The T790M mutation in EGFR kinase causes drug
resistance by increasing the affinity for ATP
Cai-Hong Yun*†, Kristen E. Mengwasser†, Angela V. Toms*†, Michele S. Woo‡, Heidi Greulich‡§, Kwok-Kin Wong‡¶,
Matthew Meyerson‡§?, and Michael J. Eck*†**
Departments of *Biological Chemistry and Molecular Pharmacology and?Pathology, Harvard Medical School, 25 Shattuck Street, Boston,
MA 02115; Departments of†Cancer Biology and‡Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115;
¶Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115; and§The Broad Institute of Harvard and Massachusetts
Institute of Technology, 320 Charles Street, Cambridge, MA 02141
Edited by Harold E. Varmus, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved December 13, 2007 (received for review
October 11, 2007)
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
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 inhib-
itors, 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
covalent binding, not as a result of an alternative binding mode.
lung cancer ? tyrosine kinase ? x-ray crystallography
tified as a cause of nonsmall cell lung cancer (1–7). The most
common oncogenic mutations are small, in-frame deletions in
exon 19 and a point mutation that substitutes Leu-858 with
arginine (L858R). These mutations likely cause constitutive
activation of the kinase by destabilizing the autoinhibited con-
formation (8, 9), which is normally maintained in the absence of
ligand stimulation. Importantly, the activating mutations have
also been found to confer sensitivity to the small molecule
first reported by Carey et al. (10) in studies with erlotinib, the
EGFR and additionally the deletion and L858R mutations
which the inhibitors compete for binding. These two effects
combine to yield the remarkable potency of gefitinib and
erlotinib against tumors and cell lines that are ‘‘addicted’’ to the
activated EGFR for survival (5, 11, 12).
Clinically, the efficacy of these TKIs is often of limited
duration because of the emergence of drug resistance conferred
by a second mutation: substitution of threonine 790 with me-
thionine (T790M) (13–15). The T790M mutation accounts for
about half of all resistance to gefitinib and erlotinib (16, 17).
Threonine 790 is the gatekeeper residue in EGFR, so named
utations in the tyrosine kinase domain of the epidermal
growth factor receptor (EGFR) have recently been iden-
because its key location at the entrance to a hydrophobic pocket
in the back of the ATP binding cleft makes it an important
determinant of inhibitor specificity in protein kinases. Substitu-
tion of this residue in EGFR with a bulky methionine has been
thought to cause resistance by steric interference with binding of
TKIs, including gefitinib and erlotinib (13–15). However, the
T790M mutant kinase remains sensitive to irreversible inhibi-
tors, including CL-387,785, EKB-569, and HKI-272 (14, 15,
18–20). These compounds closely resemble the reversible ani-
linoquinazoline inhibitors, but contain a reactive Michael-
acceptor group that forms a covalent bond with Cys-797 at the
are designed to target only this cysteine in EGFR because of
their specific noncovalent interactions in the ATP binding
pocket, which resemble those of reversible anilinoquinazoline
the T790M mutant is at odds with steric hindrance as a mech-
anism of resistance: the reversible inhibitor gefitinib and the
irreversible inhibitor EKB-569 have identical aniline substitu-
ents that are expected to bind in the gatekeeper pocket (Fig. 1),
so the same steric effects that block gefitinib binding should also
prevent the initial binding of EKB-569 (and of the related
A number of observations indicate that in addition to confer-
ring drug resistance, the gatekeeper mutation may derepress the
catalytic activity of EGFR and other kinases. A germ-line
T790M mutation has been discovered in a family with a hered-
itary predisposition to lung cancer, suggesting that this mutation
confers a growth advantage in the absence of the selective
pressure of TKIs (21). Consistent with this idea, introduction of
the T790M in tandem with the L858R mutant in NIH 3T3 cells
increases EGFR activity and enhances the transformed pheno-
type (22). Transgenic mice engineered with lung-specific expres-
sion of the T790M mutant develop lung adenocarcinomas (23),
albeit with a longer latency than those harboring the L858R or
combined L858R and T790M mutations (23, 24). The EGFR
T790M mutation was also identified in an untreated case of
Barrett’s esophagus and the corresponding adenocarcinoma
(25). Interestingly, the corresponding mutation in BCR-Abl
Author contributions: C.-H.Y., K.E.M., A.V.T., M.S.W., H.G., M.M., and M.J.E. designed
analyzed data; and C.-H.Y. and M.J.E. wrote the paper.
