Differential Inhibitor Sensitivity of Anaplastic Lymphoma Kinase Variants
Found in Neuroblastoma
Scott C. Bresler, et al.
Sci Transl Med 3, 108ra114 (2011);
DOI: 10.1126/scitranslmed.3002950
Editor's Summary
A Boost for Neuroblastoma Therapy
Crizotinib inhibits kinase activity by competing for binding with the enzyme's adenosine triphosphate (ATP)
substrate. The authors used human neuroblastoma cell lines and xenografts in mice to show that cancers with the two
most common ALK mutations, F1174L and R1275Q, are unresponsive to and effectively inhibited by crizotinib
therapy, respectively. This reduced sensitivity was caused by a heightened ATP-binding affinity in F1174L-mutated
ALK. These observations suggest that either increasing the dose of crizotinib or engineering higher-affinity inhibitors
should improve therapy for patients with this common ALK mutation. Although careful toxicity studies need to be
performed to find the maximum tolerated dose in the pediatric population, this mechanistic study provides more than a
baby step toward improving crizotinib therapy in the clinic.
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Neuroblastoma, a malignancy of the autonomic nervous system, is the most common cancer in children under 1
year of age. Nearly 10% of spontaneous neuroblastoma patients house mutations in the gene that encodes anaplastic
lymphoma kinase (ALK). The U.S. Food and Drug Administration recently approved crizotinib −−a small-molecule
inhibitor of ALK's tyrosine kinase activity and thus its cell signaling function −−for the treatment of non−small cell lung
carcinomas, and the drug is in early clinical trials for neuroblastoma. However, tumors with certain ALK mutations do
not appear to respond to crizotinib. Bresler et al. now dissect the molecular mechanisms behind the differential
crizotinib sensitivities of individual ALK mutations.
RESEARCH ARTICLE
NEUROBLASTOMA
Differential Inhibitor Sensitivity of Anaplastic
Lymphoma Kinase Variants Found in Neuroblastoma
Activating mutations in the anaplastic lymphoma kinase (ALK) gene were recently discovered in neuroblastoma, a
cancer of the developing autonomic nervous system that is the most commonly diagnosed malignancy in the first
year of life. The most frequent ALK mutations in neuroblastoma cause amino acid substitutions (F1174L and
R1275Q) in the intracellular tyrosine kinase domain of the intact ALK receptor. Identification of ALK as an oncogenic
driver in neuroblastoma suggests that crizotinib (PF-02341066), a dual-specific inhibitor of the ALK and Met tyrosine
kinases, will be useful in treating this malignancy. Here, we assessed the ability of crizotinib to inhibit proliferation
of neuroblastoma cell lines and xenografts expressing mutated or wild-type ALK. Crizotinib inhibited proliferation
of cell lines expressing either R1275Q-mutated ALK or amplified wild-type ALK. In contrast, cell lines harboring
F1174L-mutated ALK were relatively resistant to crizotinib. Biochemical analyses revealed that this reduced susceptibility of F1174L-mutated ALK to crizotinib inhibition resulted from an increased adenosine triphosphate–
binding affinity (as also seen in acquired resistance to epidermal growth factor receptor inhibitors). Thus, this
effect should be surmountable with higher doses of crizotinib and/or with higher-affinity inhibitors.
INTRODUCTION
Neuroblastoma arises in the developing autonomic nervous system
and is the most commonly diagnosed malignancy in the first year of
life. The disease shows a wide range of clinical phenotypes: Although
tumors regress spontaneously in some patients, most have aggressive
metastatic disease (1). Neuroblastoma remains a leading cause of childhood cancer mortality despite marked escalations in dose-intensive
chemoradiotherapy, and long-term survivors experience significant
treatment-related morbidity (2).
One promising therapeutic target in neuroblastoma is the anaplastic lymphoma kinase (ALK), an orphan receptor tyrosine kinase
(RTK) normally expressed only in the developing nervous system
(3). Oncogenic ALK alterations were first described in anaplastic
large cell lymphoma (4), where a chromosomal translocation leads
to production of a fusion protein with the ALK intracellular region
fused to an N-terminal fragment of nucleophosmin (NPM). Other
ALK fusion proteins are potent oncogenic drivers in a subset of non–
small cell lung cancers (NSCLCs) (5) and drive inflammatory myo-
1
Department of Biochemistry and Biophysics, Perelman School of Medicine at the
University of Pennsylvania, Philadelphia, PA 19104–6059, USA. 2Graduate Group in
Biochemistry and Molecular Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. 3Medical Scientist Training Program, Perelman School of Medicine at the University of Pennsylvania, Philadelphia,
PA 19104, USA. 4Division of Oncology and Center for Childhood Cancer Research,
Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA. 5Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia,
PA 19104, USA. 6Biostatistics and Data Management Core, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA. 7Department of Cancer Research, Pfizer Global
Research and Development, La Jolla Laboratories, La Jolla, CA 92121, USA. 8Abramson
Family Cancer Research Institute, Perelman School of Medicine at the University of
Pennsylvania, Philadelphia, PA 19104, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: mosse@email.chop.edu ( Y.P.M.);
mlemmon@mail.med.upenn.edu (M.A.L.)
fibroblastic tumors (IMTs) as well as other cancers (6). In neuroblastoma, germline activating point mutations in the intact ALK gene
were revealed by linkage analysis of a set of families with highly penetrant autosomal dominant disease (7). In addition, somatic ALK
mutations were found in ~10% of sporadic neuroblastoma cases
(7–11). The most frequently observed substitutions, together accounting for >80% of sporadic ALK mutations in neuroblastoma
samples (12), were F1174L and R1275Q, which lie in key regulatory
regions of the ALK receptor kinase domain. Mutations in the intact
ALK gene have also recently been reported in anaplastic thyroid cancer (13).
ALK tyrosine kinase activity can be inhibited by crizotinib (PF02341066), a small-molecule adenosine triphosphate (ATP)–competitive
inhibitor that selectively targets both the ALK and the Met RTKs
(14). A recent phase 1 study of crizotinib demonstrated safety and tolerability in humans, as well as tumor shrinkage or stable disease in
most patients with ALK-dependent NSCLC (15). Crizotinib is also
in early-phase clinical testing in patients with neuroblastoma. As with
other tyrosine kinase inhibitor therapies, acquired resistance to crizotinib
is already beginning to emerge (16–18). Understanding how mutations affect both kinase activity and inhibitor sensitivity is imperative
for guiding future clinical use of ALK-targeted inhibitors. Here, we
explored the ability of crizotinib to inhibit intact ALK in neuroblastoma cell line models and analyzed the effects of the two most common activating mutations seen in neuroblastoma on ALK’s tyrosine
kinase activity. We found that the F1174L mutation, although activating, reduced ALK sensitivity to crizotinib in xenograft, cell line, and
enzymatic assays, consistent with the recent surprising report of this
mutation as an acquired resistance mutation in an oncogenic ALK
fusion protein (17). Compared with the R1275Q activating mutation,
we found that an F1174L substitution increased ATP-binding affinity,
leading to crizotinib resistance that should be surmountable with higher
doses of crizotinib or new higher-affinity inhibitors.
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Scott C. Bresler,1,2,3* Andrew C. Wood,4,5* Elizabeth A. Haglund,4,5 Joshua Courtright,4,5
Lili T. Belcastro,4,5 Jefferson S. Plegaria,4,5 Kristina Cole,4,5 Yana Toporovskaya,4,5
Huaqing Zhao,5,6 Erica L. Carpenter,4,5 James G. Christensen,7 John M. Maris,4,5,8
Mark A. Lemmon,1,2† Yaël P. Mossé4,5†
RESEARCH ARTICLE
The effect of crizotinib on growth of neuroblastoma-derived
cell lines depends on ALK genomic status and the
specific mutation
To assess how the most common ALK mutations in neuroblastoma
(F1174L and R1275Q) affect intrinsic ALK activity, we expressed fulllength ALK variants in human retinal-pigmented epithelial (RPE)
cells immortalized with telomerase reverse transcriptase (hTERTRPE1). We selected RPE cells because they are derived from human
neural crest–like neuroblastomas but express no endogenous ALK
(Fig. 1A). Whereas wild-type ALK expressed in hTERT-RPE1 cells
was not detectably phosphorylated (Fig. 1A), both R1275Q- and
F1174L-mutated ALK showed robust autophosphorylation in immunoblots using an ALK pY1604-specific antibody, regardless of
the presence of serum in the medium (Fig. 1A, middle panels). Thus,
both common neuroblastoma mutations caused constitutive ALK activation to similar extents, as seen in Ba/F3 (10), NIH3T3 (9, 19), and
PC12 cells (20), as well as in numerous neuroblastoma-derived cell
lines (7, 10, 11, 19). Consistent with previous reports (19, 21), two ALK
species were always observed. Full-length ALK migrates as a 220-kD
protein, and the 140-kD species is a cleavage product lacking part
of the extracellular region (21).
