Therapeutic strategies to overcome crizotinib resistance
in non-small cell lung cancers harboring the fusion
oncogene EML4-ALK
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Citation
Katayama, R. et al. “Therapeutic strategies to overcome
crizotinib resistance in non-small cell lung cancers harboring the
fusion oncogene EML4-ALK.” Proceedings of the National
Academy of Sciences 108 (2011): 7535-7540. Web. 2 Dec.
2011. © 2011 New York Academy of Sciences
As Published
http://dx.doi.org/10.1073/pnas.1019559108
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National Academy of Sciences
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Final published version
Accessed
Thu May 26 23:33:32 EDT 2016
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http://hdl.handle.net/1721.1/67357
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Detailed Terms
Therapeutic strategies to overcome crizotinib
resistance in non-small cell lung cancers harboring
the fusion oncogene EML4-ALK
Ryohei Katayamaa,b, Tahsin M. Khana,c, Cyril Benesa,b, Eugene Lifshitsa, Hiromichi Ebia,b, Victor M. Riverad,
William C. Shakespeared, A. John Iafrateb,e, Jeffrey A. Engelmana,b,1, and Alice T. Shawa,b,c,1
a
Massachusetts General Hospital Cancer Center, Boston, MA 02129; bDepartment of Medicine, Harvard Medical School, Boston, MA 02115; cKoch Institute for
Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; dARIAD Pharmaceuticals, Cambridge, MA 02139; and
e
Department of Pathology, Massachusetts General Hospital, Boston, MA 02114
The echinoderm microtubule-associated protein-like 4 (EML4)anaplastic lymphoma kinase (ALK) fusion oncogene represents
a molecular target in a small subset of non-small cell lung cancers
(NSCLCs). This fusion leads to constitutive ALK activation with
potent transforming activity. In a pivotal phase 1 clinical trial, the
ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients
with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical
benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by
exposing a highly sensitive EML4-ALK–positive NSCLC cell line to
increasing doses of crizotinib until resistance emerged. We found
that cells resistant to intermediate doses of crizotinib developed
amplification of the EML4-ALK gene. Cells resistant to higher doses
(1 μM) also developed a gatekeeper mutation, L1196M, within the
kinase domain, rendering EML4-ALK insensitive to crizotinib. This
gatekeeper mutation was readily detected using a unique and
highly sensitive allele-specific PCR assay. Although crizotinib was
ineffectual against EML4-ALK harboring the gatekeeper mutation,
we observed that two structurally different ALK inhibitors, NVPTAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells
remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus,
we have developed a model of acquired resistance to ALK inhibitors
and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring
secondary gatekeeper mutations.
F
irst described in 2007, the oncogenic fusion kinase echinoderm
microtubule-associated protein-like 4-anaplastic lymphoma
kinase (EML4-ALK) is present in ∼4% of patients with non-small
cell lung cancer (NSCLC) (1). Chromosomal translocations involving ALK also occur in other cancers, including anaplastic
large cell lymphomas and inflammatory myofibroblastic tumors.
In all cases, the fusion partner (e.g., EML4) is believed to mediate
ligand-independent oligomerization of ALK, resulting in constitutive ALK kinase activation (2–4). In cell line and genetically
engineered mouse models, EML4-ALK serves as a potent oncogenic “driver,” and cancers with this translocation are highly
sensitive to ALK kinase inhibition (5, 6). Recently, a tyrosine
kinase inhibitor (TKI) targeting ALK, crizotinib (PF-02341066),
was examined in a phase 1 trial (7). Among 105 patients with
EML4-ALK–positive NSCLC, crizotinib showed remarkable activity, with an objective response rate of 56% and a median progression-free survival of 9.2 mo (7, 8). These results support the
notion that lung cancers harboring EML4-ALK are highly susceptible to ALK-targeted therapies and demonstrate the properties of “oncogene addiction” to ALK.
Although many patients derive substantial clinical benefit, the
development of drug resistance has curbed the impact of crizo-
www.pnas.org/cgi/doi/10.1073/pnas.1019559108
tinib in this disease. The paradigm of acquired TKI resistance has
been seen with other targeted therapy successes, including epidermal growth factor receptor (EGFR) mutant lung cancers and
v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutant
melanomas (9–16). Similar to the experience with crizotinib,
sensitive cancers ultimately develop resistance, usually within 1 y.