Conflict of interest statement: M.J.E. and M.M. are consultants for and receive research
funding from Novartis Institutes for Biomedical Research.
This article is a PNAS Direct Submission.
Data deposition: The crystallographic coordinates and structure factors have been depos-
ited in the Protein Data Bank, www.pdb.org (PDB ID codes 2JIT, 2JIU, and 2JIV).
**To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
February 12, 2008 ?
vol. 105 ?
(T315I) confers resistance to imatinib and other TKIs in the
treatment of chronic myelogenous leukemia and has also been
found to preexist in untreated CML (26, 27). The equivalent
mutation is found in v-Src (T338I) and has long been known to
confer transforming activity on c-Src (28). Despite the long
history of interest in this key residue in control of tyrosine kinase
activity, a structural understanding of its effects is lacking. To
better understand its role in inhibitor resistance and kinase
binding mode in the EGFR kinase. HKI-272 and EKB-569 are examples of irreversible inhibitors. Lapatinib and HKI-272 are thought to require the inactive
conformation of EGFR for binding because of their additional aniline substitutions.
Chemical structures of selected EGFR inhibitors. All compounds are drawn in a consistent orientation and conformation that reflects their approximate
Yun et al.
February 12, 2008 ?
vol. 105 ?
no. 6 ?
deregulation, we have studied the structural and enzymological
effects of the T790M mutation in the context of both the WT and
the L858R-mutant EGFR kinases.
T790M Mutants Bind Gefitinib with Low Nanomolar Affinity. We first
measured binding of gefitinib to the WT, L858R, T790M, and
L858R/T790M mutants by using a direct binding assay in which
intrinsic fluorescence of EGFR is quenched by titration with
the inhibitor (8). Strikingly, the T790M mutation only mod-
estly affects binding of gefitinib in the context of the L858R
mutant (Table 1). The resistant L858R/T790M double-binds
gefitinib with Kd? 10.9 nM, which is only ?4-fold weaker than
the exquisitely sensitive L858R mutant (Kd ? 2.4 nM). The
T790M mutant binds gefitinib with Kd ? 4.6 nM, nearly as
tightly as the L858R mutant and considerably tighter than the
WT kinase. The small difference in gefitinib affinity caused by
introduction of the secondary T790M mutation is in stark
contrast to the roughly two orders of magnitude differences
observed in the sensitivity of cell lines bearing the L858R vs.
L858R/T790M or exon 19 deletions with T790M mutations
(13–15, 29), and therefore cannot explain the clinically ob-
served drug resistance. We also examined binding of the
pyrrolopyrimidine compound AEE788 (Novartis Pharmaceu-
ticals), which binds in a manner similar to gefitinib despite the
difference in chemical scaffold (8). The T790M mutant has a
more dramatic effect on the affinity for AEE788, but notably
the L858R/T790M double mutant retains 18.6 nM affinity for
this compound (as compared with Kd? 1.1 nM for the L858R
mutant). The larger effect on AEE788 as compared with
gefitinib is not unexpected because the phenethylamine sub-
stituent on this inhibitor extends further into the hydrophobic
pocket that is ‘‘guarded’’ by the gatekeeper residue (8).
