact CHO-K1 cells that lack endogenous EGFR expression. In
addition to the EGFR-T766M mutant, we also replaced Thr-766 of the EGFR
by isoleucine to analyze the amino acid substitution equivalent to that
found in STI571-insensitive BCR-ABL. Transiently expressed wild-type
and mutant EGFR tyrosine kinases were stimulated in intact cells by the
addition of EGF to the culture medium. After immunoprecipitation,
wild-type and mutant EGFRs were analyzed by immunoblotting with
phosphotyrosine-specific antibody to measure autophosphorylation of the
EGFR on intracellular tyrosine residues resulting from the intrinsic
kinase activity of the receptor (17). EGF-induced stimulation of
tyrosine phosphorylation of wild-type EGFR and both mutants was
similar, indicating that the replacement of Thr-766 by isoleucine or
methionine had no significant effect on EGFR kinase activity in the
context of cellular ATP concentrations (Fig. 1C). As further
seen in Fig. 1C, pretreatment of cells with 10 nM PD153035 already strongly suppressed EGF-induced
tyrosine phosphorylation of wild-type EGFR. In stark contrast,
1000-fold higher concentrations of PD153035 only partially reduced
tyrosine phosphorylation of EGFR-T766I and had no inhibitory effect on the T766M mutant. Thus, even the modest threonine to isoleucine conversion at position 766 dramatically reduced the sensitivity of EGFR
to PD153035 in intact cells. The longer side chain of methionine in
position 766 conferred full resistance to mutant EGFR at all PD153035
concentrations tested. As evident for the T766M mutant, desensitization
to PD153035 was even more pronounced under the physiologically relevant
cellular conditions than observed in our in vitro kinase
assay. Furthermore, similar results were obtained when the quinazoline
AG1478 was used for EGFR inhibition (data not shown) (18), strongly
suggesting that substitution of methionine or isoleucine for Thr-766
desensitizes EGFR to specific 4-anilinoquinazoline inhibitors of its
kinase activity in general. These bulkier hydrophobic side chains in
position 766 might sterically interfere with the projection of the
inhibitors' 4-anilino substituents into a hydrophobic pocket located
at the nucleotide binding site of the EGFR, consistent with recent
co-crystal data of the EGFR kinase domain in complex with OSI-774 (19).
Moreover, the hydroxyl group of Thr-766 could establish hydrogen bonds
contributing to high affinity inhibitor binding. To analyze this
issue, we replaced Thr-766 by a valine residue of similar size and
compared EGF-stimulated tyrosine phosphorylation of wild-type EGFR and
the T766V mutant in transiently transfected CHO-K1 cells. As shown in
Fig. 1D, the EGFR-T766V mutant exhibited an ~10-fold
reduced sensitivity to PD153035 inhibition compared with wild-type
receptor, indicating that the hydroxyl group of Thr-766 is important
for inhibitor binding but that its absence can only partially account
for the dramatic EGFR resistance formation observed for the T766I and T766M mutants.
Signaling Capacity of Drug-resistant EGFR--
Upon ligand-induced
autophosphorylation, phosphotyrosine-dependent binding of
the adaptor proteins SHC and Grb2 couples EGFR to Sos-mediated
activation of Ras/mitogen-activated protein kinase (MAPK) signaling
(20, 21). In addition, docking proteins such as Gab1 are
tyrosine-phosphorylated upon EGFR activation and recruit additional
signal transducers into receptor-proximal multiprotein complexes (22).
To test whether these mitogenic signals can be mediated through the
EGFR-T766M mutant generated by the equivalent C to T single nucleotide
mutation as previously found in STI571-resistant BCR-ABL from advanced
CML patients (7), we utilized retroviral gene transfer to stably
express either wild-type EGFR or the T766M mutant in immortalized EF1.1
/
fibroblasts derived from EGFR knockout mice (23). By analyzing
ligand-stimulated tyrosine phosphorylation of EGFR in the presence of
different PD153035 concentrations, we first confirmed that mutation of
Thr-766 to methionine rendered EGFR resistant to PD153035 inhibition in
murine fibroblasts (Fig. 2A).
The IC50 value was shifted from below 10 nM to
more than 10 µM in agreement with our data from CHO-K1
cells presented above. Importantly, cellular tyrosine phosphorylation of both wild-type EGFR and the drug-resistant mutant was similar in
response to low, non-saturating doses of EGF (Fig. 2B). Time course experiments further revealed comparable kinetics of EGFR down-regulation upon prolonged EGF treatment (Fig. 2C).
