Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl.
ABSTRACT The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 <30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.
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ABSTRACT: We designed a series of 2-methylpyrimidine derivatives as new BCR-ABL inhibitors using scaffold-hopping strategy. These synthetic compounds exhibited significant inhibition against a broad spectrum of Bcr-Abl mutants including the gatekeeper T315I mutant. Compound 7u showed very potent kinase inhibitory activities against Bcr-Abl WT, Bcr-Abl E255K, Bcr-Abl Q252H, Bcr-Abl G250E and Bcr-Abl T315I, with IC50 values of 0.13 nM, 0.17 nM, 0.24 nM, 0.19 nM and 0.65 μM, respectively. This compound also displayed anti-proliferation activity against K562 cell line with an IC50 value of 1.1 nM, thus representing a new lead for further optimization.Science China-Chemistry 06/2013; 57(6):823-832. · 1.52 Impact Factor
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ABSTRACT: Tyrosine kinase inhibitors (TKIs) have profoundly changed the natural history of chronic myeloid leukemia (CML). However, acquired resistance to imatinib, dasatinib or nilotinib (1(st) and 2(nd) generation TKIs), due in part to BCR-ABL1 kinase mutations, has been largely described. These drugs are ineffective on the T315I gatekeeper substitution, which remains sensitive to 3(rd) generation TKI ponatinib. It has recently been suggested that the hematopoietic niche could protect leukemic cells from targeted therapy. In order to investigate the role of a stromal niche in mutation-related resistance, we developed a niche-based cell mutagenesis assay. For this purpose, ENU (N-ethyl-N-nitrosourea)-exposed UT-7 cells expressing non-mutated or T315I-mutated BCR-ABL1 were cultured with or without murine MS-5 stromal cells and in the presence of imatinib, dasatinib, nilotinib, or ponatinib. In the assays relative to 1(st) and 2(nd) generation TKIs, which were performed on non-mutated BCR-ABL1 cells, our data highlighted the increasing efficacy of the latter, but did not reveal any substantial effect of the niche. In ponatinib assays performed on both non-mutated and T315I-mutated BCR-ABL1 cells, an increased number of resistant clones were observed in the presence of MS-5. Present data suggested that T315I mutants need either compound mutations (e.g. E255K/T315I) or a stromal niche to escape from ponatinib. Using array-comparative genomic hybridization experiments, we found an increased number of variations (involving some recurrent chromosome regions) in clones cultured on MS-5 feeder. Overall, our study suggests that the hematopoietic niche could play a crucial role in conferring resistance to ponatinib, by providing survival signals and favoring genetic instability.Oncoscience. 01/2014; 1(1):57-68.
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ABSTRACT: ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.PLoS ONE 11/2014; 9(11):e112318. · 3.53 Impact Factor
A R T I C L E
Characterization of AMN107, a selective inhibitor
of native and mutant Bcr-Abl
Ellen Weisberg,1,5Paul W. Manley,2,5Werner Breitenstein,2Josef Brüggen,2Sandra W. Cowan-Jacob,2
Arghya Ray,1Brian Huntly,3Doriano Fabbro,2Gabriele Fendrich,2Elizabeth Hall-Meyers,1Andrew L. Kung,1,4
Jürgen Mestan,2George Q. Daley,4Linda Callahan,1Laurie Catley,1Cara Cavazza,1Azam Mohammed,4
Donna Neuberg,1Renee D. Wright,3D. Gary Gilliland,3and James D. Griffin1,*
1Dana-Farber Cancer Institute, Boston, Massachusetts 02115
2Novartis Institutes for Biomedical Research, Basel, CH-4002, Switzerland
3Brigham and Women’s Hospital, Boston, Massachusetts 02115
4Children’s Hospital, Boston, Massachusetts 02115
5These authors contributed equally to this work.
The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-posi-
tive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50< 30
nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants.
Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of
AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107
prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and pro-
longed survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and
patients express the 210 kDa Bcr-Abl, whereas patients with
Ph+ ALL usually express a p190 kDa Bcr-Abl protein arising
from a different breakpoint in the BCR gene (Melo et al., 1994;
Ravandi et al., 1999).
Expression of either p210 or p190 Bcr-Abl in hematopoietic
cell lines abrogates the growth factor requirements for cell pro-
liferation and survival by three major mechanisms: constitutive
activation and enhancement of mitogenic signaling (Puil et al.,
1994), reduced responsiveness to apoptotic stimuli (Bedi et al.,
1994), and altered adhesion to stroma cells and extracellular
matrix (Gordon et al., 1987). The constitutively activated tyro-
sine kinase of Bcr-Abl is essential for the transforming activity
(Lugo et al., 1990).