Crystal Structures of T790M Mutant. Crystal structures of the
T790M mutant show how inhibitors are accommodated in the
presence of the gatekeeper mutation, in both the active and
inactive conformations of the kinase. We determined struc-
tures of the T790M mutant alone and in complex with the
irreversible inhibitor HKI-272 in the inactive conformation or
in complex with AEE788 in the active conformation [see
supporting information (SI) Table 3 for crystallographic sta-
tistics]. The structure of the T790M mutant in complex with
AEE788 is shown in Fig. 2A. The compound binds in essen-
tially the same manner observed in the WT enzyme, with the
pyrrolopyrimidine core making two hydrogen bonds with the
hinge region of the kinase and the phenethylamine substituent
extending into the gatekeeper hydrophobic pocket. Compar-
ison with the binding of AEE788 to the WT enzyme reveals
only a small rotation of the phenethylamine substituent, which
is in direct contact with the mutant gatekeeper residue.
Comparison of the AEE788 complex with the structure of the
T790M mutant in the absence of inhibitor (Apo-T790M) shows
that the Met-790 side chain must adopt a different rotamer to
accommodate the inhibitor (Fig. 2B).
The irreversible inhibitor HKI-272 is a 4-(arylamino)quino-
line-3-carbonitrile compound and a potent inhibitor of both
EGFR and ErbB2 kinases (14, 30). In complex with HKI-272,
the EGFR kinase adopts an inactive conformation in which the
regulatory C-helix is displaced from its active position (Fig.
2C). The enlarged hydrophobic pocket created by the outward
rotation of the C-helix appears to be required to accommodate
the bulky aniline substituent found in HKI-272. Both HKI-272
and lapatinib contain additional aromatic groups appended to
the aniline ring (a 2-pyridinyl group in HKI-272 and a fluoro-
phenyl group in lapatinib; Fig. 1). Thus it is not surprising that
HKI-272, like lapatinib, binds the inactive conformation of the
kinase and that the overall binding mode of the two com-
pounds is similar (Fig. 2D). The quinoline core of HKI-272
forms a single hydrogen bond with the hinge region of the
kinase in a manner analogous to anilinoquinazoline com-
pounds (31, 32). The 2-pyridinyl group of HKI-272 is sur-
rounded by hydrophobic residues in the expanded pocket,
including Met-766 in the C-helix, Phe-856, and Met-790, the
mutant gatekeeper residue. The nitrile substituent of HKI-272
also approaches the gatekeeper residue (extending to ?3 Å
from the methionine side chain). In addition to these nonco-
valent interactions of HKI-272, the expected covalent bond is
formed between Cys-797 at the edge of the active site cleft and
the crotonamide Michael-acceptor group on the inhibitor,
rendering binding irreversible (Fig. 2C). Although the reso-
lution of the structure is modest, electron density for the
inhibitor and for the covalent bond is clear (SI Fig. 4).
The structure of the T790M mutant also suggests a possible
mechanism of catalytic activation. We hypothesize that the
mutation facilitates interconversion between the inactive and
active conformations via direct interaction with the Asp-Phe-Gly
sequence (DFG motif) at the base of the kinase activation loop
(see SI Figs. 5 and 6 and related discussion in SI Text). The
mutation may also enhance the stability of the active confor-
mation (relative to the inactive), as it makes favorable hydro-
phobic interactions with Met-766 and Leu-777 in the active state.
Increased ATP Affinity of the L858R/T790M Mutant Confers Drug
Resistance. The binding data and crystal structures clearly dem-
onstrate that the gatekeeper mutation does not sterically block
binding of reversible inhibitors. Why then does the T790M
mutation confer resistance? Kinetic characterization of the WT
and mutant EGFR kinases reveals a marked decrease in the
Michaelis-Menten constant (Km) for ATP in the drug-resistant
L858R/T790M mutant as compared with the drug-sensitive
activates EGFR, but also reduces the apparent affinity for ATP
(Table 2). Strikingly, the T790M mutation restores the ATP
affinity to near WT levels in the L858R/T790M double mutant
(Km[ATP]? 8.4 ?M, as compared with Km[ATP]? 148 ?M for the
L858R mutant). In isolation, the T790M mutation does not
significantly affect ATP affinity. We cannot explain structurally
why the T790M mutation increases ATP affinity in the context
of the L858R mutant, but not in the context of the WT enzyme.