Thus, wild-type EGFR and the PD153035-insensitive mutant showed the same activation and desensitization characteristics under
physiologically relevant conditions in intact cells.

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Fig. 2.
Characterization of PD153035-insensitive
EGFR-T766M in stably transfected fibroblasts. A,
control-infected EF1.1 / fibroblasts devoid of EGFR expression or
EF1.1 / cells stably expressing either wild-type EGFR or the
EGFR-T766M mutant were serum-starved for 24 h. Following
preincubation with the indicated concentrations of PD153035 for 15 min,
cells were stimulated for 5 min with 10 ng/ml EGF prior to lysis. EGFR
was immunoprecipitated with mAb108.1 and analyzed for tyrosine
phosphorylation by immunoblotting with monoclonal anti-phosphotyrosine
antibody ( PY, upper panels). In parallel, the
amount of EGFR in immunoprecipitates was analyzed using polyclonal
anti-EGFR antibody (lower panels). B,
serum-starved EF1.1 cells expressing either wild-type EGFR or the
EGFR-T766M mutant were stimulated with the indicated concentrations of
EGF for 5 min prior to cell lysis. EGFR was then immunoprecipitated and
analyzed as described in panel A. C,
after serum starvation, EGFR- and EGFR-T766M-expressing EF1.1
fibroblasts were treated for the indicated times with 10 ng/ml EGF.
Upon cell lysis, EGFR was isolated by immunoprecipitation and further
analyzed as described in panel A.
|
|
To characterize EGFR-proximal signaling in wild-type and mutant
receptor-expressing cells, we examined EGF-stimulated tyrosine phosphorylation of the adaptor proteins SHC and Gab1. These cellular tyrosine kinase substrates play essential roles in receptor tyrosine kinase-mediated signal transmission. As shown in Fig.
3, A and B,
EGF-induced SHC and Gab1 tyrosine phosphorylation was abrogated when
cells expressing wild-type EGFR were preincubated with 1 µM PD153035, whereas neither signaling event was affected
in fibroblasts expressing the EGFR-T766M mutant upon inhibitor
pretreatment. Moreover, similar sets of proteins, including the adaptor
protein Grb2, were found to co-precipitate with SHC or Gab1 in both
cell lines, demonstrating that the Thr-766 to methionine mutation did not alter EGFR-proximal signaling steps in intact cells (Fig. 3,
A and B).

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Fig. 3.
Signaling capacity of PD153035-insensitive
EGFR-T766M. Control-infected EF1.1 / fibroblasts devoid of
EGFR expression or EF1.1 / cells stably expressing either wild-type
EGFR or the EGFR-T766M mutant were serum-starved for 24 h.
Following preincubation with the indicated concentrations of PD153035
for 15 min, cells were stimulated for 5 min with 10 ng/ml EGF prior to
lysis. A, lysates were subjected to immunoprecipitation with
polyclonal anti-SHC antiserum. After SDS-PAGE, immunoblotting was
performed with anti-phosphotyrosine antibody ( PY,
upper panel) and monoclonal anti-Grb2 antibody (lower
panel). In parallel, the amount of immunoprecipitated SHC was
analyzed with monoclonal anti-SHC antibody (middle panel).
B, lysates were immunoprecipitated using polyclonal
anti-Gab1 antiserum. Subsequent immunoblotting was performed with
anti-phosphotyrosine antibody ( PY, upper
panel) and monoclonal anti-Grb2 antibody (lower panel).
In parallel, the amount of immunoprecipitated Gab1 was probed with
anti-Gab1 antibody (middle panel). C, total cell
lysates from EF1.1 / fibroblasts stably expressing either wild-type
EGFR or the EGFR-T766M mutant were resolved by gel electrophoresis and
then analyzed by immunoblotting with monoclonal anti-phospho-ERK1/2
antibody. D, cells were pretreated with 1 µM
PD153035 for 15 min prior to stimulation with 10 ng/ml EGF where
indicated. After 1 h, total cell lysates were prepared and
induction of c-Fos protein expression was analyzed by immunoblotting
with polyclonal anti-c-Fos antibody.