For example, most patients with EGFR-mutant NSCLC will have
disease progression after 9–12 mo of EGFR TKI monotherapy.
In about one-half of cases, resistance arises due to the acquisition
of a secondary gatekeeper mutation (T790M) within the EGFR
(13, 15, 16). This mutation leads to substitution of a threonine in
the ATP-binding pocket with a bulky methionine residue, which
hinders drug binding. In about 10–20% of cases, resistance arises
as a result of focal amplification of the MET proto-oncogene (9,
16, 17). Importantly, preclinical modeling of acquired resistance to
EGFR TKIs has proven invaluable, not only for accurately predicting the resistance mechanisms that emerge in the clinic, but
also for assessing new therapeutic strategies to overcome resistance (12, 16–22).
Recently, one NSCLC patient with acquired resistance to crizotinib was described (23). In this patient who relapsed after 5 mo
of treatment, molecular analyses revealed that the resistant tumor
cells harbored two secondary mutations within the kinase domain
of EML4-ALK, C1156Y, and the gatekeeper mutation L1196M.
These mutations occurred independently in distinct subclones
of the resistant tumor. Studies of Ba/F3 cells engineered to
express EML4-ALK harboring either mutation suggested that
these mutants were resistant to crizotinib as well as resistant to a
more potent ALK TKI, termed 2,4-pyrimidinediamine derivative
(PDD). Thus, strategies to overcome this type of resistance have
not yet been established. In addition, because there are no laboratory models of acquired resistance via this mechanism, it
remains unknown if cancers that develop these resistance mutations remain addicted to ALK kinase activity and will be highly
sensitive to other therapeutics targeting ALK.
In this study, we have explored resistance to crizotinib by generating and characterizing a unique cell line model of acquired
resistance. In this model of acquired resistance, EML4-ALK is
amplified and harbors the L1196M gatekeeper mutation. We
show that cells expressing the L1196M mutant form of EML4ALK are resistant to crizotinib, remain addicted to ALK signaling,
Author contributions: R.K., J.A.E., and A.T.S. designed research; R.K., T.M.K., E.L., H.E.,
V.M.R., W.C.S., and A.J.I. performed research; R.K., C.B., V.M.R., W.C.S., and A.J.I. contributed new reagents/analytic tools; R.K., T.M.K., C.B., A.J.I., J.A.E., and A.T.S. analyzed data;
and R.K., J.A.E., and A.T.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence may be addressed. E-mail: jengelman@partners.org or
ashaw1@partners.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1019559108/-/DCSupplemental.
PNAS | May 3, 2011 | vol. 108 | no. 18 | 7535–7540
MEDICAL SCIENCES
Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved March 23, 2011 (received for review December 27, 2010)
cells, we detected a C → A substitution at nucleotide 3586 of
EML4-ALK variant 1 (Fig. 2B) that was not detected in the parental cells. This 3586C → A substitution results in a leucine →
methionine change within the ALK TK domain, corresponding to
the L1196M gatekeeper mutation previously reported in a patient
with acquired resistance to crizotinib (23). We did not detect any
other TK mutations including the C1156Y substitution, which was
also reported in the same patient (23). Consistent with the notion
that this L1196M mutation confers resistance to crizotinib, Ba/F3
cells engineered to express this mutant were also resistant to
crizotinib, whereas cells expressing wild-type EML4-ALK were
sensitive (SI Appendix, Fig. S2). Notably, in the DNA sequence
tracings, the peak corresponding to the mutation is approximately
one-half to one-third as high as the wild-type peak (Fig. 2B). This
suggests that only a fraction of the amplified EML4-ALK fusion
genes acquire the secondary resistance mutation.
These resistant models were developed by stepwise increases
in drug concentration until fully resistant cells emerged. Because
both amplification and mutation were identified in H3122 CR cells,
we sought to investigate the temporal relationship between gene
amplification and secondary mutation in acquired crizotinib resistance. Thus, we analyzed H3122 CR0.6 cells, the intermediate,
partially resistant predecessors of H3122 CR cells that were resistant to 600 nM crizotinib. By Western blotting analysis, ALK
protein level and p-ALK were up-regulated in H3122 CR0.6 cells
(SI Appendix, Fig. S3). FISH analysis demonstrated amplification
of the EML4-ALK fusion gene in the H3122 CR0.6 cells (Fig. 2A).