We and others have shown that RNA interference (RNAi) knockdown of ALK, or pharmacological inhibition of ALK kinase activity,
has an antiproliferative effect in several ALK-mutated neuroblastoma
cell lines (7, 9–11), associated with G1 arrest and increased apoptosis
(10). To exploit this observation clinically, we assessed the cytotoxicity
of the ALK/Met inhibitor crizotinib (PF-02341066) in cell models for
neuroblastoma. We first analyzed the ability of crizotinib to inhibit
substrate-adherent growth of a panel of 18 human neuroblastoma–
derived cell lines. The 18 cell lines were chosen to represent ALK genomic status in primary tumors, and have all previously been well
characterized. Analysis of concentration-response curves across a fourlog crizotinib dose range (1 to 10,000 nM) revealed significant differences in IC50 (median inhibitory concentration) that correlated with
ALK status. As illustrated in Fig. 1B, cell lines harboring an ALK aberration (amplification or mutation) were significantly more sensitive
to growth inhibition by crizotinib than those with wild-type ALK status (P = 0.001, two-sided exact Wilcoxon-Mann-Whitney test). To ensure that crizotinib cytotoxicity in this assay reflected ALK inhibition,
we confirmed that the drug reduced phospho-ALK (pALK) levels (see
below). In addition, because crizotinib also inhibits Met kinase activity
(14), it was important to exclude Met inhibition as a mechanism. We
confirmed that none of the crizotinib-inhibited neuroblastoma cell
lines displayed significant phospho-Met levels (fig. S1). Moreover,
RNAi knockdown of Met in a panel of cell lines with altered ALK
genomic status had no growth-inhibitory effect (table S1). The enhanced
crizotinib sensitivity of almost all neuroblastoma cell lines harboring
ALK mutation or amplification (Fig. 1B) strengthens the argument
for ALK inhibition as a useful therapeutic strategy in neuroblastoma.
Mutation-specific stratification of crizotinib sensitivity in neuroblastoma cell lines (Fig. 1B) may have significant implications for clinical
use of this drug. Cell lines expressing F1174L-mutated ALK were marginally significantly less sensitive to growth inhibition by crizotinib
than those expressing R1275Q-mutated ALK (P = 0.067, two-sided
exact Wilcoxon-Mann-Whitney test), justifying further investigation.
This result was initially unexpected, because the F1174L and R1275Q
mutations appear to promote similar degrees of constitutive ALK
activation (Fig. 1A), and small interfering RNA (siRNA) knockdown of either variant inhibits growth of the relevant cell lines (7).
Assuming a similar oncogenic driver role for both ALK variants, the
data in Fig. 1B suggest that the F1174L mutation may also promote
crizotinib resistance. Indeed, an F1174L mutation was recently found
to cause acquired crizotinib resistance in a patient with an IMT driven
by constitutively active RANBP2-fused ALK (17). The F1174L substitution may therefore combine the characteristics of an activating
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RESULTS
Fig. 1. Constitutive activity and inhibitor sensitivity of F1174L and R1275
ALK mutants. (A) Immunoblots of total ALK and pALK in hTERT-RPE1 cells
infected with retroviruses directing expression of wild-type (WT) or mutated ALK. Lower panel is actin loading control. (B) Proliferation of neuroblastoma cell lines over 72 hours of incubation with 333 nM crizotinib.
Growth inhibition ± SD is reported for at least three independent experiments. P values were calculated for marked comparisons with twosided exact Wilcoxon-Mann-Whitney tests. Cell lines were as follows (left
to right): WT ALK-amplified (NB1); R1275Q (NB1643, LAN5); F1174L (SH-SY5Y,
KELLY, NBSD, SMS-SAN); WT ALK, normal copy number (NB1691, NB-EBc1,
IMR5, NB16, NLF, IMR32, NBLS, SKNBE2C, NGP, SKNAS, SKNFI).
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RESEARCH ARTICLE
mutation and a resistance mutation, understanding of which is crucial
for developing ALK-targeted therapy.
The F1174L mutation reduces crizotinib sensitivity
of ALK autophosphorylation
The reduced crizotinib sensitivity of cell lines and xenografts harboring F1174L-mutated ALK prompted us to analyze the effects of this
drug on constitutive ALK activity in representative cell lines: NB1643
cells (which express R1275Q ALK) and SH-SY5Y cells (which express
F1174L ALK). Crizotinib treatment abrogated Y1604 phosphorylation
of ALK in both cell lines, but at different doses (Fig. 3). In NB1643
(R1275Q) cells, ALK phosphorylation was essentially abolished at
100 nM crizotinib (IC50, ~10 nM), whereas complete inhibition of
F1174L ALK phosphorylation in SH-SY5Y cells required almost
1 mM crizotinib (IC50, ~50 nM). Akt phosphorylation followed similar trends (fig. S2), consistent with a previous report that ALK inhibition promotes apoptosis in neuroblastoma cell lines (10). Crizotinib
resistance of cells harboring the ALK F1174L mutation therefore appears to arise from less potent inhibition of constitutive ALK activity
and thus of downstream survival signaling.
F1174L and R1275Q mutations promote
autophosphorylation of the ALK kinase domain
To understand why F1174L- and R1275Q-mutated ALK have different crizotinib sensitivities and how the mutations enhance kinase
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Tumor growth driven by F1174L-mutated ALK also shows
reduced sensitivity to crizotinib in vivo
Using xenograft models of neuroblastoma, we next asked whether
the two ALK variants show differential crizotinib sensitivity in vivo.
We tested a pharmacologically relevant crizotinib dose (100 mg/kg
per day for 4 weeks) against serially passaged human neuroblastoma
cells xenografted in the flank of CB17 immunodeficient mice (14).
As shown in Fig. 2A, crizotinib caused tumor regression within
3 weeks in all NB1643 xenografts (R1275Q-ALK), and complete regression was sustained over the fourth week of dosing (P < 0.0001).
Assessment of pALK levels by immunoblotting NB1643 xenograft
lysates harvested after 48 hours of crizotinib administration (4 hours
after final dose) confirmed inhibition of ALK phosphorylation (Fig. 2A,
inset). Consistent with the differential sensitivity suggested in vitro,
xenografts harboring F1174L-ALK (SH-SY5Y and NBSD) were substantially less responsive to crizotinib. No tumor regression was observed in crizotinib-treated SH-SY5Y xenografts (Fig. 2B), although
tumor growth was significantly delayed (P < 0.0001) and the time
taken to reach the study endpoint (tumor volume ≥1.5 cm3) was
extended by an average of 7.7 days (P < 0.0001). Crizotinib also failed
to reduce tumor volume (P = 0.29) in NBSD (F1174L) xenografts (Fig.
2C), although the time to reach the study endpoint was again extended by a mean of 3.7 days (P = 0.04). These results argue that tumors driven by F1174L-mutated ALK are significantly less sensitive to
crizotinib in vivo. Crizotinib treatment led to complete tumor regression in NB1 xenografts with amplified, overexpressed wild-type ALK
(Fig. 2D), but not in NB-EBc1 or SKNAS xenografts that have no
amplification (Fig. 2, E and F). Crizotinib treatment delayed tumor
growth (P < 0.0001) in NB-EBc1 xenografts (low pALK levels, but
no mutation or amplification), but had no detectable effect (P = 0.70)
in SKNAS xenografts, which express low levels of wild-type ALK and
show no detectable pALK (Fig. 2F).
Fig. 2. Crizotinib activity in vivo for WT and mutated ALK. Subcutaneously implanted neuroblastoma tumors were monitored in CB17 scid
mice treated with crizotinib (solid red lines) or vehicle (dashed blue
lines). Tumor volume (left panels) is displayed as mean ± SEM. Study
end points for survival analysis (right panels) were tumor volume ≥1.5cm3
or treatment-related death. (A) NB1643 (R1275Q) xenografts: inset shows
immunoblot of ALK and pALK. (B) SH-SY5Y (F1174L). (C) NBSD (F1174L). (D)
NB1 (WT amplified with strong pALK staining). (E) NB-EBc1 (WT, with weak
pALK staining). (F) SKNAS (WT, undetectable pALK). Statistical treatment
is described in Materials and Methods.
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activity, we turned to in vitro studies of the ALK tyrosine kinase domain (ALK-TKD), purified from baculovirus-infected Sf9 cells as a
hexahistidine-tagged protein. To generate fully dephosphorylated (in-
Fig. 3. Inhibition of constitutive ALK autophosphorylation by crizotinib.
Representative immunoblots of ALK autophosphorylation in neuroblastoma cell lines after treatment with different crizotinib concentrations (0 to 10 mM). (A and B) Whole-cell lysates were immunoblotted
for pALK (using pY1604 antibody), total ALK, and actin (loading control) for (A) NB1643 cells (R1275Q ALK) and (B) SH-SY5Y cells (F1174L
ALK). Downstream signaling molecules are analyzed in fig. S2. (C) Quantification of pALK levels (220-kD species) as a function of crizotinib
concentration.
active) ALK-TKD, we used YopH phosphatase treatment to reverse
spontaneous autophosphorylation arising during production (Supplementary Methods). For comparative studies, fully autophosphorylated
ALK-TKD was generated by clustering the protein on lipid vesicles
bearing NTA-Ni head groups (DOGS-NTA-Ni), thus imitating ligandinduced ALK dimerization. This method has been used to promote
assembly and activation of other receptor fragments, including epidermal growth factor receptor (EGFR)–TKD (22, 23), and markedly
enhances the rate of ALK-TKD autophosphorylation (Fig. 4A).