We also find that the T790M mutation activates the kinase
?5-fold as compared with the WT enzyme (Table 2); this
catalytic activation of the T790M mutant likely explains its
presence as a germ-line mutation in a family predisposed to lung
cancer (21). Although the L858R/T790M mutant has a modestly
decreased kcatrelative to the L858R mutant, it is still much more
active than the WT enzyme and also exhibits a 5-fold higher
kcat/Km[ATP]than the L858R mutant (Table 2).
Because TKIs such as gefitinib must compete with ATP for
binding to the kinase active site, the enhanced ATP affinity is
expected to decrease the apparent inhibitor potency. In the
L858R/T790M mutant this ‘‘Kmeffect’’ combines with the small
difference in binding affinity for gefitinib to dramatically de-
Table 1. Inhibitor dissociation constants for the WT and mutant
Gefitinib AEE788Gefitinib AEE788
35.3 ? 0.4
4.6 ? 0.1
2.4 ? 0.1
10.9 ? 0.6
5.3 ? 0.3
27.6 ? 0.7
1.1 ? 0.1
18.6 ? 0.5
The ratio Kd/Km[ATP]provides a relative estimate of inhibitor potency.
www.pnas.org?cgi?doi?10.1073?pnas.0709662105 Yun et al.
crease inhibitor sensitivity at cellular concentrations of ATP.
The expected potency of gefitinib (calculated Ki
a function of ATP concentration for the L858R and L858R/
T790M mutants in Fig. 3A. Whereas the L858R mutant main-
tains low-nanomolar sensitivity to gefitinib at cellular ATP
concentrations (?1 mM), the L858R/T790M mutant does not.
This predicted loss of inhibitor sensitivity in the L858R/T790M
mutant is confirmed by direct in vitro measurement of enzyme
inhibition by gefitinib at 10 ?M vs. 1 mM concentrations of ATP.
The L858R/T790M mutant is sensitive to gefitinib at 10 ?M
ATP, but resistant at 1 mM, whereas the L858R mutant is
effectively inhibited even at the higher concentration, which
approximates the cellular level of ATP (Fig. 3 B and C). We also
observe this effect in other assays using polyE4Y or the signaling
adapter Shc as an EGFR substrate and in the presence of either
Mn2?or Mg2?(data not shown). We conclude that the clinically
observed resistance of L858R/T790M mutant stems largely from
app) is plotted as
its enhanced affinity for ATP (as compared with the inhibitor-
sensitive L858R mutant) and not from a steric block of inhibitor
binding as previously hypothesized.
The present work highlights the extent to which the compro-
mised ATP affinity of the EGFR mutants renders them suscep-
tible to inhibition (at least for the L858R, G719S, and exon19
deletions that have been studied to date) (8, 10). The T790M
mutation merely restores ATP affinity to the level of the WT
kinase. In effect, the diminished ATP affinity of the oncogenic
mutants open a ‘‘therapeutic window,’’ which renders them more
easily inhibited relative to the WT EGFR and other kinases on
which the inhibitors might have activity. The T790M secondary
mutation effectively closes this window by restoring ATP affinity
to WT levels.
The activating nature of the gatekeeper mutation is not
unique to EGFR, as indicated by its effect on Src and its
presence in chronic myelogenous leukemia before imatinib
treatment. The Kmeffect may be more idiosyncratic; note that
the T790M mutation has little effect on Kmin the context of
the WT EGFR kinase (Table 2). For the Abl T315I mutant, we
measure Km[ATP]? 1.8 ?M as compared with Km[ATP]? 6 ?M
for the WT Abl kinase (data not shown), this change in ATP
affinity is expected to have only a modest effect on inhibitor
potency. Also, it is clear that the T315I gatekeeper mutation
in BCR-Abl directly blocks binding of imatinib and other
compounds with a similar binding mode.