However, they did not harbor a secondary ALK resistance mutation (Fig. 2B). To determine whether L1196M is present at a low
level in a subpopulation of H3122 CR0.6 cells, we developed a
highly sensitive gatekeeper mutation-specific PCR assay that can
detect the L1196M mutation when it represents at least 1% of the
mutated ALK alleles (SI Appendix, Fig. S4). Using this assay, we
detected L1196M in H3122 CR cells and in all 11 clones derived
from single H3122 CR cells (SI Appendix, Fig. S5), but not in the
H3122 CR0.6 cells, the parental H3122 cells, or the other NSCLC
cell lines harboring EML4-ALK (Fig. 2C and SI Appendix, Fig. S5).
These results suggest that the L1196M mutation is present in each
cell of the resistant H3122 CR cells and that there is a stepwise
model of acquired resistance involving amplification of the target,
which confers resistance to 600 nM crizotinib, followed by sec-
1000 nM
M
non
1%
50
H3122 CR
crizotinib(6hr)
100 nM
H3122
crizotinib (6hr)
crizotinib (200nM), n=704
300 nM
C
B
100
pALK
(pY1604)
ALK
pAKT
(pS473)
H460
BT474
SKBR3
H3122 CR
H522
H1299
A549
panAKT
0
H3122
cell number (% of control)
A
30 nM
EML4-ALK Gene Is Amplified and Mutated in H3122 CR Cells. To determine whether gene amplification underlies the elevated protein
level of EML4-ALK in H3122 CR cells, we performed fluorescence in situ hybridization (FISH) analysis. Relative to parental
H3122 cells, H3122 CR cells showed an increase in the number of
rearranged EML4-ALK genes per cell (Fig. 2A).
Because the phosphorylation of EML4-ALK in H3122 CR cells
is not impacted by 1 μM crizotinib (Fig. 1C), we hypothesized
that resistant cells might also harbor a mutation within ALK. We
prepared cDNA and examined the entire coding sequence of
EML4-ALK in H3122 parental and resistant cells. In the resistant
1000 nM
M
non
and is highly sensitive to treatment with crizotinib. To explore
mechanisms of crizotinib resistance, we generated resistant
H3122 clones by exposing the sensitive parental cells to increasing
concentrations of crizotinib for 4 mo. We maintained cells with
intermediate crizotinib resistance, referred to as H3122 CR0.6, in
600 nM of crizotinib. The fully resultant cells, H3122 crizotinibresistant (H3122 CR) cells, were maintained in 1 μM of crizotinib.
H3122 CR cells were as resistant to crizotinib as other cancer
cell lines that did not harbor ALK gene rearrangements (IC50 > 1
μM; Fig. 1A and SI Appendix, Fig. S1A). In addition, we assessed
704 established cancer cell lines derived from a wide variety of
tumor types for sensitivity to crizotinib as part of our automated
platform to examine drug sensitivity across multiple cell lines
(24). Whereas H3122 parental cells segregated with the most
sensitive cell lines, the H3122 CR cells segregated with the highly
resistant cell lines (Fig. 1B). Unlike the parental H3122 cells, the
H3122 CR cells maintained ALK phosphorylation in the presence
of crizotinib (Fig. 1C). Accordingly, AKT and ERK phosphorylation were not suppressed by crizotinib in the resistant cells.
Notably, H3122 CR cells also contained higher total protein
levels of EML4-ALK compared with the parental line (Fig. 1C).
300 nM
Generation and Biochemical Characterization of Crizotinib-Resistant
Cells. The NSCLC cell line H3122 expresses EML4-ALK variant 1
100 nM
Results
30 nM
and are highly sensitive to other structurally distinct ALK TKIs
as well as to Hsp90 inhibition. On the basis of these results, we
propose two therapeutic strategies for overcoming acquired resistance to crizotinib, particularly when mediated by secondary
mutations within the ALK TK domain.