Our initial investigations with MnCl2-containing assay conditions
used in previous ALK-TKD studies (24–26) suggested only a threefold increase in kinase activity upon autophosphorylation. This contrasts starkly with the ~100- to 200-fold activity increase typically seen
upon activation loop autophosphorylation of other kinases from the
insulin receptor (IR) family (27–29). The anomaly arises because the
previously used high (nonphysiological) Mn2+ concentrations both increase unphosphorylated ALK-TKD activity (fig. S3, A to C)—as reported
for other RTKs (30, 31)—and reduce activity of fully autophosphorylated ALK-TKD (fig. S3D). We therefore used a more physiological
10 mM MgCl2 in subsequent studies (and no Mn2+). Under these
conditions, autophosphorylation promoted strong ALK-TKD activation (see below), as did the F1174L and R1275Q mutations (Fig. 4B
and fig. S3E).
Native gel electrophoresis (Fig. 4B) showed that autophosphorylation of dephosphorylated ALK-TKD was substantially accelerated by the F1174L and R1275Q mutations, confirming that these
mutations activate the isolated kinase domain. Mobility of ALK-TKD
in native gels was increased by autophosphorylation, with successive
autophosphorylation events giving rise to four differently phosphorylated species over a period of 0.5 to 20 min at 37°C. Whereas unphosphorylated wild-type ALK-TKD was still detectable in Fig. 4B after
4 min, this species disappeared completely within 1 min for both mutated forms. Similarly, the first pALK-TKD species persisted until
at least 8 min for wild-type protein, but only until ~2 min for F1174L
ALK-TKD and ~3 min for R1275Q ALK-TKD. These results are shown
graphically in fig. S4. Using analytical ultracentrifugation, we ruled
out the possibility that increased dimerization of mutated TKDs enhances their autophosphorylation (fig. S5). ALK-TKD remained monomeric in solution regardless of mutation. The increased ALK-TKD
autophosphorylation rates promoted by the F1174L and R1275Q mutations therefore reflect elevated basal kinase activity.
Wild-type and mutated ALK-TKDs have similar
inhibitor selectivity profiles
The relative crizotinib resistance of F1174L-mutated ALK observed
in cell-based and xenograft studies prompted us to compare its inhibitor selectivity profile with those of wild-type and R1275Q-mutated
ALK. We reasoned that the F1174L mutation might alter the drugbinding site so that certain ATP-competitive compounds (such as
crizotinib) are less effective inhibitors of this variant than of R1275Q
or wild type. To establish inhibitor selectivity profiles for wild-type,
F1174L, and R1275Q ALK-TKD, we assessed the ability of overlapping panels of 320 well-characterized kinase inhibitors to inhibit their
in vitro autophosphorylation (see the Supplementary Methods). As
shown in fig. S6, the inhibition profile of F1174L-mutated ALK-TKD
was essentially identical to those for wild-type and R1275Q-mutated
ALK-TKD. Thus, the F1174L mutation does not appear to change the
relative abilities of different inhibitors to bind ALK-TKD.
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RESEARCH ARTICLE
Autophosphorylation alone is sufficient for
maximal ALK-TKD activation
To better understand the reduced crizotinib sensitivity of F1174Lmutated ALK, we analyzed kinase activity more quantitatively. As a
baseline for these studies, it was important first to establish the full
range of wild-type ALK-TKD activity levels—in fully activated and
unactivated states, respectively. We monitored tyrosine phosphorylation of a peptide corresponding to the ALK activation loop, with
sequence ARDIYRASYYRKGGCAMLPVK [“YYY” peptide (25)].
Table 1 lists kinetic parameters (kcat, Km, peptide, and Km, ATP) for
wild-type ALK-TKD in both inactive (unphosphorylated) and activated (fully autophosphorylated) states.
As shown in Fig. 4C and Table 1, autophosphorylation elevated
ALK-TKD catalytic efficiency primarily through a 45-fold increase in
kcat (P < 0.0001), from 9.32 ± 0.85 min−1 (unphosphorylated) to 424 ±
63 min−1 (fully phosphorylated), accompanied by a 1.6-fold decrease in
Km, peptide. Values for Km, ATP were higher than reported from Mn2+containing assays (24–26) and were not significantly altered by ALKTKD autophosphorylation (Table 1 and Fig. 4D). This contrasts with
other RTKs, where activation loop autophosphorylation reduces Km for
both peptide and ATP substrates under similar (MnCl2-free) assay conditions (28, 29, 32). Unlike other members of its family, ALK therefore
does not appear to be autoinhibited by pseudosubstrate-like interaction
of its unphosphorylated activation loop with the active site.
Although autophosphorylation increases ALK-TKD activity, we wondered
whether TKD dimerization might play
an additional allosteric activating role, as
seen with EGFR (23). Whereas clustering
EGFR-TKD on the surface of vesicles containing DOGS-NTA-Ni lipids promoted
significant activation (Fig. 4E), no activating effect was seen when fully phosphorylated pALK-TKD was similarly treated
(Fig. 4F). These data argue against an allosteric activation mechanism for ALK
and suggest that autophosphorylation is
sufficient for its maximum activation.
Fig. 4. Analysis of ALK-TKD activation in vitro. (A) Separation of differently autophosphorylated ALK-TKD
species by native gel electrophoresis to monitor autophosphorylation at 25°C in the absence (top) and
presence (bottom) of vesicles containing 10% DOGS-NTA-Ni (100 mM total lipid), 10 mM MgCl2, and
2 mM ATP. (B) Autophosphorylation of WT and mutated ALK-TKD (10 mM) with saturating ATP (2 mM)
and 10 mM MgCl2 at 37°C. Results are quantified in fig. S4. (C) Rate of 32P incorporation at 25°C into
substrate peptide (see Materials and Methods) for autophosphorylated ALK-TKD (10 nM) and unphosphorylated ALK-TKD (500 nM) as peptide substrate concentration is increased. ATP concentration was
2 mM. (D) Km, ATP determination for autophosphorylated (10 nM) and unphosphorylated (500 nM)
ALK-TKD at fixed peptide substrate concentration (1 mM). (E) Enhancement of EGFR-TKD kinase (1 mM)
by vesicles containing increasing molar percentages of DOGS-NTA-Ni (10 mM DOGS-NTA-Ni, 100 to
200 mM total lipid). (F) Effect on autophosphorylated ALK-TKD (1 mM) activity of adding DOGS-NTA-Ni
vesicles. Data are shown as means ± SEM from at least three independent experiments. All experiments
except those in (B) were performed at 25°C.
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F1174L and R1275Q mutations
constitutively activate ALK-TKD and
display different kinetic profiles
As reported in Table 1, the F1174L and
R1275Q mutations both cause significant
increases in kcat even without autophosphorylation. The F1174L mutation increased kcat by ~40-fold (P = 0.0013) to
365 ± 61 min−1, close to the maximum
measured for fully phosphorylated wildtype protein. The R1275Q mutation had a
more modest effect, increasing kcat of unphosphorylated ALK-TKD by just 12fold (P = 0.0001). Whereas the R1275Q
mutation left Km, peptide unaltered, the
F1174L mutation increased Km, peptide by
about threefold. These two neuroblastoma mutations therefore promoted similar
(10-fold) increases in catalytic efficiency
(kcat/Km, peptide) of unphosphorylated
ALK-TKD (Fig. 5A and Table 1) in the
presence of saturating ATP. Autophosphorylation further increased catalytic efficiency (kcat/Km, peptide) for both R1275Q
and F1174L ALK-TKD variants (Fig. 5E).
For R1275Q, this resulted largely from an
increase (about threefold) in kcat. For F1174L,
phosphorylation reduced Km, peptide by about
sevenfold (Table 1). In addition to being
constitutively activated, F1174L ALK-TKD
may be slightly “superactivated” upon full
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RESEARCH ARTICLE
RESEARCH ARTICLE
Table 1. Kinetic parameters of ALK-TKD. Data are shown as means ± SEM of at least three independent experiments. Details of analysis are described in the Supplementary Methods. P values quoted in the text were determined with an unpaired t test.
Kinase
kcat
(min−1)
Km, peptide
(mM)
kcat/Km, peptide
(min−1 mM−1)
Km, ATP*
(mM)
kcat/Km, ATP
(min−1 mM−1)
Wild type
9.32 ± 0.85
2.88 ± 0.42
3.41 ± 0.44
0.134 ± 0.007
69.7
R1275Q
119 ± 13
2.56 ± 0.32
46.8 ± 1.9
0.326 ± 0.033
364
F1174L
365 ± 61
9.18 ± 1.43
39.7 ± 2.8
0.127 ± 0.011
2870
Phospho–wild type
424 ± 63
1.80 ± 0.17
237 ± 35
0.159 ± 0.012
2660
Phospho-R1275Q
347 ± 15
1.39 ± 0.10
252 ± 24
0.248 ± 0.015
1400
Phospho-F1174L
436 ± 51
1.25 ± 0.20
357 ± 25
0.109 ± 0.001
3980
autophosphorylation for the particular peptide substrate used here
(Fig. 5B), with a catalytic efficiency that was ~30% higher than measured for wild-type (P = 0.0342) or R1275Q (P = 0.0327) pALK-TKD.