(A) Superposition of EGFR T790M/AEE788 complex (yellow) and WT/AEE788 complex [light blue; drawn from PDB ID code 2J6M (8)]. Dashed lines indicate
hydrogen bonds to the kinase hinge region that are preserved in both complexes. The location of the T790M mutation is indicated. (B) Superposition of EGFR
T790M/AEE788 complex (yellow) and apo-T790M structure (green). Note the alternate side-chain conformation of Met-790 in the presence of the inhibitor. (C)
between Cys-797 and the crotonamide Michael acceptor of HKI-272. (D) The structure of the T790M mutant in complex with HKI-272 (yellow) is superimposed
inactive conformation and the inhibitors bind in a similar manner, with a single hydrogen bond to the hinge (dashed lines) and with their aniline substituents
extending into the enlarged hydrophobic pocket that is characteristic of the inactive conformation.
Crystal structures of the EGFR T790M mutant show that inhibitors are readily accommodated in the active and inactive conformations of the kinase.
Table 2. Enzyme kinetic parameters of WT and mutant EGFR
5.2 ? 0.2
5.9 ? 0.1
148 ? 4
8.4 ? 0.3
Yun et al.
February 12, 2008 ?
vol. 105 ?
no. 6 ?
Our findings explain the puzzling observation that irreversible
anilinoquinazoline inhibitors (and closely related irreversible
compounds such as HKI-272) maintain efficacy against the
T790M resistance mutant, and additionally they are important
for understanding the nature of resistance and possible avenues
to the development of more effective drugs. The fact that the
T790M substitution confers resistance by increasing the affinity
for ATP, rather than by simply sterically interfering with inhib-
itor binding, means that T790M is a ‘‘generic’’ resistance mutant;
To our knowledge, this mechanism of drug resistance, resistance
conferred by a mutation that increases affinity for a competing
physiologic substrate, has not been previously documented in a
clinical context. Interestingly, a distinct, but related, effect has
recently been described in a mutant of the mitotic kinesin KSP
(also called Eg5). The KSP mutant was discovered in a labora-
tory screen and confers drug resistance by an allosteric mech-
anism involving enhanced affinity for ATP (33).
As a class, irreversible inhibitors can overcome T790M resis-
tance through covalent binding; once covalently bound, they are
no longer in a competitive, reversible equilibrium with ATP. A
number of such compounds are currently in clinical trials in
oncology, including HKI-272, but none have yet received ap-
proval. One concern with covalent inhibitors is the potential for
toxicity caused by off-target effects. At least 10 kinases in
equivalent to Cys-797 in EGFR, so it will be important to
understand the activity of available irreversible agents against
these kinases in particular, which include Tec family kinases,
JAK3, and other kinases important for hematopoietic develop-
ment and immune function. However, our results indicate that
irreversible binding is not required for effective inhibition of the
T790M mutant. A reversible inhibitor that binds with sufficient
affinity to outcompete ATP should work as well. Calculations
similar to those shown in Fig. 3A indicate that reversible
inhibitors with affinity of ?200 pM or tighter against the T790M
mutant should be effective.
of the human EGFR and bearing the WT sequence or the T790M and L858R
mutations were expressed and purified by using a baculovirus/insect cell
system as described (8). Crystals of the T790M mutant were obtained in 0.1 M
Hepes (pH 7.5), 21% PEG6000, 0.3 M NaCl, and 5 mM tris(2-carboxyethyl)-
cocrystallization in 0.1 M Hepes (pH 7.0), 0.2 M Li2SO4, 28% PEG3350, and 5
mM TCEP. T790M/AEE788 complex crystals were made by soaking the apo-
T790M crystals in 300 ?M AEE788 inhibitor overnight.