<0
0.2
2
0.2 - 0.5
0.5 - 0.75
> 0.75
pERK
ERK
Actin
Fig. 1. H3122 CR cells are resistant to crizotinib. (A) Cell lines were seeded in 96-well plates and treated with 1 μM of crizotinib for 72 h. Cell survival was
analyzed using the CellTiter-Glo viability assay. In contrast to the parental H3122 cells, which are highly sensitive to crizotinib (red bar), H3122 CR cells (blue
bar) are as insensitive to crizotinib as non-ALK rearranged cell lines. (B) Pie chart representation of the sensitivity of H3122 and H3122 CR cells compared with
704 other human cancer cell lines assessed by an automated cell viability assay to 200 nM crizotinib. Viability at 72 h was calculated as a ratio of viable
crizotinib-exposed cells to viable DMSO-treated cells. The color scheme corresponds to the relative inhibitory effect of treatment. The chart depicts the most
sensitive cell lines in order of decreasing sensitivity (from top to bottom). (C) H3122 parental and H3122 CR cells were treated with crizotinib at the indicated
concentration for 6 h. Cell extracts were immunoblotted to detect the indicated proteins.
7536 | www.pnas.org/cgi/doi/10.1073/pnas.1019559108
Katayama et al.
A
H3122
B
Forward
H3122 CR
H3122 CR0.6
Reverse
C
water
H2228
MGH006
M
H3122 CR0.6
3122 CR
H3
H3122
22 CR0.6
H312
H3122
H3122 CR
Leu (CTG)
Met (ATG)
Katayama et al.
B
si-ALK
A549
si-control
si-ALK
si-ALK
ol
si-contro
H3122
CR
ol
si-contro
H3122
100
pALK
(pY1604)
50
ALK
H3122
H3122 CR
si-A
ALK
si-c
cntl
Actin
ALK
si-A
0
si-c
cntl
the gatekeeper L1196M mutation causes resistance by steric interference with crizotinib binding (23). Thus, although resistant to
crizotinib, this mutant ALK may be sensitive to structurally distinct ALK kinase inhibitors. To address this hypothesis, we tested
the activity of the ALK inhibitor NVP-TAE684, a 5-chloro-2,4diaminophenylpyrimidine, in parental and resistant H3122 cells.
As shown in Fig. 4, NVP-TAE684 markedly reduced cell survival
in both sensitive H3122 and H3122 CR cells, but had little to no
effect on the viability of other, non-ALK–dependent cancer cell
lines (Fig.4A and SI Appendixx, Fig. S1B). Within the panel of 704
established cancer cell lines assessed in the automated platform
(24), the vast majority were largely insensitive to 200 nM NVPTAE684; however, 1% of lines displayed marked sensitivity to
NVP-TAE684. Both the H3122 and the H3122 CR cell lines
segregate with the highly sensitive cell lines (Fig. 4B). This is in
sharp contrast to the resistance of H3122 CR cells to crizotinib
A
si-A
ALK
NVP-TAE684 and AP26113 Overcome Crizotinib Resistance in H3122 CR
Cells. Based on the crystal structure of the kinase domain of ALK,
cntl
si-c
H3122 CR Cells Are Addicted to ALK. To determine if H3122 CR cells
continue to require EML4-ALK for their viability, we used siRNAs
targeting ALK to knock down EML4-ALK. In both H3122 parental and H3122 CR cell lines, ALK-specific siRNA decreased expression of EML4-ALK and potently suppressed cell growth (Fig.
3 A and B). As expected, the ALK siRNA had no effect on the
growth of KRAS mutant A549 cells (Fig. 3 A and B). These results
demonstrate that resistant H3122 CR cells expressing EML4-ALK
L1196M remain addicted to ALK signaling.
(Fig. 1B). In contrast to crizotinib, NVP-TAE684 treatment of
H3122 CR cells suppressed phosphorylation of ALK, AKT, and
ERK and induced marked apoptosis (Fig. 4C and SI Appendix,
Fig. S6). Similarly, NVP-TAE684 potently suppressed the survival
of Ba/F3 cells expressing the EML4-ALK L1196M mutant (SI
Appendix, Fig. S7A). However, in this system, the potency of
NVP-TAE684 against mutant EML4-ALK was slightly reduced
relative to native EML4-ALK, with IC50 values of 2.7 versus 1.2
nM, respectively.
cell number (% of control)
ondary gatekeeper mutation that renders cells resistant to 1 μM
crizotinib.
A549
Fig. 3. Resistant H3122 CR cells are oncogene-addicted. (A) The indicated
cell lines were transfected with small interfering RNA (siRNA) against ALK
(siALK) or control siRNA, and cell viability was measured after 72 h. (B) Immunoblotting of protein lysates 72 h after knockdown showing reduced
levels of phosphorylated ALK and total ALK in siALK-transfected cells.