Imatinib mesylate (Gleevec, STI571; Novartis Pharma AG) is
a drug targeting the tyrosine kinase activity of Bcr-Abl (Buch-
dunger et al., 2001) and is an effective therapy for CML. After
a median 19 months of treatment, newly diagnosed patients
show an estimated 97% complete hematological response
(CHR) and 76% complete cytogenetic response (CCR; no de-
tectable Ph+ cells) (O’Brien et al., 2003). However, in ALL or in
CML patients who have progressed to either the accelerated
or blastic phases of the disease, response rates to imatinib
Chronic myelogenous leukemia (CML) constitutes about 15%
of adult leukemias and annually affects 1–2 people per
100,000. The disease progresses in three phases (O’Dwyer et
al., 2002): the initial chronic phase, which has a median dura-
tion of 4–6 years, is a clonal myeloproliferative disorder charac-
terized by a massive accumulation of functional granulocytes
and immature myeloid cells in blood, marrow, and spleen. In
untreated patients, the disease may progress via an ac-
celerated phase, characterized by the appearance of undif-
ferentiated blast cells in blood and bone marrow, to a terminal
blast crisis phase of the disease. In the blastic phase, for which
median survival is 18 weeks, more than 30% of the blood and
bone marrow cells are blasts, and myeloid precursors may also
form tumors in the lymph nodes, skin, and bone (Kantarjian
and Talpaz, 1988).
The underlying cause of CML is the BCR-ABL oncogene,
which results from a reciprocal t(9;22) chromosome transloca-
tion in a hematopoietic stem cell (Deininger et al., 2000). This
fusion gene encodes a chimeric Bcr-Abl protein, in which the
tyrosine kinase activity of Abl is constitutively activated. CML
S I G N I F I C A N C E
Imatinib is an effective therapy for chronic phase CML, but advanced stage CML and Ph+ ALL patients frequently relapse due to
the development of resistance caused by point mutations within the kinase domain of Bcr-Abl. New Abl kinase inhibitors with higher
potency against native and imatinib-resistant mutants of Bcr-Abl could have substantial clinical utility. AMN107 is a high-affinity
inhibitor that targets many imatinib-resistant mutants of Bcr-Abl, and which may therefore be useful in CML, reducing the incidence
of resistant mutants, and in the treatment of imatinib-resistant disease.
CANCER CELL : FEBRUARY 2005 · VOL. 7 · COPYRIGHT © 2005 ELSEVIER INC.DOI 10.1016/j.ccr.2005.01.007 129
A R T I C L E
Figure 1. Effects of AMN107 on Bcr-Abl-expressing cell lines in vitro
A: The chemical structures of imatinib and AMN107.
B: Comparison of effects of 3-day treatment of Bcr-Abl-expressing cell lines with imatinib and AMN107 (upper panels), in the presence and the absence
of WEHI conditioned medium as a source of IL-3 (lower panels). Bars are SEM, n = 2. In all dose-response curves, viable cell counts are represented as
percent of control cells for each drug dose, and were determined by trypan blue exclusion assay.
C: Inhibition of Bcr-Abl autophosphorylation by AMN107 in Bcr-Abl-expressing cells. Autophosphorylation was determined by Bcr-Abl immunoprecipitation,
followed by a pTyr immunoblot. Treatment with AMN107 was carried out in both the absence and the presence of IL-3.
D: Dose-response curves generated for combinations of AMN107 and imatinib against Ba/F3.p210 (upper panels), demonstrating synergy (lower panel).
Combination studies using Ba/F3.p210 were performed in duplicate; shown are the data for one representative experiment.
therapy are significantly decreased and, of those who initially
respond to treatment, many relapse within 12 months. Relapse
is often associated with point mutations in Bcr-Abl that reduce
the binding affinity of imatinib, or occasionally with amplifica-
tion of the BCR-ABL gene (Gorre et al., 2001; Cowan-Jacob et
al., 2004; le Coutre et al., 2000; Weisberg and Griffin, 2000;
Mahon et al., 2000; Campbell et al., 2002; Hochhaus et al.,
2002; Morel et al., 2003). Thus, there is a need for additional
Bcr-Abl tyrosine kinase inhibitors that are more potent and
active against imatinib-resistant Bcr-Abl mutants.
The molecular details of the interaction of imatinib with the
Abl kinase domain have been revealed from crystal structures
of complexes (Schindler et al., 2000; Nagar et al., 2003;
Cowan-Jacob et al., 2004), and further supported by an analy-
sis of the effects on binding of point mutations in the protein
(Cowan-Jacob et al., 2004). Based upon this structural data,
we hypothesized that more potent and selective compounds
could be designed by incorporating alternative binding groups
for the N-methylpiperazine group, while retaining an amide
pharmacophore to maintain the H-bond interactions to Glu286
and Asp381 (Manley et al., 2004). This approach resulted in
the discovery of AMN107 (Figure 1A), and here we detail the
characterization of this molecule in vitro and in experimental
Bcr-Abl-driven models of leukemia in mice. AMN107 has supe-
rior potency to imatinib as an inhibitor of Bcr-Abl in vitro and
in vivo. Furthermore, we report on the efficacy of AMN107 in
inhibiting some imatinib-resistant mutant forms of the kinase
both in vitro and in vivo.