Reduced Km for ATP explains the relative resistance of
F1174L-mutated ALK to crizotinib
The Km, ATP values listed in Table 1 (and Fig. 5C) suggest one explanation for the reduced crizotinib sensitivity of cell lines and xenografts
expressing F1174L-mutated ALK. Km, ATP for the F1174L mutant was
2.3-fold lower than that of R1275Q (P = 0.0007) when autophosphorylated (Fig. 5, D and F), and 2.6-fold lower (P = 0.0045) in the
dephosphorylated form (Fig. 5, C and F). This trend was maintained
when the assay was repeated at a higher peptide concentration of 2 mM
(fig. S7 and table S2). These data suggest that the F1174L mutation
enhances the ATP-binding affinity of ALK, which in turn will reduce
potency of any ATP-binding site competitor (such as crizotinib) at cellular ATP concentrations, as seen for drug resistance mutations in some
other RTKs (33).
To test this hypothesis, we compared the in vitro crizotinib sensitivity of the recombinant ALK-TKD variants at two different ATP concentrations (Fig. 5, G and H). At 0.2 mM ATP (significantly lower
than cellular levels), IC50 values (measured for 50 nM unphosphorylated protein) were similar for R1275Q and F1174L ALK-TKD (Fig. 5G
and table S3). However, at a more physiological ATP concentration
of 2 mM, F1174L ALK-TKD was significantly less sensitive (P = 0.0128)
to crizotinib inhibition (IC50 = 130 nM) than R1275Q ALK-TKD (IC50 =
84.6 nM). A similar difference was also seen with autophosphorylated
proteins (table S4), as anticipated from Table 1. Wild-type ALK-TKD
resembled the F1174L variant in its relative insensitivity to crizotinib
inhibition in vitro (tables S3 and S4), consistent with the finding that
the drug does not inhibit growth of neuroblastoma cells that express
wild-type ALK at normal levels (Figs. 1B and 2E). However, NB1 cells
that express the receptor at very high levels because of an ALK amplification were sensitive to crizotinib (Figs. 1B and 2D), indicating that
inhibitor response may also be affected by issues of trafficking, subcellular localization, and expression level (19).
The correlation between the increased ATP-binding affinity of
F1174L-mutated ALK and its reduced sensitivity to crizotinib argues that
this effect will be general, affecting the response to all ATP-competitive
inhibitors. We tested this hypothesis with two other inhibitors. As shown
in fig. S8 and table S5, in vitro inhibition of ALK-TKD by staurosporine
is also impaired by the F1174L mutation compared with that seen for
R1275Q-mutated ALK-TKD. Moreover, the ALK/insulin-like growth
factor 1 receptor (IGF-1R)/IR inhibitor GSK1838705A (34) was substantially less effective at inducing cytotoxicity in neuroblastoma cell
lines expressing F1174L ALK than those expressing R1275Q ALK
(table S6). Like crizotinib, GSK1838705A only showed significant
in vivo activity against R1275Q xenografts (fig. S9). These data, and
the Km, ATP values reported in Table 1, therefore suggest that F1174Lmutated ALK will be less sensitive to all ATP-competitive inhibitors.
Structural changes can explain the increased ATP-binding
affinity in F1174L-mutated ALK
Recent crystal structures of ALK-TKD (24, 26) revealed unexpected
similarities with EGFR-TKD that are useful for understanding and
predicting the consequences of ALK mutations. In the inactive ALK
and EGFR TKD structures, key autoinhibitory interactions between
the crucial aC helix and a short a helix at the beginning of the activation loop (magenta in Fig. 6) displace the aC helix from the position it must adopt in the “active” kinase. Intriguingly, the location
of R1275 in ALK coincides closely with that of L837 in EGFR (L861 in
pro-EGFR), where mutation to glutamine activates EGFR in NSCLC
(35). The R1275 side chain contributes directly to interactions between
the magenta activation loop helix and the aC helix (Fig. 6B), stabilizing the autoinhibited ALK-TKD conformation. Replacing R1275 with
a glutamine, or phosphorylating nearby Y1278 in the activation loop
(Fig. 6B), will disrupt these autoinhibitory interactions and thus activate ALK.
F1174 lies at the C terminus of the aC helix (Fig. 6B), and its side
chain contributes to the small well-packed hydrophobic “core” between aC and the activation loop. A reduction in the size of the
F1174 side chain will disrupt this packing, weakening autoinhibitory
interactions and thus allowing ALK-TKD to adopt its active configuration. F1174 also makes another important interaction that may explain its effects on ATP binding. As shown in Fig. 6C, F1174 is in direct
van der Waal’s contact with F1271 of the crucial “DFG” motif, the aspartate of which (D1270) coordinates a Mg2+ ion involved in ATP binding. Reducing the size of the F1174 side chain would remove one
structural restraint on the DFG motif, which may allow D1270 to adopt
a position with more optimal Mg2+ coordination geometry, slightly
increasing ATP-binding affinity (that is, reducing Km, ATP). By contrast,
crizotinib makes no interactions with the DFG motif (Fig. 6C, lower
panel) and does not come within 4 Å of the D1270 side chain, so
altering the DFG motif position should not affect crizotinib binding.
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*Determined at 1 mM peptide.
RESEARCH ARTICLE
These observations provide a structural hypothesis for how the F1174L
mutation can increase the affinity of ALK-TKD for ATP without affecting its affinity for ATP-competitive inhibitors such as crizotinib,
A
80
B
Unphosphorylated
300
staurosporine, and GSK1838705A, leading to relative resistance. By
contrast, the R1275 side chain is more than 7 Å away from the ATPbinding site, so its mutation should not directly affect ATP binding.
Phosphorylated
DISCUSSION
min–1
min–1
200
40
F1174L
R1275Q
Wild-type
20
0
0
0.0
0.5
1.0
1.5
[Peptide] (mM)
D
1.0
Fraction of Vmax
0.0
2.0
0.8
0.6
0.4
F1174L
R1275Q
Wild-type
0.2
0.0
0.0
E
500
1.0
[ATP] (mM)
1.5
F
0.6
0.4
pF1174L
pR1275Q
pwild-type
0.2
Km, ATP (mM)
0.4
300
200
0
0.5
1.0
[ATP] (mM)
1.5
2.0
Unphosphorylated
Phosphorylated
0.3
0.2
0.1
0.0
1.2
H
0.2 mM ATP
1.0
0.8
0.6
F1174L
R1275Q
0.4
0.2
0.0
–8.0
–7.5
Wild-type
F1174L
R1275Q
ALK-TKD variant
F1174L
R1275Q
ALK-TKD variant
–7.0
–6.5
Log [crizotinib]
–6.0
1.2
Fractional activity
Wild-type
Fractional activity
2.0
0.8
0.0
100
G
1.0
1.5
[Peptide] (mM)
0.0
2.0
Unphosphorylated
Phosphorylated
400
kcat/Km, peptide
(min–1mM–1)
0.5
0.5
1.0
Fraction of Vmax
C
pF1174L
pR1275Q
pwild-type
100
2 mM ATP
1.0
0.8
0.6
F1174L
R1275Q
0.4
0.2
0.0
–8.0
–7.5
–7.0
–6.5
Log [crizotinib]
–6.0
Fig. 5. Comparison of WT ALK-TKD with F1174L and R1275Q variants in vitro. (A and B) Rates of 32P
incorporation into YYY peptide at saturating ATP (2 mM) for (A) unphosphorylated WT (500 nM) or
mutated (50 nM) ALK-TKD and (B) phosphorylated WT and mutated ALK-TKD (all at 10 nM). (C and D)
Km, ATP determination (with YYY peptide fixed at 1 mM) for (C) unphosphorylated WT (500 nM) or
mutated (50 nM) ALK-TKD and (D) pALK-TKD variants (all at 10 nM). (E) Comparison of catalytic efficiencies (kcat/Km, peptide) for unphosphorylated ALK-TKD and pALK-TKD variants. (F) Comparison of
Km, ATP values. (G and H) Inhibition of unphosphorylated F1174L and R1275Q ALK-TKD (50 nM) by
crizotinib in peptide phosphorylation assays ([peptide] is 0.5 mM) at 0.2 mM ATP (G) and 2 mM ATP
(H). All data are shown as means ± SEM from at least three independent experiments. Experiments were
performed at 25°C.
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Genetic studies have firmly established
ALK as a tractable molecular target in
neuroblastoma, as well as several other
human malignancies including NSCLC.
We established here that neuroblastoma
cell lines driven by ALK mutation or amplification were sensitive to crizotinib, an
orally bioavailable ATP-competitive ALK
inhibitor. Neuroblastoma cell lines and
xenografts that express R1275Q-ALK,
one of the two most commonly occurring
mutations (12), were highly sensitive to
crizotinib. By contrast, those expressing
F1174L-mutated ALK (the other of the
two most common mutations) were resistant to crizotinib in vitro and in vivo.
We showed that the reduced sensitivity
of F1174L-expressing cell lines can be explained, at least in part, by an increased ATPbinding affinity compared with R1275Q,
as manifested by Km, ATP values, and this reduces the potency of several ATP-competitive
inhibitors.