PNAS | May 3, 2011 | vol. 108 | no. 18 | 7537
MEDICAL SCIENCES
Fig. 2. EML4-ALK is both amplified and mutated in resistant H3122 CR cells. (A) Dual-color FISH [ALK-N-terminal (green)/ALK-C-terminal (red)] analysis was
performed on H3122 parental, H3122 CR0.6, and H3122 CR cells. Arrows indicate the split ALK C-terminal region (EML4-ALK). Both H3122 CR0.6 and H3122 CR
cells demonstrate amplification of EML4-ALK. (B) Secondary L1196M (gatekeeper) mutation in resistant H3122 CR cells. Shown are electrophoretograms of
EML4-ALK cDNA from H3122 parental, H3122 CR0.6, and H3122 CR cells. The 1903C → A mutation within exon 23 results in a L635M substitution that
corresponds to the L1196M gatekeeper mutation of ALK. (C) Detection of the gatekeeper mutation using an L1196M mutation-specific PCR assay. Shown is
the amplified 160-bp product in H3122 CR cells after 35 PCR cycles. This product is not detected in parental H3122 or H3122 CR0.6 cells or in other EML4-ALK–
positive cell lines (H2228, MGH006).
300 nM
3 nM
10 nM
30 nM
100 nM
non
30 nM
100 nM
300 nM
10 nM
non
50
pERK
BT474
ERK
< 0.2
0.2 - 0.5
0.5 - 0.75
> 0.75
E
Actin
H3122 CR
H3122
3 nM
10 nM
30 nM
100 nM
300 nM
crizotinib (1uM)
non
3 nM
10 nM
30 nM
100 nM
300 nM
crizotinib (1uM)
H460
H522
SKBR3
H1299
A549
H3122 CR
pAKT
(pS473)
panAKT
D
non
AP26113 (6hr)
100
pALK
(pY1604)
50
pAKT
(pS473)
AP26113 (6hr)
ALK
BT474
SKBR3
H460
A549
H1299
H522
0
H3122 CR
panAKT
AKT
H3122
cell number (% of control)
H3122 CR
TAE684 (6hr)
pALK
(pY1604)
ALK
H3122
ce
ell number (% of c o
ontrol)
TAE684 (200nM), n=704
100
0
H3122
TAE684 (6hr)
C
3 nM
B
A
pERK
ERK
Actin
Fig. 4. Two different ALK kinase inhibitors, NVP-TAE684 and AP26113, overcome crizotinib resistance in H3122 CR cells. (A) Cell lines were seeded in 96-well
plates and treated with 100 nM of TAE684 for 72 h. Cell survival was analyzed using the CellTiter-Glo viability assay. Both parental H3122 cells (red bar) and
H3122 CR cells (blue bar) show marked sensitivity to TAE684. Non-ALK rearranged cell lines (A549, H1299, SKBR3, H522, H460, and BT474 cells) show minimal
growth inhibition with TAE684. (B) Pie chart representation of the sensitivity of H3122 and H3122 CR cells compared with 704 other human cancer cell lines
assessed by an automated cell viability assay to 200 nM TAE684. Viability at 72 h was calculated as a ratio of viable crizotinib-exposed cells to viable DMSOtreated cells. The chart depicts the most sensitive cell lines in order of decreasing sensitivity (from top to bottom). Both parental H3122 and H3122 CR cells are
among the 1% of the most sensitive cell lines to TAE684. (C) Suppression of ALK signaling by TAE684 in resistant H3122 CR cells. H3122 parental and resistant
cells were exposed to increasing concentrations of TAE684 for 6 h. Cell lysates were immunoblotted to detect the indicated proteins. (D) Sensitivity of H3122
CR cells to AP26113. Cell lines were seeded in 96-well plates and treated with 300 nM of AP26113 for 72 h. Cell survival was analyzed using the CellTiter-Glo
viability assay. Both parental H3122 cells (red bar) and H3122 CR cells (blue bar) show marked sensitivity to AP26113, compared with non-ALK rearranged cell
lines. (E) Suppression of ALK signaling by AP26113 in resistant H3122 CR cells. H3122 parental and resistant cells were exposed to increasing concentrations of
AP26113 for 6 h. Cell lysates were immunoblotted to detect the indicated proteins.