CANCER CELL : FEBRUARY 2005
A R T I C L E
Table 1. Comparison of imatinib and AMN107 for effects on autophosphorylation and proliferation in cells
Imatinib (IC50in nM) AMN107 (IC50in nM)
Kinase (cell type) AutophosphorylationProliferationAutophosphorylationProliferation
wt-32D + IL-3
wt-Ba/F3 + IL-3
p210 Bcr-Abl (32D)
p210 Bcr-Abl (Ba/F3)
p190 Bcr-Abl (Ba/F3)
E255K Bcr-Abl (Ba/F3)
E255V Bcr-Abl (Ba/F3)
T315I Bcr-Abl (Ba/F3)
F317L Bcr-Abl (Ba/F3)
M351T Bcr-Abl (Ba/F3)
F486S Bcr-Abl (Ba/F3)
M244V Bcr-Abl (Ba/F3)
L248R Bcr-Abl (Ba/F3)
Q252H Bcr-Abl (Ba/F3)
Y253H Bcr-Abl (Ba/F3)
E279K Bcr-Abl (Ba/F3)
E282D Bcr-Abl (Ba/F3)
V289S Bcr-Abl (Ba/F3)
L384M Bcr-Abl (Ba/F3)
G250E Bcr-Abl (Ba/F3)
PDGFR-α + PDGFR-β (A31)
PDGFR-β (Tel Ba/F3
c-Kit exon13 mutant (GIST882)
c-Kit del 560-561 (Ba/F3)
NPM-Alk (Ba/F3-NPM-Alk cl. 1)
Akt (Ba/F3-MyrAkt, cl. 21)
194 ± 7, n = 94
470 ± 59, n = 15
466 ± 59, n = 7
220 ± 36, n = 12
122 ± 15, n = 3
2108 ± 367, n = 3
6499 ± 666, n = 13
>10000, n = 20
818 ± 99, n = 10
595 ± 63, n = 10
1230 ± 121, n = 10
74 ± 11, n = 11
96 ± 12, n = 7
27, n = 1
>10000, n = 6
>10000, n = 4
6140 ± 449, n = 14
7384 ± 766, n = 5
334 ± 37, n = 23
272 ± 27, n = 21
80 ± 35, n = 13
649 ± 52, n = 18
20 ± 1, n = 53
43 ± 15, n = 3
60 ± 19, n = 5
21 ± 2, n = 5
33 ± 4, n = 3
150 ± 12, n = 3
246 ± 36, n = 8
>10000, n = 12
41 ± 5, n = 8
31 ± 4, n = 8
43 ± 6, n = 4
71 ± 7, n = 20
200 ± 13, n = 19
27 ± 3, n = 3
3720 ± 920, n = 5
>10000, n = 2
6134 ± 228, n = 3
>10000, n = 15
9.2 ± 0.3, n = 3
12 ± 2, n = 3
8 ± 2, n = 6
25 ± 2, n = 49
>6000, n = 3
6368 ± 892, n = 11
7393 ± 157, n = 3
1583 ± 236, n = 14
1285 ± 180, n = 13
2728 ± 676, n = 7
3100*wst, n = 1
>20000*wst, n = 1
2900*wst, n = 1
17700*wst, n = 1
9900*wst, n = 1
1300*wst, n = 1
1400*wst, n = 1
2800*wst, n = 1
4800*wst, n = 1
566 ± 107, n = 3
681 ± 72, n = 12
>10000, n = 15
80 ± 6, n = 11
33 ± 3, n = 10
87 ± 4, n = 4
39wst, n = 1
919wst, n = 1
16wst, n = 1
751wst, n = 1
75wst, n = 1
39wst, n = 1
7wst, n = 1
39wst, n = 1
219wst, n = 1
39 ± 4, n = 8
120 ± 6, n = 15
27 ± 2, n = 2
57 ± 7, n = 18
160 ± 12, n = 17
26 ± 1, n = 4
>10000, n = 6
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>10000, n = 1
>10000, n = 1
>10000, n = 4
>10000, n = 4
>3000AB, n = 3
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000, n = 1
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000AB, n = 2
>3000, n = 2
The influence of compounds on kinase autophosphorylation or cell viability was calculated as percentage inhibition. Dose-response curves were used to calculate IC50
values, expressed as mean ± SEM, n = number of experiments. The antiproliferative activity was assessed using either the ATPlite assay kit (Perkin-Elmer) or, where
indicated (AB), the Alamar Blue assay kit (Biosource International Inc.).wst, WST-1 reagent (Roche), as previously described (Azam et al., 2003), was used to generate
IC50 values. *, values as reported previously for imatinib (Azam et al., 2003)
normal human myeloid and erythroid progenitor cells (assayed
as CFU-GM [colony-forming unit, granulocyte/macrophage]
and BFU-E [burst-forming unit, erythroid]) at concentrations
%100 nM, but resulted in w50% reduction in colony numbers
at 1 ?M (Supplemental Data). In preliminary mouse tolerability
studies, AMN107 (50 or 150 mg/kg p.o. b.i.d. for 14 days) was
well tolerated, and there were no significant reductions in blood
erythrocytes, reticulocytes, leukcocytes, or platelets.