The R1275Q mutation in ALK resembles oncogenic EGFR mutations found in
NSCLC, such as L834R (L858R in proEGFR), increasing both kcat and Km, ATP
(36, 37). By contrast, the F1174L mutation
in ALK appears to combine the characteristics of an activating mutation and a
resistance mutation, increasing kcat while
maintaining a wild-type–like Km, ATP. The
F1174L mutation thus resembles the drugresistant EGFR L834R/T766M double mutation (L858R/T790M in pro-EGFR) that
has a reduced Km, ATP (33, 37). The F1174L
mutation has emerged not only as a builtin resistance mechanism in neuroblastoma as described here but also as an escape
mechanism in ALK-translocated tumors
treated with crizotinib (17).
Despite the resistance described here,
our results indicate that patients harboring the F1174L ALK mutation may benefit from treatment with ATP-competitive
ALK inhibitors in some circumstances. For
crizotinib itself, an increase in dosage to
overcome the relative difference in Km, ATP
compared with R1275Q-mutated ALK may
be possible, although it remains unclear
whether the exposures necessary to achieve
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60
Fig. 6. Structural basis for ALK-TKD activation by F1174L and R1275Q mutations. (A) Cartoon
representations of inactive ALK-TKD (26) from Protein Data Bank (PDB) entry 3LCS (cyan), inactive EGFR-TKD (23) from PDB entry 2GS7 (gray), and active EGFR from PDB entry 1M14
(green). The activation loop is colored magenta in each structure. Positions of F1174 (red) and
R1275 (blue) are marked in ALK-TKD; their structural equivalents V745 and L837 are marked on
EGFR (inactive and active). (B) Detail of interactions between the short activation loop helix
(magenta) and helix aC in inactive ALK-TKD (left) and inactive EGFR-TKD (right). (C) Close-up of relationship between F1174 side chain, the
“DFG motif,” and ATP/drug-binding site. In the upper panel, taken from PDB entry 3LCT (26), which contains bound adenosine diphosphate
(ADP), an Mg2+ ion (not reported in this structure) was placed based on its location in the active IR TKD. Lower panel (with bound crizotinib)
was taken from PDB entry 2XP2.
the biochemical effect in tumor tissue will be achievable in the
clinic. Crizotinib was well tolerated in adult patients with refractory
lung cancer (15), suggesting that a therapeutic window exists and that
pediatric phase 1 studies should seek to define a true maximum tolerated dose in an effort to circumvent the de novo resistance caused
by the F1174L mutation. The absence of an appropriate active-site
cysteine precludes use of the irreversible inhibitor approach being investigated for EGFR (38). Alternative strategies include development
of therapeutic ALK antibody reagents for patients whose tumors harbor
the F1174L mutation and/or ATP-competitive inhibitors that can overcome the Km, ATP effects by having higher affinity for ALK while retaining selectivity.
MATERIALS AND METHODS
In vitro tumor growth inhibition
In vitro crizotinib activity was evaluated in 18 neuroblastoma cell
lines with the real-time cell electronic sensing (RT-CES) system
(ACEA Biosciences). Cell lines were plated at 5000 to 30,000 cells
per well depending on growth kinetics, and the drug was added at
1 to 10,000 nM after 24 hours. IC50 was calculated from the cell
index in triplicate samples after 72 hours of incubation. Growth inhibition at 333 nM crizotinib was calculated with the following formula:
% inhibition = 100 × (cell index control − cell index treatment)/cell
index control. Because of noncomparable maximum growth inhibition
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RESEARCH ARTICLE
RESEARCH ARTICLE
In vitro protein and phosphoprotein detection
Each cell line was grown to 70 to 80% confluence, treated with crizotinib
at the noted concentration (ranging from 0 to 10,000 nM) for 2 hours,
and washed twice with ice-cold phosphate-buffered saline. Whole-cell
lysates were then analyzed by immunoblotting as described (7) with
antibodies to ALK (1:1000; Cell Signaling), pALK Tyr1604 (1:1000; Cell
Signaling), and actin (1:2000; Santa Cruz). Immunoblots were quantified with ImageJ (National Institutes of Health).
In vivo tumor growth inhibition
CB17 scid female mice (Taconic Farms) were used to propagate subcutaneously implanted neuroblastoma tumors. Tumor diameters were
measured twice per week with electronic calipers, and tumor volumes
were calculated with the spheroid formula, (p/6)×d3, where d represents mean diameter. Once tumor volume exceeded 200 mm3, mice
were randomized (n = 10 per arm) to receive crizotinib (100 mg/kg
per dose) or vehicle (acidified water) daily by oral gavage for 4 weeks.
Mice were maintained under protocols and conditions approved by
our institutional animal care and use committee. Mice were killed when
tumor volume exceeded 1500 mm3. A mixed-effects linear model was
used to assess tumor volume over time between treatment and vehicle
groups, controlling for tumor size at enrollment. Survival analysis was
performed with the log-rank test with progression defined as tumor
volume exceeding 1500 mm3 or treatment-related death.
In vivo protein and phosphoprotein detection
Mice harboring subcutaneously implanted NB1643 neuroblastoma tumors were randomized once tumor volume exceeded 300 mm3 (n = 3
per arm) to receive crizotinib or vehicle as described above for 2 days.
Mice were killed 4 hours after the final dose, and tumors were immediately snap-frozen in liquid nitrogen and pulverized for extraction of
whole-cell lysates with 100 ml of extraction buffer (Invitrogen) containing protease inhibitor (Sigma), phosphatase inhibitors (Sigma),
and phenylmethylsulfonyl fluoride. Lysates were sonicated, rotated for
1 hour at 4°C, and clarified by centrifugation. Protein concentration
was normalized with the Bradford method, and lysates (200 mg) were
subjected to immunoblotting as outlined above.
Recombinant protein expression and purification
A construct encoding ALK residues 1090 to 1416, together with an
N-terminal hexahistidine tag, was used to express hexahistidinetagged recombinant ALK-TKDs in Sf9 cells as described in the Supplementary Methods.
Native gel kinase assays
Native gel electrophoresis was used to monitor autophosphorylation
progress for 10 mM ALK-TKD in 100 mM Hepes (pH 7.4), 150 mM
NaCl, 2 mM dithiothreitol, 2 mM ATP, and 10 mM MgCl2, either free
in solution or in the presence of lipid vesicles containing 10% DOGSNTA-Ni (100 mM total lipid). Aliquots (10 ml) were taken at each
time point, quenched by adding EDTA to 20 mM and placing on
ice, and then subjected to electrophoresis on 7.5% tris-glycine native
gels at 100 V for ~14 hours. Gels were stained with Coomassie brilliant blue R-250.
Peptide phosphorylation assays
Analysis of substrate phosphorylation by ALK-TKD used a peptide
mimic of the ALK activation loop with the following sequence: biotinARDIYRASYYRKGGCAMLPVK (CanPeptide), referred to as YYY peptide (25). Assay details (with Mg2+ as divalent cation) and analysis of
inhibition are described in the Supplementary Methods. Spontaneous
autophosphorylation is negligible at the ALK-TKD concentrations used
for assays.
SUPPLEMENTARY MATERIAL
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Methods
Fig. S1. Met and pMet protein expression in neuroblastoma cell lines.
Fig. S2. Crizotinib inhibits ALK autophosphorylation and downstream signaling in neuroblastoma
cell lines.
Fig. S3. Effects of MnCl2 on ALK-TKD assays.
Fig. S4. Progress of ALK-TKD autophosphorylation as assessed in native gels.
Fig. S5. Sedimentation equilibrium ultracentrifugation analysis of ALK-TKD variants.
Fig. S6. Inhibition of ALK-TKD by inhibitors in the Enzo ScreenWell and EMD InhibitorSelect
1 to 3 collections.
Fig. S7. Confirmation of Km, ATP values of ALK-TKD variants at different near-saturating peptide
concentrations.
Fig. S8. The F1174L mutation also increases IC50 for staurosporine inhibition.
Fig. S9. The ALK/IGF-1R inhibitor GSK1838705A promotes regression of xenografts containing
R1275Q mutations, but not F1174L or wild-type ALK.
Table S1. Effect of siRNA knockdown of ALK and Met on proliferation of neuroblastoma cell lines.
Table S2. Dependence of Km, ATP on peptide concentration for unphosphorylated ALK-TKD
variants.
Table S3. Crizotinib inhibition of unphosphorylated ALK-TKD in vitro at two different ATP
concentrations.
Table S4. IC50 measurements for crizotinib inhibition of phosphorylated ALK-TKD variants (10 nM).
Table S5. IC50 measurements for in vitro staurosporine inhibition of ALK-TKD variants.
Table S6. IC50 measurements for inhibition of neuroblastoma cell lines by the ALK/InsR/IGF-1R
inhibitor GSK1838705A.