Structure Determination and Refinement. Diffraction data were collected at
G719S structure [Protein Data Bank (PDB) ID code 2itn] (8) for apo-T790M and
(9) for the T790M/HKI-272 structure. CNS/simulated-annealing (36) was then
adjustment of the model. Repeated rounds of manual refitting and crystal-
lographic refinement were performed by using COOT (37) and refmac5 (38).
Inhibitors were modeled into the closely fitting positive Fo ? Fc electron
density and then included in the following refinement and fitting cycles.
Topology and parameter files for the inhibitors were generated by using
Enzyme Kinetic Assays, Inhibition Assays, and Data Analysis. EGFR kinetic
parameters were determined in triplicate by using the ATP/NADH coupled
assay system in a 96-well format as described (8). The reaction mixture con-
tained 0.5 mg/ml BSA, 2 mM MnCl2, 1 mM phospho(enol) pyruvic acid (PEP;
Sigma-Aldrich; catalogue no. P7002), 1 mM TCEP, 0.1 M MOPS 7.5, 5 mM
reaction mixture volume of pyruvate kinase/lactic dehydrogenase enzymes
at cellular concentrations of ATP. (A) The calculated Ki
mutant and L858R/T790M double mutant are plotted versus ATP concentra-
tion, using experimentally measured values for the Km[ATP] (Table 2) and
double mutant (solid line) as ATP concentrations approach cellular levels (?1
mM). (B) Inhibition of L858R mutant EGFR kinase by gefitinib in the presence
of 10 ?M (black squares) or 1.0 mM ATP (red circles). (C) Inhibition of the
squares) or 1.0 mM ATP (red circles). In B and C, the in vitro kinase activity of
the indicated EGFR mutant was measured in the presence of the indicated
concentrations of gefitinib by using an EGFR Tyr-1173 autophosphorylation
site peptide (ENAEYLRVA) as substrate.
The drug resistance of T790M secondary mutation is manifested only
appfor the L858R single
www.pnas.org?cgi?doi?10.1073?pnas.0709662105Yun et al.
fromrabbitmuscle(Sigma-Aldrich;catalogueno.P-0294),0.5mMNADH,and Download full-text
0.5 ?M EGFR kinase; ATP at varied concentration was added last to start the
reaction. Steady-state initial velocity data were drawn from the slopes of the
A340curves and fit to the Michaelis-Menten equation to determine Vmand Km
values. To assure that our derived kcatparameters reflected concentrations of
preparation by titration of the samples with the tight binding inhibitor
gefitinib or AEE788 (see below).
Inhibition assays were carried out by using the same kinetic assay method,
with 10 mM MgCl2 and 1.25 mM EGFR autophosphorylation site peptide
(ENAEYLRVA) as the phospho-acceptor substrate. The ATP concentration was
fixed at 10 ?M or 1 mM, and the indicated concentrations of the inhibitors
were added before the addition of ATP.
Binding Constant Assay and Calculation of the Ki
estimate the active enzyme concentration as described (8). Fluorescence mea-
appValues. The equilibrium
surements were carried out in a nitrogen-sparged buffer containing 20 mM
Km,ATPwere used to calculate the Ki
appvalues using the following equation
app? Ki?1 ? ?ATP?/Km,ATP?,
assuming that the Kdvalues obtained in the binding assays are equal to Ki
under the condition of the above kinetic assays.
ACKNOWLEDGMENTS. We thank G. Caravotti (Novartis) and S. Rabindran
(Wyeth Pharmaceuticals) for the compounds AEE788 and HKI-272, respec-
tively; R. Copeland and C. Walsh for comments on the manuscript; and Y. Li
and F. Poy for technical assistance. This work was supported by National
Institutes of Health Grants CA080942 (to M.J.E.) and CA116020 (to M.M.).
M.J.E. is the recipient of a Scholar Award form the Leukemia and Lymphoma
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