AMN107 potently inhibited proliferation of cells transformed
by activated mutants of Arg, Kit, PDGFRα, and PDGFRβ (Table
1 and Supplemental Data), but had no significant effect on the
viability or proliferation of Ba/F3 cells rendered factor-indepen-
dent through expression of activated forms of erbB2, Flt3, Met,
Ret, IGF-1R, or NPM-ALK, and several other tyrosine kinases
at concentrations %3 ?M (Table 1). Taken together, these data
suggest that AMN107 selectively inhibits Bcr-Abl, Kit, and
PDGFR tyrosine kinases, but does not significantly affect any
of the kinases required for IL-3 signaling, such as JAK2, or a
AMN107 selectively inhibits proliferation
of Bcr-Abl-expressing cells and inhibits
AMN107 inhibited the proliferation of Ba/F3 cells expressing
p210- and p190-Bcr-Abl, or K562 and Ku-812F cells with IC50
values %12 nM (Figure 1B and Table 1). AMN107 was at least
10-fold more potent than imatinib against Bcr-Abl expressing
cell lines, but like imatinib, did not inhibit untransformed Ba/F3
cells growing in IL-3 at concentrations %6 ?M. Ba/F3 cells
expressing p210 and p190 Bcr-Abl could also be partially res-
cued from the inhibitory effects of up to 1 ?M AMN107 when
cultured in the presence of IL-3 (Figure 1B). At concentrations
of AMN107 higher than 1 ?M, cells died even in the presence
of IL-3 (Figure 1B and Supplemental Data). Inhibition of cell
growth by AMN107 was associated with induction of apoptosis
(Supplemental Data). AMN107 did not reduce the formation of
CANCER CELL : FEBRUARY 2005 131
A R T I C L E
Figure 2. Effects of AMN107 on imatinib-resistant and wild-type Bcr-Abl-expressing cells in vitro
A and B: Treatment of Bcr-Abl mutant-expressing and wild-type Bcr-Abl-expressing Ba/F3 cells with imatinib versus AMN107. A: Upper panel: Imatinib
treatment; bars are SEM, n = 3. Lower panel: AMN107 treatment, n = 3. B: Left panel: Imatinib treatment. Right panel: AMN107 treatment, n = 2.
C: Inhibition of Bcr-Abl autophosphorylation by AMN107 in imatinib-resistant Bcr-Abl-expressing cells. Bcr-Abl autophosphorylation was determined as in
broad variety of other receptor tyrosine kinases or tyrosine ki-
nase oncogenes. Furthermore, in cell-free assays, AMN107 at
concentrations <3 ?M had no significant effect on transphos-
phorylation catalyzed by the GST-fusion kinase domains of
CDK-1, FGFR-1, Flt-3, HER-1, IGF-1R, InsR, c-Met, PKA, PKB,
c-Src, Tie-2, or VEGFR-2 (data not shown).
Imatinib and AMN107 were compared quantitatively via cap-
ture ELISA for their effects on cellular Bcr-Abl autophosphory-
lation activity and on Bcr-Abl-dependent cell proliferation (Ta-
ble 1). AMN107 (IC5020–60 nM) was consistently more potent
than imatinib (IC50120–470 nM) in inhibiting Bcr-Abl tyrosine
kinase activity in cell lines. In the presence of IL-3, AMN107
inhibited autophosphorylation but no longer inhibited cell via-
bility (Figures 1B and 1C).
Exposure of Ba/F3.p210 (Figure 1D) or Ba/F3.p190 (data not
shown) cells to both AMN107 and imatinib simultaneously across
a range of concentrations resulted in synergistic cytotoxicity.
von Bubnoff et al., 2002; Branford et al., 2002; Roche-Les-
tienne et al., 2002). A series of Ba/F3 cell lines stably express-
ing E255V, T315I, F317L, M351T, F486S, G250E, M244V,
L248R, Q252H, Y253H, E255K, E279K, E282D, V289S, and
L384M Bcr-Abl mutants were generated after transfection with
expression plasmids containing each mutant (Azam et al., 2003).