|
|
We next examined whether the EGFR-T766M could also trigger MAPK signal
transduction and gene expression in a PD153035-insensitive manner. To
assay for activation of ERK MAP kinases, lysates from EGF-treated cells
were subjected to immunoblotting with antibody specific for dually
phosphorylated ERK1 and ERK2. As shown in Fig. 3C,
micromolar concentrations of PD153035 abrogated ERK phosphorylation in
wild-type EGFR but not in EGFR-T766M-expressing fibroblasts. Moreover,
although pretreatment with 100 nM PD153035 strongly suppressed autophosphorylation of wild-type EGFR (Fig. 2A),
this inhibitor concentration was not yet sufficient to block
EGF-induced ERK activation, indicating that the remaining residual EGFR
activation is still sufficient to trigger the ERK pathway. Finally,
EGF-induced expression of the immediate-early gene, c-fos,
was investigated; we again found that the threonine to methionine
mutation had conferred PD153035-resistance to this EGFR-mediated
signaling event (Fig. 3D). Thus, our results show that the
equivalent C to T nucleotide change as found in STI571-resistant
BCR-ABL fully restores EGFR-mediated signaling in the presence of a
selective 4-anilinoquinazoline inhibitor. These findings further
demonstrate that none of the kinases downstream of EGFR were affected
by the quinazoline compound PD153035, confirming the high specificity
of this EGFR inhibitor.
Mitogenic Responses Mediated through Wild-type and
Inhibitor-resistant EGFR--
Targeted cancer therapy employing
selective EGFR inhibitors primarily aims at suppressing tumor cell
proliferation. To quantify how the anti-proliferative effect of a
specific quinazoline inhibitor is diminished if EGFR had acquired drug
resistance, we performed thymidine incorporation assays to measure the
EGF-mediated proliferative responses in both wild-type and T766M mutant
EGFR-expressing cell lines. PD153035 pretreatment inhibited
EGF-stimulated DNA synthesis mediated through wild-type EGFR with an
IC50 value of about 50 nM (Fig.
4), whereas half-maximal inhibition of
EGF-triggered thymidine incorporation through the EGFR-T766M mutant
occurred at about 50-fold higher PD153035 concentrations of around 2.5 µM. Because even up to 10 µM of the
inhibitor were without effect on EGFR-T766M tyrosine phosphorylation in
intact cells, this finding reveals that cellular targets of PD153035
distinct from EGFR are involved in EGF-triggered cell cycle
progression. These unknown secondary targets are inhibited at much
higher concentrations of PD153035, again demonstrating the high
selectivity of this specific EGFR blocker and further implying that
analogous EGFR mutations might render cancer patients resistant to
treatment with selective EGFR blockers.

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Fig. 4.
Effects of PD153035 on thymidine
incorporation triggered through wild-type and inhibitor-resistant
EGFR. EF1.1 / fibroblasts stably expressing either wild-type
EGFR or the EGFR-T766M mutant were serum-starved for 24 h. Cells
were then pretreated with the indicated concentrations of PD153035 for
15 min prior to stimulation with 10 ng/ml EGF. After 14 h of
incubation, cells were pulse-labeled with
methyl-[3H]thymidine for 4 h, and its incorporation
into DNA was determined. Basal thymidine incorporation in
Me2SO-treated, unstimulated cells was set to 1. Data shown
represent the mean ± S.D. for triplicate samples.
|
|
Quinazoline-resistant EGFR Mutant as a Tool for Target
Validation--
We reasoned that co-expression of inhibitor-resistant
EGFR should complement for endogenous, inhibitor-sensitive EGFR upon PD153035 treatment and thereby provide a novel tool for
chemical-genetic validation of EGFR function. To test this, we used the
EGFR-dependent ERK MAPK activation upon stimulation of G
protein-coupled receptors as a model system (10). Upon transient
expression of hemagglutinin (HA) epitope-tagged ERK2, COS-7 cells were
stimulated with either 10 µM LPA to activate its cognate
G protein-coupled receptor or 1 ng/ml EGF. HA-ERK2 was then
immunoprecipitated and analyzed by immunoblotting with
activation-specific antibody recognizing dually phosphorylated ERK2. In
agreement with published data (10), pretreatment of cells with 1 µM PD153035 strongly suppressed LPA-induced and blocked
EGF-triggered HA-ERK2 activation (Fig. 5,
upper two panels). Similar inhibition of LPA- and
EGF-induced ERK2 activity by PD153035 was observed when wild-type EGFR
was co-expressed (Fig. 5, middle two panels). But, in
striking contrast, co-transfection of the PD153035-resistant EGFR-T766M
mutant fully restored both LPA- and EGF-triggered HA-ERK2 activation in
the presence of 1 µM PD153035 (Fig. 5, lower two
panels). Thus, this result confirms that specific inhibition of
EGFR by PD153035 suppresses LPA-stimulated ERK activation and
establishes inhibitor-resistant EGFR as a useful tool for target
validation in signal transduction analysis.