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39. Acknowledgments: We thank members of the Lemmon, Mossé, and Maris laboratories for valuable discussions, and Pfizer for their gift of crizotinib. Funding: This
work was funded in part by the U.S. Army Peer-Reviewed Medical Research Program
(W81XWH-10-1-0212/3 to M.A.L. and Y.P.M.), NIH grant R01-CA140198 (Y.P.M.), the
Carly Hillman Fund (Y.P.M.), the Alex’s Lemonade Stand Foundation (J.M.M.), and the
Abramson Family Cancer Research Institute (J.M.M.). S.C.B. was supported by an NIH
Training Grant in Structural Biology (T32-GM008275). A.C.W. was supported by a fellowship from the St. Baldrick’s Foundation. Author contributions: S.C.B. designed and performed in vitro studies of ALK-TKD, analyzed and interpreted the data, and drafted the
manuscript. A.C.W. designed and performed cellular and in vivo experiments, analyzed
and interpreted the data, and drafted the manuscript. E.A.H., J.C., L.T.B., J.S.P., K.C., Y.T.,
and H.Z. made important contributions to cellular and in vivo experiments and/or data
analysis. E.L.C., J.G.C., J.M.M., Y.P.M., and M.A.L. guided study design, implementation,
and interpretation; provided conceptual direction; and wrote the manuscript with S.C.B.
and A.C.W. Competing interests: J.G.C. is an employee of Pfizer, the developer of crizotinib.
J.M.M. and Y.P.M. are authors on a patent related to the discovery of ALK mutations filed
by the Children’s Hospital of Philadelphia (WO/2009/103061 or PCT/US2009/034288:
Methods and Compositions for Identifying, Diagnosing, and Treating Neuroblastoma).
The other authors declare that they have no competing interests.
Submitted 21 July 2011
Accepted 30 September 2011
Published 9 November 2011
10.1126/scitranslmed.3002950
Citation: S. C. Bresler, A. C. Wood, E. A. Haglund, J. Courtright, L. T. Belcastro, J. S. Plegaria,
K. Cole, Y. Toporovskaya, H. Zhao, E. L. Carpenter, J. G. Christensen, J. M. Maris, M. A. Lemmon,
Y. P. Mossé, Differential inhibitor sensitivity of anaplastic lymphoma kinase variants found in
neuroblastoma. Sci. Transl. Med. 3, 108ra114 (2011).
www.ScienceTranslationalMedicine.org
9 November 2011
Vol 3 Issue 108 108ra114
10
Downloaded from stm.sciencemag.org on November 9, 2011
RESEARCH ARTICLE
www.sciencetranslationalmedicine.org/cgi/content/full/3/108/108ra114/DC1
Supplementary Materials for
Differential Inhibitor Sensitivity of Anaplastic Lymphoma Kinase
Variants Found in Neuroblastoma
Scott C. Bresler, Andrew C. Wood, Elizabeth A. Haglund, Joshua Courtright, Lili T.
Belcastro, Jefferson S. Plegaria, Kristina Cole, Yana Toporovskaya, Huaqing Zhao, Erica
L. Carpenter, James G. Christensen, John M. Maris, Mark A. Lemmon,* Yaël P. Mossé*
*To whom correspondence should be addressed. E-mail: mosse@email.chop.edu (Y.P.M.);
mlemmon@mail.med.upenn.edu (M.A.L.)
Published 9 November 2011, Sci. Transl. Med. 3, 108ra114 (2011)
DOI: 10.1126/scitranslmed.3002950
The PDF file includes:
Methods
Fig. S1. Met and pMet protein expression in neuroblastoma cell lines.
Fig. S2. Crizotinib inhibits ALK autophosphorylation and downstream signaling
in neuroblastoma cell lines.
Fig. S3. Effects of MnCl2 on ALK-TKD assays.
Fig. S4. Progress of ALK-TKD autophosphorylation as assessed in native gels.
Fig. S5. Sedimentation equilibrium ultracentrifugation analysis of ALK-TKD
variants.
Fig. S6. Inhibition of ALK-TKD by inhibitors in the Enzo ScreenWell and EMD
InhibitorSelect 1 to 3 collections.
Fig. S7. Confirmation of Km, ATP values of ALK-TKD variants at different nearsaturating peptide concentrations.
Fig. S8. The F1174L mutation also increases IC50 for staurosporine inhibition.
Fig. S9. The ALK/IGF-1R inhibitor GSK1838705A promotes regression of
xenografts containing R1275Q mutations, but not F1174L or wild-type ALK.
Table S1. Effect of siRNA knockdown of ALK and Met on proliferation of
neuroblastoma cell lines.
Table S2. Dependence of Km, ATP on peptide concentration for unphosphorylated
ALK-TKD variants.
Table S3. Crizotinib inhibition of unphosphorylated ALK-TKD in vitro at two
different ATP concentrations.
Table S4. IC50 measurements for crizotinib inhibition of phosphorylated ALKTKD variants (10 nM).
Table S5. IC50 measurements for in vitro staurosporine inhibition of ALK-TKD
variants.
Table S6. IC50 measurements for inhibition of neuroblastoma cell lines by the
ALK/InsR/IGF-1R inhibitor GSK1838705A.
References
SUPPLEMENTARY METHODS
DNA constructs and retrovirus production. Mutated and wild-type ALK cDNAs were cloned
into the pCMV-XLS vector and subcloned into the retroviral vector MigR1, which also directs
EGFP expression. Infection of retinal pigment epithelial cells (RPE1) that express telomerase
(hTERT-RPE1) was performed as follows: Phoenix Ampho cells (Oribigen – RVC-10001) were
plated ~ 500,000 cells in a 6 well plate in DMEM media with 10% FBS, 1% Pen/Strep,
Gentamicin. Twenty-four hours after plating (~50% confluent), Phoenix cells were transfected
with the ALK expression constructs using a 6:1 dilution of Fugene:plasmid DNA, and viruscontaining medium was harvested 48 hours later. hTERT-RPE1 cells were plated at ~500,000
cells per well in a 6 well plate, and growth medium was replaced with ʻvirus cocktailʼ (2 ml
growth medium, 1ml filtered (0.45µm) viral media from Phoenix cells, 4µg/ml Polybrene (Santa
Cruz)) and incubated overnight. Viral medium then replaced with fresh growth medium, and
hTERT-RPE1 cells were incubated for 48 hours before sorting by flow cytometry for EGFPpositive cells.
Production and purification of recombinant ALK-TKD proteins.
DNA encoding ALK residues 1090-1416, together with an N-terminal hexahistidine tag, was
subcloned into pFastBac-1 (Invitrogen) for expression of histidine-tagged recombinant ALKTKDs in Sf9 cells. Recombinant baculovirus was generated using the Bac-to-Bac system
(Invitrogen). Sf9 cells were infected with recombinant virus for 3 days at 27˚C, harvested by
centrifugation, and lysed by sonication. His-tagged protein was recovered from the lysis
supernatant using NTA-Ni-agarose beads (Qiagen). After elution from NTA-Ni beads, ALK-TKD
was dephosphorylated by incubation with 1µM YopH phosphatase (S1) for 12 hours at 4˚C to
reverse spontaneous autophosphorylation that occurs within Sf9 cells during expression. ALKTKD was then flowed over a cation exchange column (to remove YopH), exchanged into buffer
containing 1M (NH4)2SO4, and applied to a butyl sepharose column (GE Healthcare). Protein
was eluted with a 20 column-volume linear gradient to 0M (NH4)2SO4. ALK-TKD fractions were
pooled and concentrated, and gel filtered using a Superdex 200 column (GE Healthcare)
equilibrated in 25mM HEPES pH 7.4, 150mM NaCl, 4mM DTT. Mass spectrometry confirmed
that dephosphorylation of the starting material was complete. Protein concentrations were
determined by absorbance at 280nm using the calculated extinction coefficient 39440cm-1M-1.
Phosphorylated ALK-TKD for peptide-based assays was produced in 100mM HEPES pH 7.4,
150mM NaCl, 2mM DTT,10mM MgCl2, 2mM ATP in the presence of lipid vesicles containing
10% DOGS-NTA-Ni (Avanti Polar Lipids) in a background of dioleoylphosphatidylcholine (Avanti
Polar Lipids) prepared as described (S2) with a final concentration of 10µM DOGS-NTA-Ni
(100µM total lipid). Full ALK-TKD autophosphosphorylation was achieved within 50mins (Fig.
4A, lower panel) under these conditions at 25˚C. Reactions were quenched where necessary by
addition of EDTA to a final concentration of 20mM, followed by desalting under the conditions
described above for gel filtration. EGFR-TKD was prepared as described (S2). Mass
spectrometry-based phosphopeptide analysis confirmed that ALK-TKD autophosphorylation
occurs at the expected sites within the activation loop, as well as additional sites.
In vitro protein and phosphoprotein detection.
Each neuroblastoma cell line was cultured in ten T75 flasks under standard cell culture
conditions. At 70-80% confluence crizotinib was added to cell culture medium to achieve a
designated final concentration at one of nine doses ranging from 0nM to 10,000nM. Cells were
incubated for 2 hours with drug, then collected, pelleted, and washed twice with ice cold
phosphate-buffered saline (PBS). Whole cell lysates were then harvested, separated and
2
immunoblotted as previously described (S3). The following antibodies were used, according to
the supplierʼs instructions: anti-ALK (1:1,000; Cell Signaling, 3333), anti-phospho-ALK Tyr 1604
(1:1,000, Cell Signaling, 3341), anti-STAT3 (1:1,000; Cell Signaling, 9132), antiphospho-STAT3
Tyr 705 (1:1,000; Cell Signaling, 9145), anti-AKT (1:1,000; Cell Signaling, 9272), anti-phosphoAKT Ser 473 (1:1,000; Invitrogen, 44-621G), anti-p44/42 MAPK (ERK1/2) (1:1,000; Cell
Signaling, 4695), anti-phospho-p44/42 MAPK (ERK 1/2) (1:1,000; Cell Signaling, 9101), antiactin (1:2,000; Santa Cruz, sc-1616).