As reported, imatinib effectively inhibited proliferation of Ba/
F3 cells expressing nonmutated Bcr-Abl, but was substantially
less active against cells expressing any of these Bcr-Abl point
mutants (IC50 values R1 ?M; Figure 2A, 2B, and Table 1)
(Cowan-Jacob et al., 2004). In contrast, AMN107 inhibited pro-
liferation of Ba/F3 cells expressing G250E, E255K(V), F317L,
M351T, F486S, M244V, L248R, Q252H, Y253H, E255K, E279K,
E282D, V289S, and L348M Bcr-Abl mutants at <1 ?M concen-
trations (Table 1). However, the T315I mutant remained resis-
tant to AMN107 below 10 ?M. Also, four of these mutants had
IC50values >500 nM (E255K[V], L248R, Y253H), indicating in-
termediate sensitivity. AMN107-induced cytotoxicity of Ba/F3
cells expressing E255V, F317L, M351T, and F486S mutants
could be rescued by the addition of IL-3, suggesting that the
critical target was probably Bcr-Abl itself (Supplemental Data).
Similarly, AMN107, in contrast to imatinib, potently inhibited
the tyrosine autophosphorylation of the E255K, E255V, F317L,
M351T, and F486S Bcr-Abl mutants with mean IC50values of
AMN107 selectively inhibits the proliferation of imatinib-
resistant Bcr-Abl-expressing cells and
autophosphorylation of imatinib-resistant Bcr-Abl mutants
Imatinib and AMN107 were compared for effects on cells ex-
pressing point mutants of Bcr-Abl associated with imatinib re-
sistance in patients (Hofmann et al., 2002; Shah et al., 2002;
CANCER CELL : FEBRUARY 2005
A R T I C L E
Figure 3. Abl-AMN107 complex
A: Superposition of AMN107 (magenta) bound
(orange), and imatinib (green)
bound to Abl (yellow). H bonds within the
complex are depicted as
dashed red lines, whereas those in the imatinib
complex are shown in black. The variability in
the positions of side chains from the C-helix (top
right corner) is due to crystal contacts that influ-
ence the position of the N-terminal lobe of the
kinase. The methyl-imidazole group of AMN107
packs in a hydrophobic pocket formed by
these residues with the nitrogen exposed to
B: Superposition of parts of the backbone struc-
tures of imatinib-Abl (yellow), AMN107-AblM351T
(orange), and AMN107-Abl (cyan). The inhibi-
tors are shown in green, magenta and blue,
respectively. The small black arrows show the
shifts within helix E and the preceding loop, DE.
150, 246, 41, 31, and 43 nM, respectively (Figure 2C and Table
1). These effects were not associated with a decrease in Abl
or Bcr-Abl protein levels (Figure 2C and Supplemental Data).
Autophosphorylation of the T315I mutant was unaffected by
AMN107. Overall, these results indicate that many imatinib-
resistant Bcr-Abl mutants are relatively or absolutely more sen-
sitive to AMN107.
AMN107 also inhibited ligand-induced cellular PDGFR ki-
nase activity and the growth of cells whose proliferation is de-
pendent on activated forms of PDGFR with mean IC50values
of 71 and 57 nM (Table 1). In addition, AMN107 inhibited con-
stitutively activated (autophosphorylated) c-Kit, harboring gain-
of-function mutations in exon-13 (GIST882 cells) or exon-11
(juxtamembrane domain deletion 560–561, expressed in Ba/F3
cells), with mean IC50values of 200 nM and 27 nM, respec-
tively (Table 1). The inhibition of these cellular kinase activities
was well correlated with the effects on cellular viability and cell
proliferation, and the results are comparable with those ob-
tained with imatinib (Table 1 and Supplemental Data).
Crystallographic structural analysis
of AMN107-Abl complexes
Structural analysis of the binding of AMN107 and imatinib to
Abl can explain the differential sensitivity of Abl point mutations
to these two compounds. The binding modes of AMN107 to
the tyrosine kinase domains of both wild-type Abl and the
AblM351Tmutant were elucidated from the crystal structures of
the complexes (Figure 3). Additional details of the structure of
Abl in complex with AMN107 are described elsewhere (P.W.M.,
unpublished data). Here we report two structures of AMN107
in complex with AblM351Tsolved in two different space groups,
such that common differences between the structures and the
wild-type Abl-AMN107 complex probably result from the muta-
tion and not from artifacts such as crystal contacts. In all of
the structures, AMN107 was found to bind to the inactive con-
formation of Abl as observed for imatinib (Nagar et al., 2003).
In the Abl-AMN107 structure, the helix containing Met351 (resi-
dues 338–358) is locked into position by the methionine side-
chain hooking into a cavity adjacent to Leu364. However,
CANCER CELL : FEBRUARY 2005133
A R T I C L E
Figure 4. In vivo investigation of AMN107 against 32D.p210 and 32D-E255V cells
A: 32D.p210 cells following 3 days of treatment with increasing concentrations of imatinib or AMN107. Bars are SEM, n = 4.
B: Mean plasma concentrations of AMN107-base following a single oral dose of either 20 mg/kg or 75 mg/kg to naïve mice. Different groups (n = 4) of
female OF1 mice received a single oral dose of 20 mg/kg in the respective formulation (based on the free base). The AMN107-base was formulated in
10% N-methyl pyrrolidone/90% PEG200 (v/v). At the allotted times, mice were sacrificed, blood and tissue removed, and the concentration of compound
determined by reversed-phase HPLC/MS-MS analysis. Bars are SEM, n = 4. The limit of quantitation (LOQ) was set to 5 ng/ml. Antiproliferative IC50against
Ba/F3 Bcr-Abl p210 cells (0.022 ?M) is inserted.