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Fig. 5.
Reconstitution of PD153035-suppressed ERK
MAPK activation by co-expression of EGFR-T766M. COS-7 cells were
transiently transfected with pcDNA3-HA-ERK2 expression plasmid
(0.25 µg/well) plus either empty expression vector or pLXSN
expression plasmids encoding EGFR or EGFR-T766M (0.25 µg/well).
Following serum starvation for 24 h, cells were pretreated with 1 µM PD153035 for 15 min and then stimulated with 10 µM LPA or 1 ng/ml EGF for 7 min. After lysis, HA-ERK2 was
immunoprecipitated and analyzed by immunoblotting with monoclonal
anti-phospho-ERK1/2 antibody (panels 1, 3, and
5). Filters were reprobed with polyclonal anti-ERK2 antibody
(panels 2, 4, and 6).
|
|
 |
DISCUSSION |
Our results identify Thr-766 as a critical structural determinant
controlling inhibitor sensitivity of the EGFR. Importantly, introduction of bulkier hydrophobic side chains at this position fully
preserved the cellular kinase activity of the EGFR in the presence of
selective kinase inhibitors, indicating potential mechanisms of
molecular resistance formation as previously found for BCR-ABL from
STI571-treated CML patients. In addition to the Thr-315 to isoleucine
substitution in the Abl kinase domain described by Gorre et
al. (7), previous work has established that the corresponding
threonine residues in the cytoplasmatic protein kinases p38 and Src are
critical for their sensitivities to the pyridinylimidazole inhibitor
SB203580 and the pyrazolopyrimidine derivative PP1, respectively (24,
25). Thus, a shared structural feature emerges from our work and
published data that define an active-site threonine residue conserved
in a subset of protein kinases as critical for kinase inhibition by
small molecular weight inhibitors belonging to different compound
classes. Remarkably, although the majority of protein kinases have a
larger, hydrophobic residue in the equivalent position, most of the
selective and highly potent inhibitors have been identified for
the comparably small subgroup of protein kinases possessing a threonine
at this site. Published co-crystal structures of protein kinases from the latter group reveal that inhibitor moieties always extend into a
hydrophobic cavity at the ATP-binding site, which cannot be occupied by
ATP itself (8, 19, 26). These additional inhibitor-kinase interactions
allow high affinity binding, which is abrogated when the conserved
threonine residue located in the hydrophobic pocket is replaced by a
more space-filling amino acid. It further appears that this particular
structure-activity relationship has influenced the development of
specific tyrosine kinase inhibitors for cancer therapy, because most of
these therapeutics in clinical testing selectively target receptor
tyrosine kinases possessing a threonine in the conserved position (or a
valine of comparable size as is present in fibroblast growth
factor receptor and vascular endothelial growth factor receptor) (3,
4). In this context, our results raise the issue whether all of these
receptor tyrosine kinase targets might be susceptible to resistance
formation when required for the proliferation of genetically unstable
tumor cells, because even minimal genetic alterations could abrogate
small molecule inhibitor binding, as demonstrated for the EGFR in this study. In addition to substitutions equivalent to the Thr-315 to
isoleucine exchange initially detected in STI571-resistant BCR-ABL,
other kinase domain mutations could also confer inhibitor resistance to
receptor tyrosine kinases as recently reported for the Abl kinase
domain from relapsed CML patients (27, 28). Our findings emphasize the
importance of protein structure analysis for drug development and
reveal further obstacles but also new opportunities for better,
target-specific therapies for cancer.
 |
ACKNOWLEDGEMENTS |
We thank M. Sibilia and E. Wagner for
providing EF1.1
/
fibroblasts. We also thank T. Herget, B. Klebl,
and M. Stein-Gerlach for critical reading of the manuscript.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
49-89-550-65-356; Fax: 49-89-550-65-461; E-mail:
daub@axxima.com.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211158200
 |
ABBREVIATIONS |
The abbreviations used are:
CML, chronic myeloid
leukemia;
EGFR, epidermal growth factor receptor;
ERK, extracellular
signal-regulated protein kinase;
G6PDH, glucose-6-phosphate
dehydrogenase;
LPA, lysophosphatidic acid;
MAPK, mitogen-activated
protein kinase;
HA, hemagglutinin;
CHO, Chinese hamster ovary;
mAb, monoclonal antibody.
 |
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