Quantification of native gels.
Quantification of autophosphorylation progress employed ImageJ (NIH). Background was
subtracted using a rollerball radius of 50 pixels. Using the gel analysis tool, the intensity of each
band in the native gel was determined and normalized to the total intensity in each respective
lane. Data were plotted as fraction of total intensity in each lane vs. reaction time.
Sedimentation equilibrium analytical ultracentrifugation.
ALK-TKD proteins were diluted to 10 and 16.5µM in 25mM HEPES pH 7.4, 150mM NaCl, 2mM
DTT, and subjected to sedimentation equilibrium ultracentrifugation at 13,000, 19,000, and
25,000 rpm in a Beckman Optima XL-A ultracentrifuge. Representative data from 13,000 rpm at
10µM are shown in fig. S5. Data are plotted as ln(abs280) vs (r2 -r02)/2, where r is the radial
distance of the sample and r0 is the radial distance of the meniscus. The slope of the line is
proportional to the weight-average molecular weight of the species in the sample. Data were
analyzed using Sedphat (http://analyticalultracentrifugation.com).
Inhibitor profiling using commercially available kinase inhibitor screens.
Kinase inhibitor screens containing 320 (overlapping) commercially available and wellcharacterized ATP-competitive inhibitors (Enzo Life Sciences ScreenWell and EMD
InhibitorSelect Kinase Inhibitor Libraries 1-3) were used to compare the inhibitor selectivities of
wild-type, R1275Q, and F1174L ALK-TKD. ALK TKD (2µM) was incubated with ATP (200µM,
corresponding to the approximate Km,ATP of ALK-TKD at 10mM MgCl2) in 96-well format with
50µM of each inhibitor (DMSO only in the negative control wells, or TAE-684 in the positive
control wells) in a final reaction volume of 10µl. After incubation at room temperature, reactions
were quenched with by adding EDTA to a final concentration of 20mM in 25mM HEPES pH 7.4,
150mM NaCl, 4mM DTT, and spotted onto nitrocellulose membranes in triplicate. Membranes
were immunoblotted with anti-phosphotyrosine (PY20, 1:1000, Biomol), detecting with HRPconjugated α-mouse IgG (1:1000, GE Healthcare).
In vitro kinase assays.
Analysis of substrate phosphorylation by ALK-TKD employed a peptide mimic of the ALK
activation loop with sequence: biotin-ARDIYRASYYRKGGCAMLPVK (CanPeptide), referred to
as YYY peptide (S4). Autophosphorylated ALK-TKD was used at 10nM in these assays,
whereas unphosphorylated kinase was studied at 500nM (wild-type) or 50nM (R1275Q and
F1174L), concentrations at which spontaneous autophosphorylation is negligible. Assays
monitored incorporation of 32P from γ-32P ATP included in trace amounts (~20µCi per
experiment) in the reactions. Comparisons with other peptides confirmed that peptide
biotinylation does not affect kinetic parameters, and that reaction rates were linear with respect
to both enzyme concentration and time under the assay conditions used (fig. S3E,F). Peptide
concentrations were determined by absorbance at 280nm using a calculated extinction
coefficient 3960cm-1M-1.
3
Assays were performed in 100mM HEPES pH 7.4, 150mM NaCl, 2mM DTT, 10mM
MgCl2, and 0.5mg/mL BSA at 25˚C, and were initiated by mixing equal volumes of solutions
containing ALK-TKD and substrates respectively (at twice the desired final concentration). For
determination of kcat and Km, peptide, peptide concentration was varied from 0 to 2mM, and ATP
was saturating (2mM). For determination of Km, ATP, peptide was present at a fixed concentration
of 1 or 2mM, and ATP was varied from 0.015625 to 2mM. Aliquots were taken at each time
point, spotted onto pieces of phosphocellulose paper (Upstate Biotechnology), and immediately
quenched with 0.5% phosphoric acid followed by extensive washing and drying with acetone.
Incorporated radioactivity was measured by scintillation counting with appropriate background
correction. Initial rates (determined at <10% product formation) were calculated using measured
apparent γ-32P ATP specific activity, normalized to enzyme concentration, and fit to the
Michaelis-Menten equation (vo = vmax[S]/(Km+[S])) using GraphPad Prism 4.0. The enzyme was
assumed to have one active site per molecule, therefore kcat = vmax at saturating ATP
concentrations.
For experiments with added vesicles, which were prepared essentially as described
(S2), the appropriate amount of vesicle solution (10µM DOGS-NTA-Ni, 100 or 200µM total lipid
depending on the molar percentage of DOGS-NTA-Ni used) was added to the 2x protein
mixture, and the reaction was performed and processed as above. Control experiments were
performed with vesicles containing only dioleoylphosphatidylcholine (200µM total lipid). Enzyme
concentration was held fixed at 1µM, with 2mM peptide and 2mM ATP present. Experiments
using EGFR-TKD were performed with poly-Glu4Tyr (Sigma) instead of YYY peptide. Data are
reported in Table 1 and tables S2-S5 as the mean of individual experiments (in at least
triplicate) ± SEM. p values were determined using an unpaired t-test in GraphPad Prism 4.0.
In vitro analysis of ALK-TKD inhibition.
Inhibitor was added to the 2x substrate mixture prior to initiating reactions with ALK-TKD
addition, so that final reactions represented a log2 dilution series from 0 to 25,600nM inhibitor.
Protein concentration was held at 50nM, and assays were performed as described above. The
concentration of ʻYYYʼ peptide was held fixed at 0.5mM, and ATP was present at 0.2mM or
2mM. Background counts from a no-enzyme control were subtracted, the data were normalized
to the 0nM inhibitor reaction and were fit to a sigmoidal dose-response (variable slope) equation
in GraphPad Prism 4.0. Data are reported as means of individual experiments (in at least
triplicate) ± SEM. p values were determined using an unpaired t-test in GraphPad Prism 4.0.
4
Figure S1. Met and pMet protein expression in neuroblastoma cell lines.
Immunoblots showing native Met and phospho-Met expression in 7 neuroblastoma cell lines, as
well as native HeLa cells and HeLa cells treated with hepatocyte growth factor (HGF), the ligand
for Met. F1174L-mutated ALK is expressed in NBSD and KELLY cells. R1275Q ALK is
expressed in NB1643 cells. NB1 cells have amplified wild-type ALK, and IMR32 cells express
wild-type ALK at normal levels. NLF and SKNAS cells harbor no ALK alteration (and do not
appear to express ALK).
5
Figure S2. Crizotinib inhibits ALK autophosphorylation and downstream signaling in
neuroblastoma cell lines.
Indicated cell lines were treated with the noted crizotinib concentrations and prepared for
immunoblotting of whole cell lysates as described in Supplementary Methods. Diminution of
pAkt levels approximately parallels that of ALK phosphorylation, with pAkt levels maximally
reduced by ~100nM crizotinib in NB1 cells, ~333nM in NB1643 cells, but not until 10,000nM in
SH-SY5Y cells. Substantial reduction of pSTAT3 levels requires higher crizotinib concentrations
in all three cell lines, and pERK remains detectable even at 10,000nM crizotinib in NB1643 and
SH-SY5Y cells. These data are consistent with previous reports that inhibition (or knockdown)
of mutated ALK promotes apoptosis of neuroblastoma cell lines (S5). The differences in
ʻsharpnessʼ of pAkt and pERK inhibition between NB1 and NB1643/SH-SY5Y cells appear to
reflect receptor expression levels (which are very high in NB1).
.
6
Figure S3. Effects of MnCl2 on ALK-TKD assays.
(A) MnCl2 increases the rate of phosphotransfer catalyzed by unphosphorylated ALK-TKD
(250nM). Results are representative of two independent experiments performed with ʻYFFʼ
peptide (0.5mM) in the presence of 50µM ATP. (B) Km, ATP for wild-type unphosphorylated ALKTKD is reduced to 21.3±4.33µM by the addition of 10mM MnCl2 (measured at 1mM ʻYYY”
peptide, 250nM ALK-TKD, mean ± SEM of four independent experiments), compared with a
value of 134±7µM in the presence of 10mM MgCl2 alone (500nM ALK-TKD and 1mM ʻYYYʼ
peptide). (C) The presence of MnCl2 (5mM) diminishes the apparent activating effects of
F1174L and R1275Q mutations, as assessed using native gel electrophoresis to monitor
progress of autophosphorylation. In addition, increased basal activity of unphosphorylated ALKTKD is promoted by MnCl2. All reactions were performed at 37˚C with 10µM ALK-TKD and 1mM
ATP. (D) MnCl2 (10mM) in addition to MgCl2 (10mM) reduces activity of fully
autophosphorylated wild-type ALK-TKD (100nM) using 0.5mM ʻYYYʼ peptide as the
phosphoacceptor with 0.2mM ATP present. Results are representative of two independent
experiments. (E) Initial rates in the final optimized conditions were linear with time, shown here
with unphosphorylated proteins (500nM for wild-type, 50nM for R1275Q and F1174L), 2mM
ʻYYYʼ peptide, and saturating ATP (2mM). Data are shown as mean ± SEM for at least 3
independent experiments. (F) Reaction rates in the final optimized conditions were also linear
with enzyme concentration from 0 to 0.7µM for unphosphorylated wild-type ALK-TKD in the
presence of 1mM ʻYYYʼ peptide and saturating ATP (1mM).