We also tested a second, structurally distinct ALK kinase inhibitor AP26113. A new highly selective small molecule inhibitor
of ALK, AP26113 exhibits about fivefold greater potency in vitro
compared with crizotinib (SI Appendix, Table S1A). Like NVPTAE684, AP26113 was highly active against both sensitive and
resistant H3122 cells, decreasing cell growth (Fig. 4D and SI Appendix, Fig. S1C), suppressing ALK phosphorylation (Fig. 4E), and
inducing apoptosis (SI Appendix, Fig. S6). Higher doses of AP26113
were required to completely suppress ALK in the resistant cells
because there were greater levels of ALK/p-ALK as a result of gene
amplification. AP26113 was also active in Ba/F3 cells expressing
either native or mutant EML4-ALK (IC50’s were 10 and 24 nM,
respectively) (SI Appendix, Fig. S7B). Consistent with the Ba/F3
data, the potency of AP26113 against mutant EML4-ALK was
slightly reduced relative to native EML4-ALK as determined by its
capacity to decrease p-ALK in the H3122 and H3122 CR cells, with
IC50 values of 7.4 versus 16.8 nM, respectively (SI Appendix, Fig.
S8). These results suggest that AP26113 can overcome crizotinib
resistance mediated by the gatekeeper L1196M mutation. Indeed,
7538 | www.pnas.org/cgi/doi/10.1073/pnas.1019559108
we observed impressive in vivo activity of both NVP-TAE684 and
AP26113, but not of crizotinib, against the H3122 CR cells grown as
xenograft tumors in vivo (SI Appendix, Fig. S9).
Hsp90 Inhibitor 17-AAG Overcomes Crizotinib Resistance in H3122 CR
Cells. In a recent phase 2 study, the Hsp90 inhibitor IPI-504
demonstrated clinical activity in three of three patients with ALKpositive NSCLC and induced rapid degradation of EML4-ALK in
vitro (25). Hsp90 inhibitors also induced tumor regression in a
genetically engineered mouse model of lung cancer driven by
EML4-ALK (5). Therefore,we evaluated the efficacy of the Hsp90
inhibitor 17-AAG in EML4-ALK L1196M-expressing cells. As
shown in Fig. 5A, 17-AAG potently suppressed cell growth in both
parental H3122 and H3122 CR cells, but not in other non-ALK–
dependent lung cancer lines (SI Appendix, Fig. S1D). However, in
contrast to NVP-TAE684 and AP26113, 17-AAG also inhibited
the viability of SKBR3 and BT474 cell lines, both of which harbor
HER2 amplification and are known to be sensitive to Hsp90 inhibition (26, 27). In Ba/F3 cells, 17-AAG was active against both
Katayama et al.
ALK
50
pAKT
(pS473)
panAkt
H522
H460
A549
H1299
BT474
SKBR3
pERK
H3122
0
ERK
Actin
Fig. 5. The Hsp90 inhibitor 17-AAG can overcome crizotinib resistance in
H3122 CR cells. (A) Cell lines were seeded in 96-well plates and treated with
10 nM of 17-AAG for 72 h. Cell survival was analyzed using CellTiter-Glo.
Both parental H3122 cells (red bar) and H3122 CR cells (blue bar) show
sensitivity to 17-AAG. (B) Suppression of ALK signaling by 17-AAG in resistant H3122 CR cells. H3122 parental and resistant cells were exposed to
increasing concentrations of 17-AAG for 6 h. Cell lysates were immunoblotted to detect the indicated proteins.
native and mutant EML4-ALK to a similar extent, but showed no
activity in parental Ba/F3 cells (SI Appendix, Fig. S7C). 17-AAG
treatment decreased both p-ALK level and ALK protein expression with similar potencies in the parental and resistant cells (Fig.
5B). These results suggest that Hsp90 inhibition might represent
an alternative therapeutic strategy for overcoming acquired resistance to crizotinib due to acquisition of a resistance mutation.
Discussion
In tumors dependent on a driver kinase such as BCR-ABL or
mutant EGFR, secondary mutation within the kinase domain is
a common mechanism of acquired drug resistance. The most
frequent resistance mutation involves the gatekeeper residue.