C: Kaplan-Meier plot of survival for 32D.p210-injected Balb/c mice treated with either vehicle or AMN107.
D: Kaplan-Meier plot of survival for 32D-E255V-injected Balb/c mice treated with either vehicle or AMN107.
within AblM351T, this hook is absent, and the whole helix can
translate up to 1 Å along its axis. This translation slightly shifts
the residues in the loop preceding helix E, which are involved
in contacting the SH2 domain in the assembled inactive state
(Nagar et al., 2003), and also form part of the myristate bind-
we developed mouse models of CML in which tumor burden
was quantified by noninvasive imaging of the luminescent tu-
mor cells. Murine 32D.p210 cells were engineered to stably ex-
press firefly luciferase and evaluated for their responsiveness
to imatinib and AMN107 in vitro: AMN107 inhibited the prolifer-
ation of 32D.p210 cells in vitro with a mean IC50of 9 nM (ima-
tinib IC50300 nM; Table 1 and Figure 4A).
Sublethally irradiated NOD-SCID mice were then inoculated
with these cells and noninvasive imaging was used to serially
assess tumor burden. Mice with established leukemia were di-
vided into cohorts with equivalent tumor burden, and oral ad-
ministration was initiated with AMN107 or vehicle (Figure 5).
Mice with 32D.p210-Luc+ leukemia treated with AMN107 (100
mg/kg/day) showed a profound and rapid reduction in tumor
burden (Figure 5). After 4 doses, there was a one-log reduction
in overall leukemia burden in AMN107-treated mice, in com-
parison to a 1.5 log increase in tumor burden in vehicle-treated
mice. The results suggested that AMN107 could reduce the
accumulation of leukemic cells in marrow, spleen, lymph node
areas, and liver.
To determine if this ability to suppress leukemic cell growth
AMN107 prolongs survival of mice
with Bcr-Abl+ leukemias
The pharmacokinetic properties of AMN107 were evaluated
following single administration of a solution of either 20 or 75
mg/kg in 10% NMP/90% PEG300 by gavage to naïve female
Balb/c mice. Sample analysis was based on an HPLC-MS
method with LOQ 0.01 ?M, and pharmacokinetic parameters
were calculated from the concentration versus time profiles.
AMN107 was orally bioavailable and well absorbed, with mean
plasma levels of 5.6, 5.4, and 0.4 ?M, or 29, 30, and 25 ?M,
at 2, 8, and 24 hr following administration of either 20 mg/kg
(AUC0–24h82 hr/?mol/l) or 75 mg/kg (AUC0–24h641 hr/?mol/l),
respectively (details are presented in Figure 4B and Table 2).
To directly assess the in vivo antitumor efficacy of AMN107,
CANCER CELL : FEBRUARY 2005
A R T I C L E
Table 2. Pharmacokinetic parameters of AMN107/AA-salt following single oral administration of either 20 mg/kg or 75 mg/kg to naïve mice
PK parameters20 mg/kg plasma75 mg/kg plasma75 mg/kg bone marrow 75 mg/kg liver
AUC0–24h, dose([h·µmol/l] [mg/kg])
17.92 ± 2.71
0.39 ± 0.18
47.90 ± 14.34
29.69 ± 7.80
21.05 ± 3.14
9.20 ± 2.22
51.11 ± 2.85
26.18 ± 10.57
Area under the plasma concentration versus time curve (AUC) was calculated from the mean concentrations by linear/log trapezoidal rule using noncompartmental
analysis (WinNonlin, Pharsight). The pharmacokinetic parameters Cmax, Clast, tmax, and tlast were determined by inspection of the data.
would prolong survival, AMN107 was administered to a larger
cohort of Balb/c mice at an oral dose of 75 mg/kg/day over a
16-day period, commencing three days after injection of
32D.p210 cells. Vehicle-treated animals (19/20) developed a le-
thal disease (median survival 16 days, range 15–36 days), char-
acterized by splenomegaly (Figure 4C). One control mouse
failed to develop signs of leukemia, but was included in the
survival analysis. Treatment with AMN107 resulted in the sur-
vival of 15/20 animals over 105 days of observation.
Five of the AMN107-treated mice either died or were sacri-
ficed; median survival was not reached. All remaining mice
were sacrificed at the planned end of the study (day 105), and
were censored in the survival analysis. Body weights and
spleen weights were available for 17/20 AMN107-treated mice,
and were found to be within the normal range (median body
weight 21.9 g, median spleen weight 0.085 g; spleen as a % of
body weight: 0.4%). In comparison, vehicle-treated mice had a
median body weight of 16.6 g and a median spleen weight of
0.51 g (spleen as % of body weight: 3.2%). Using the Wilcoxon
rank sum test, these parameters differed significantly between
the vehicle control and the AMN107-treated mice, with p val-
ues < 0.0001 for body weight, = 0.0001 for spleen weight, and
<0.0001 for spleen as a percentage of body weight. p values
were two-sided, and were obtained from the normal approxi-
mation to the Wilcoxon. Treatment with AMN107 was associ-
ated with a significant prolongation of survival, p < 0.00001.