7
Figure S4. Progress of ALK-TKD autophosphorylation as assessed in native gels.
(A) Species corresponding to unphosphorylated (0P), singly phosphorylated (1P) and those with
2 (2P), 3 (3P) or 4 (4P) phosphates respectively are tracked with time as denoted in the key –
for wild-type, R1275Q, and F1174L ALK-TKD (10µM) with 2mM ATP and 10mM MgCl2 as
shown in Fig. 4B. In (B), the kinetics of appearance/disappearance of each different
phosphospecies are directly compared across the three ALK-TKD variants. All data are shown
as mean ± SEM of three independent experiments.
8
Figure S5. Sedimentation equilibrium ultracentrifugation analysis of ALK-TKD variants.
Purified wild-type, F1174L, and R1275Q ALK-TKD were subjected to sedimentation equilibrium
analytical ultracentrifugation to measure molecular weight and assess propensity to selfassociate (which might be affected by activating mutations). Proteins were centrifuged at three
different speeds (13,000, 19,000 and 25,000 r.p.m.) at two different concentrations (10µM and
16.5µM). Efforts were made to globally fit data to models assuming either a single species or
self-associating species, and best fits suggested a single species with molecular weight of
38.2kDa (wild-type), 42.4kDa (R1275Q) and 39.6kDa (F1174L). Data from representative
experiments performed at 10µM ALK-TKD (at 13,000 r.p.m.) are plotted as ln(abs280) against (r2ro2)/2. This representation yields a straight line for a single species, with slope proportional to
molecular weight. The data (and fits) suggest that – at concentrations in the 10-20µM range –
each ALK-TKD variant is monomeric, excluding promotion of dimerization as a mechanism of
TKD activation by these neuroblastoma mutations.
9
Figure S6. Inhibition of ALK-TKD by inhibitors in the Enzo ScreenWell and EMD
InhibitorSelectTM 1 to 3 collections.
Inhibitor screens using a 96-well plate autophosphorylation assay were performed as described
in Supplementary Methods. Several new hits were identified that could be used as scaffolds for
future ALK inhibitors. The left-hand arrays correspond to the Enzo ScreenWell panel of 80
kinase inhibitors. Others employed EMD InhibitorSelect Libraries 1-3 as marked. Inhibition was
analyzed as described in Supplementary Methods by monitoring autophosphorylation of ALKTKD variants (2µM) with 0.2mM ATP in anti-pY immunoblots (shown in inverse grey scale).
DMSO negative controls are boxed in green. TAE-684 positive inhibition controls are boxed in
red. No significant difference in inhibitor selectivity profile is apparent when comparing wild-type,
F1174L and R1275Q ALK-TKD, suggesting that the mutations do not substantially alter the
nature of the ATP binding site. Components of Enzo Screen-WellTM are listed at
http://www.enzolifesciences.com/BML-2832/kinase-inhibitor-library, and those of EMD
InhibitorSelectTM are listed at http://www.emdchemicals.com/life-science-research.
10
Figure S7. Confirmation of Km, ATP values of ALK-TKD variants at different nearsaturating peptide concentrations.
(A) Since using a truly saturating level of peptide in Km, ATP assays was not feasible, we
determined Km, ATP values for unphosphorylated ALK-TKD variants in the presence of 2mM
peptide. Results show a very similar profile to that determined at 1mM peptide (Fig. 5C), with
Km, ATP values very slightly increased. The comparative profile for the different ALK-TKD variants
(500nM for wild-type, 50nM for mutants) is maintained. (B) Direct comparison of Km, ATP values
determined in assays employing 1mM and 2mM peptide. Data are shown as mean ± SEM of
three independent experiments, and are also shown in table S2.
11
Figure S8. The F1174L mutation also increases IC50 for staurosporine inhibition.
The ability of staurosporine to inhibit ALK-TKD in vitro was analyzed as described for crizotinib
in Figure 5 G and H. The IC50 for staurosporine inhibition with 0.2mM ATP (A) is slightly higher
for F1174L-mutated ALK-TKD than for the R1275Q variant (see table S5). As with crizotinib, this
difference is enhanced when experiments were performed in the presence of 2mM ATP (B),
more closely resembling cellular concentrations. All data are shown as the mean ± SEM from at
least three independent experiments. The reduced Km,ATP for F1174L ALK-TKD also appears to
increase IC50 for staurosporine inhibition.
12
Figure S9. The ALK/IGF-1R inhibitor GSK1838705A promotes regression of xenografts
containing R1275Q mutations, but not F1174L or wild-type ALK.
GSK1838705A is another ATP-competitive ALK kinase inhibitor that also inhibits the insulin
receptor and IGF-1R (S6). GSK1838705A also showed significant single agent activity against
the NB1643 cell line (R1275Q) xenografted into CB17 scid-/- mice, but not against WT or
F1174L xenografts. By contrast, the IGF-1R inhibitor GSK1904529A (S7) had no significant
effect. Mice were randomized to GSK1838705A 60mg/kg/day once daily, GSK1904529A
30mg/kg/day once daily, or with vehicle (N=10/arm) following xenotransplantation and
establishment of a tumor at 200 mm3. GSK1838705A caused complete regression of NB1643
xenografts within two weeks. Dosing was continued for a further two weeks, during which time
tumors remained in macroscopic remission.
13
Table S1. Effect of siRNA knockdown of ALK and Met on proliferation of neuroblastoma
cell lines.
ALK siRNA
Met siRNA
Relative proliferation
KELLY EBC1 SKNAS
NLF
(F1174L) (WT)
(WT)
(WT)
0.190
0.728
0.930
0.729
0.772
0.421
0.797
0.439
Proliferation of the KELLY cell line harboring a F1174L mutation is inhibited by siRNA
knockdown of ALK, but shows less inhibition with Met knockdown. The remaining three cell
lines harbor wild-type normal copy number ALK. Cells were plated overnight and then
transfected in triplicate with a pool of four siRNA targeting each kinase (Thermo Scientific
Kinase siGenome Library), with positive (siPLK1) and negative (GAPD) controls. Substrate
adherent growth was monitored for 100 hours using the Roche xCELLigence system. The area
under the curve was calculated for each siRNA triplicate and mock transfection. % relative
growth = AUC of the siKinase/AUC siControl.
Table S2. Dependence of Km, ATP on peptide concentration for unphosphorylated ALKTKD variants. (Data are shown as mean ± SEM for at least 3 independent
experiments).
kinase
WT
R1275Q
F1174L
Km, ATP, 1mM peptide
(mM)
0.134 ± 0.007
0.326 ± 0.033
0.127 ± 0.011
Km, ATP, 2mM peptide
(mM)
0.181 ± 0.004
0.460 ± 0.024
0.179 ± 0.022
Table S3. Crizotinib inhibition of unphosphorylated ALK-TKD in vitro at two different
ATP concentrations. (Data are shown as mean ± SEM for at least 3 independent
experiments).
kinase
WT
R1275Q
F1174L
IC50, 2 mM ATP
(nM)
147 ± 6
84.6 ± 8.0
130 ± 10
IC50, 0.2 mM ATP
(nM)
-80.4 ± 4.8
88.5 ± 6.4
14
Table S4. IC50 measurements for crizotinib inhibition of phosphorylated ALK-TKD
variants (10 nM).
kinase
pWT
pR1275Q
pF1174L
IC50
(nM)
93.3
67.3
111.0
Table S5. IC50 measurements for in vitro staurosporine inhibition of ALK-TKD variants.
(Data are shown as mean ± SEM for at least 3 independent experiments).
kinase
R1275Q
F1174L
IC50, 0.2mM ATP
(nM)
227 ± 32.0
271 ± 26.7
IC50, 2.0mM ATP
(nM)
404 ± 25.6
545 ± 39.5
Table S6. IC50 measurements for inhibition of neuroblastoma cell lines by the
ALK/InsR/IGF-1R inhibitor GSK1838705A.
ALK
status
R1275Q
F1174L
F1174L
F1174L
WT amp
WT,
normal
copy
number
Cell line
NB1643
NBSD
KELLY
SH-SY5Y
IMR5
SKNFI
NGP
NB16
NLF
IC50 for
GSK1838705A(nM)
124
10000
605
781
7911
10000
10000
10000
10000
GSK1838705A is an ATP-competitive inhibitor of the ALK, insulin receptor, and IGF-1R tyrosine
kinases GSK1838705A (S6, S8).
15
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16