Amino acid substitutions at this position hinder drug binding and
thereby confer high-level resistance to many tyrosine kinase
inhibitors. Other mechanisms of acquired drug resistance involve
gene amplification of the kinase target or activation of alternative signaling pathways to bypass the need for kinase activation
(10, 28). Identification of the genetic alterations underlying acquired drug resistance has fostered the development of targeted
agents for patients with TKI resistance. For example, two secondgeneration TKIs—dasatinib and nilotinib—have efficacy against
most of the known imatinib-resistant mutations and are approved
for patients with imatinib-resistant chronic myeloid leukemia
(CML) (29, 30). However, these drugs are not active against the
gatekeeper T315I mutation within ABL (31). Similarly in EGFRmutant lung cancer, second-generation, irreversible EGFR
inhibitors are being explored to treat cancers that develop resistance via the gatekeeper T790M mutation (22, 32).
EML4-ALK–positive NSCLC represents another tyrosine kinasedriven cancer that is highly responsive to TKI therapy. Recently,
two studies have reported the identification of secondary resistance mutations within the ALK TK domain in patients who
relapsed on crizotinib (23, 33). One of the mutations identified
was the gatekeeper L1196M substitution, analogous to T315I in
ABL and T790M in EGFR (23). In this study, we generated a cell
line model of acquired crizotinib resistance by exposing sensitive,
EML4-ALK–positive H3122 cells to increasing concentrations
of crizotinib. The resistant cells harbor both amplification of
EML4-ALK and the same L1196M gatekeeper mutation that was
identified in a patient with acquired resistance. Notably, we did
not identify any other ALK mutations in this cell line, including
C1156Y. Because we did not use more sensitive technologies such
as deep sequencing, we cannot exclude the possibility that a small
fraction of resistant cells harbor distinct secondary mutations such
Katayama et al.
as C1156Y. However, this seems unlikely because we observed the
L1196M mutation and not the C1156Y mutation in all of the
clones derived from single cells from the H3122 CR cell line.
Although amplification of ALK is a known oncogenic event
in pediatric neuroblastoma (34), amplification of ALK fusion
oncogenes has not been reported. Analysis of partially resistant
H3122 cells (H3122 CR0.6) that harbor EML4-ALK amplification
(but no gatekeeper mutation) suggests a step-wise evolution of
acquired resistance involving gene amplification followed by point
mutation. This model is also supported by the observation that
only a fraction of the amplified ALK fusion genes (per cell) harbor
L1196M (Fig. 2C). In other TKI-resistant cancers, target amplification preceding acquisition of secondary mutations has not
been reported in vitro or in vivo. It remains unknown if this twostep mechanism is unique to EML4-ALK–positive NSCLC or if
it is a product of the methodology used to develop resistance in
the laboratory.
The detection of gatekeeper and other acquired mutations in
resistant tumor samples is clinically important, but can be technically challenging. The EGFR T790M resistance mutation, for
example, is often difficult to detect on standard sequence tracings and may require more sensitive techniques such as deep
sequencing for detection (19, 35, 36). This problem may be due
to contamination of the tumor specimen with noncancerous cells
(e.g., stromal or inflammatory cells). Alternatively, for some
tumors, only a small percentage of the EGFR alleles per cell may
harbor the T790M mutation (termed “allelic dilution”), or only
a small percentage of resistant cells in the tumor specimen might
have T790M-mediated resistance. Notably, the finding of both
EML4-ALK amplification and L1196M mutation in the CR cells
suggests that allelic dilution also could ultimately contribute to
difficulty in detecting resistance mutations in cancers with acquired resistance to crizotinib. In this study, we developed an
L1196M-mutation specific PCR assay to detect potentially low
levels of the gatekeeper L1196M mutation in partially resistant
H3122 CR0.6 cells. This assay is highly sensitive with a detection
limit of 1% or less (SI Appendix, Fig. S4) and requires only 30 ng
genomic DNA. Thus, this assay might serve as the basis for
a clinical diagnostic test for L1196M in biopsy samples from
crizotinib-resistant patients.
In both CML and EGFR-mutant lung cancer, resistant tumors
with gatekeeper mutations have proven refractory to secondgeneration, more potent TKIs. One recent report suggests that
the gatekeeper mutation in ALK might behave similarly; compared with control Ba/F3 cells expressing native EML4-ALK, cells
expressing mutant EML4-ALK L1196M were markedly less sensitive to a potent PDD (23). In contrast, we have found that crizotinib resistance mediated by the gatekeeper L1196M mutation
can be effectively overcome by two different ALK TKIs. Importantly, one of these—AP26113—is under clinical development
and is scheduled to begin clinical testing within the year. In H3122
CR cells, the improved cellular activity of AP26113 compared
with crizotinib is likely based on both its enhanced potency against
ALK and its increased activity against L1196M-mutated ALK.