The log rank test was used to assess differences in survival.
The effects of AMN107 were also evaluated in a bone mar-
row transplant model. Bone marrow cells from normal Balb-c
mice were transduced with Bcr-Abl and transferred to suble-
thally irradiated hosts. Such mice develop a reproducible mye-
loproliferative disease similar to human CML. Groups of mice
(n = 12) were treated with vehicle (control), imatinib (125 mg/
kg/day in two divided doses), or AMN107 (75 mg/kg daily) by
oral gavage, which was started on day 8 following transplant.
Mice were sacrificed when moribund as assessed by consis-
tent standard criteria.
The animals in the control group developed splenomegaly
and marked leukocytosis as observed previously (Mohi et al.,
2004), and all were sacrificed by day 18 posttransplantation
(Figure 6). There was a significantly prolonged survival in mice
treated with either imatinib or AMN107, all of which were alive
at the study end point 20 days after transplantation (p < 0.001,
Figure 6A). Disease burden, as evidenced by spleen weights at
the time of sacrifice, was compared for mice treated with ima-
tinib and AMN107. There was a significant reduction in tumor
bulk in mice treated with AMN107 (p < 0.001, Figure 6D). In
contrast, imatinib failed to control spleen weights despite pro-
longed survival at 20 days.
AMN107 prolongs survival of mice with leukemia
due to imatinib-resistant mutants of Bcr-Abl
The results shown in Figures 4–6 demonstrate that AMN107 is
effective in prolonging survival in mice with Bcr-Abl+ leuke-
mias. To determine if AMN107 would also prolong survival in
mice with imatinib-resistant Bcr-Abl+ leukemias, we replaced
native Bcr-Abl with E255V Bcr-Abl in both the 32D.p210 cell
line and BMT models. The E255V mutant is known to be resis-
tant to imatinib (Hofmann et al., 2002; Shah et al., 2002; von
Bubnoff et al., 2002; Table 1 and Figure 2). An oral dose of 75
mg/kg/day AMN107 administered over a 16-day period, com-
mencing three days after injection of parental 32D.p210-E255V
Figure 5. Efficacy of AMN107 against 32D.p210-
and 32D-E255V-Luc+ cells in vivo
Left panel: Bioluminescence of vehicle- or
AMN107-treated mice. Right panel: Quantita-
tion of AMN107 effects against 32D.p210-Luc+
cells in vivo. Bars are SEM, n = 5.
CANCER CELL : FEBRUARY 2005135
A R T I C L E
Figure 6. AMN107 treatment prolongs survival and decreases tumor burden in an imatinib-resistant Bcr-Abl mutant BMT model
A–C: Kaplan-Meier plots demonstrating survival for mice transplanted with marrow transduced with wild-type Bcr-Abl (A), the imatinib-resistant Bcr-Abl
mutant E255V (B), and the imatinib-resistant Bcr-Abl mutant M351T (C). A significant difference in survival was demonstrated between mice treated with
AMN107 and imatinib (log-rank test, p < 0.001 for the E255V and M351T mutants).
D–F: Scatter diagrams demonstrating spleen weights for mice treated with either placebo, imatinib, or AMN107 for mice transplanted with marrow trans-
duced with wild-type Bcr-Abl (D), the imatinib-resistant Bcr-Abl mutant E255V (E), or the imatinib-resistant Bcr-Abl mutant M351T (F). Horizontal bars represent
median values. Comparisons between values for mice treated with imatinib and AMN107 demonstrate a significant reduction in spleen weights for
AMN107-treated animals (p values shown).
cells, resulted in a delayed onset of leukemia in drug-treated
mice versus vehicle-treated controls (Figure 4D). The Wilcoxin
p value for a difference in time of onset of tumor development
A similar trial was performed for mice transplanted with bone
marrow transduced with the imatinib-resistant E255V mutant
of BCR-ABL, and treated with vehicle, imatinib, or AMN107.