In vitro kinase assays revealed that wild-type and L1196Mmutated ALK showed similar Km values for ATP binding (30.7
and 27.2 μM, respectively). However, crizotinib was about 10-fold
less potent against L1196M ALK compared with wild-type ALK
(Ki of 8.2 and 0.7 nM, respectively), consistent with its diminished
efficacy against the L1196M ALK in cell lines (SI Appendix, Table
S1B). In contrast, AP26113 was ∼10-fold more potent against
ALK than crizotinib in vitro, and unlike crizotinib, it was similarly
potent against the L1196M mutant (Ki of 0.09 and 0.08 nM,
respectively).
In addition to studying ALK TKIs, we have shown that EML4ALK harboring the gatekeeper mutation is an Hsp90 client
protein similar to wild-type EML4-ALK and sensitive to Hsp90
inhibition. Notably, cell lines expressing either native EML4PNAS | May 3, 2011 | vol. 108 | no. 18 | 7539
MEDICAL SCIENCES
300 nM
100 nM
30 nM
10 nM
300 nM
non
100 nM
10 nM
non
H3122 CR
17AAG (24hr)
pALK
(pY1604)
H3122 CR
ce
ell number (% of control)
100
30 nM
H3122
17AAG (24hr)
3 nM
B
3 nM
A
ALK or EML4-ALK L1196M were even more sensitive to 17AAG than two Hsp90-dependent, HER2-amplified breast cancer
lines (Fig. 5A). Therefore, patients with crizotinib resistance due
to an acquired L1196M mutation may derive benefit from two
different ALK-targeted strategies: more potent ALK TKIs such
as AP26113 or any of a number of Hsp90 inhibitors already in
clinical trials. Notably, although gatekeeper mutations are particularly common in EGFR-mutant lung cancer, the frequency of
L1196M in EML4-ALK–positive NSCLC has yet to be established. Because secondary mutations such as the gatekeeper
mutation may not represent the predominant mechanism of acquired crizotinib resistance, additional studies are needed to
elucidate other mechanisms of resistance. The results of these
studies will be critical to selecting the best therapeutic strategies
for targeting TKI resistance in the clinic.
Materials and Methods
See SI Appendix for the description of the cell culture conditions, generation
of H3122 CR cells, survival assays, fluorescence in situ hybridization, immunoblotting, apoptosis assay retroviral infection, quantitative RT-PCR (qPCR),
siRNA transfection, xenograft study, and statistical analyses.
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Reagents. Crizotinib and NVP-TAE684 were purchased from ChemieTek and
17-AAG was purchased from Selleck. AP26113 was provided by ARIAD
Pharmaceuticals that makes AP26113 available to qualified academic laboratories free of charge for verification and reproduction of the results
reported in this paper. Each compound was dissolved in DMSO for cell culture
experiments. Control or ALK siRNA was from Invitrogen, and HiPerfect reagent was from Qiagen.
Isolation of Genomic DNA Preparation and L1196M Mutation-Specific PCR.
Genomic DNA was isolated from cell pellets with a DNeasy kit (QIAGEN)
according to the manufacturer’s protocol. Exon 23 of ALK was PCR-amplified
from genomic DNA using Pfu Ultra II (Agilent Technologies) and sequenced
bidirectionally by Sanger dideoxynucleotide sequencing with the primers
described in SI Appendix. ALK-Exon23 or L1196M mutation-specific qPCR
was performed by a LightCycler 480 (Roche Diagnostics) with CYBR Green
Master Mix (Roche). Primer sequences are provided in SI Appendix.
ACKNOWLEDGMENTS. This study was supported in part by a V Foundation
for Cancer Research Translational Grant (to J.A.E. and A.T.S.), by the Charles
W. and Jennifer C. Johnson Koch Institute Clinical Investigator Award (to
A.T.S.), by National Institutes of Health K08 Grant CA120060-01 (to J.A.E.), by
the Sig Adler Lung Cancer Research Fund, by the Massachusetts General
Hospital Thoracic Oncology Fund, and by a Japan Society for the Promotion
of Science Postdoctoral Fellowship for Research Abroad from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan (to R.K.).
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