Animals in the vehicle-treated control group (12 mice) again
developed myeloproliferative disease, as did the mice treated
with imatinib, and all were sacrificed by day 15 posttransplan-
tation (Figure 6B). In contrast, treatment with AMN107 signifi-
cantly prolonged survival in mice transplanted with the E255V
mutant, with 7/12 mice still alive at the study endpoint, 17 days
posttransplant (p < 0.001, Figure 6B). Tumor burden was again
compared between mice treated with imatinib or AMN107, and
a significant reduction in spleen weight was demonstrated for
those animals treated with AMN107 (Figure 6E). Similar results
were obtained when the M351T mutant was tested in mice
treated with imatinib or AMN107, with significantly prolonged
survival in AMN107-treated mice (p < 0.001, Figure 6C), and a
significant reduction in spleen weight in mice treated with
AMN107 (Figure 6F). Liver weights and white cell counts (wcc)
were also used as a determinant of tumor burden in mice trans-
planted with native, E255V, and M351T-Bcr-Abl, and, similar to
spleen, a significant reduction was observed in AMN107-
treated mice (Supplemental Data). Taken together, these results
show that AMN107 can prolong survival of mice with two dif-
ferent imatinib-resistant mutants of Bcr-Abl. However, in both
murine models, survival of AMN107-treated mice with native
Bcr-Abl appeared to be superior to that of mice with either
E255V or M351T mutants, consistent with the fact that these
mutants are 27- and 2-fold less sensitive to AMN107 than is
native Bcr-Abl (Table 1).
Imatinib, an orally administered drug that inhibits the tyrosine
kinase activity of Bcr-Abl and of the c-Kit and PDGF receptors,
has proven to be an effective treatment for CML- and c-Kit-
positive gastrointestinal stromal tumors (GISTs) (Buchdunger et
al., 1996; Druker et al., 1996; Carroll et al., 1996; Buchdunger
et al., 2000; Demetri et al., 2002). In chronic phase CML, ima-
tinib induces complete hematologic remissions in almost all
patients, and in a substantial number produces cytogenetic re-
sponses (O’Brien et al., 2003). However, resistance to imatinib
occurs in a small number of chronic phase patients, with some
CANCER CELL : FEBRUARY 2005
A R T I C L E
patients relapsing after months or years of treatment. Chronic
phase CML patients who achieve a 1000-fold reduction in
BCR-ABL transcript levels have a negligible risk of disease
progression over the subsequent 12 months; this level of cyto-
genetic response has been achieved in 39% of patients receiv-
ing a standard dose regimen of imatinib (O’Brien et al., 2003).
In contrast, patients diagnosed with Ph+ ALL, as well as many
patients with more advanced stage CML (accelerated phase
and blast crisis), fail to achieve a complete cytogenetic re-
sponse and frequently develop resistance to therapy and re-
lapse (Sawyers et al., 2002; Ottmann et al., 2002).
Resistance to imatinib often results from the emergence of
clones expressing mutant forms of Bcr-Abl that exhibit a de-
creased sensitivity toward inhibition by imatinib. These include
G250E, Y253H, E255K(V), T315I, F317L, and M351T (Branford
et al., 1999; Gorre et al., 2001; Barthe et al., 2001; Hochhaus
et al., 2001; Barthe et al., 2002; Ricci et al., 2002), and more
than 30 such mutants have now been isolated from patients
(Hochhaus and La Rosee, 2004). Crystallographic studies re-
vealed that imatinib binds to Bcr-Abl by filling a pocket created
in the ATP binding site by the DFG motif of the activation loop
being displaced from the position that it occupies in the cata-
lytically active conformation of the enzyme (Schindler et al.,
2000; Nagar et al., 2003). Point mutations of Bcr-Abl have been
characterized as either those that destabilize this inactive pro-
tein conformation, or those that sterically impede direct con-
tact between the protein and imatinib (Shah et al., 2002; Corbin
et al., 2003; Cowan-Jacob et al., 2004). In general, point muta-
tions affecting residues in close contact with imatinib confer a
greater degree of resistance than those affecting the stability
of the protein conformation.
A number of strategies to prevent the emergence of resistant
clones have been proposed, including combination of imatinib
with other agents (Krystal, 2001). Additive/synergistic effects
have been achieved when imatinib was combined with stan-
dard chemotherapeutic agents such as interferon α, daunoru-
bicin, cytosine arabinoside, and homoharringtonine (Thiesing
et al., 2000; Tipping et al., 2002). Agents that disrupt signaling
pathways associated with Bcr-Abl or that lead to accelerated
catabolism of the Bcr-Abl protein have also been tested for
synergy with imatinib (Sun et al., 2001; Gorre et al., 2002; Hoo-
ver et al., 2002; Nimmanapalli et al., 2002; Klejman et al., 2002;
Nakajima et al., 2003; Warmuth et al., 2003). These alternative
strategies are being clinically evaluated alone and in combina-
tion with imatinib.
There has also been increasing interest in identifying new
Bcr-Abl inhibitors with greater potency than imatinib, or that
retain the ability to inhibit imatinib-resistant point mutants to
Bcr-Abl (Mow et al., 2002; La Rosee et al., 2002; Shah et al.,
2004; O’Hare et al., 2004). Dual Abl and Src kinase inhibitors
have the potential attraction that Src may be involved in signal-
ing by Bcr-Abl (Danhauser-Riedl et al., 1996). However, it does
not appear that Lyn, hck, or Fgr are important for myeloid dis-
ease in mice (Hu et al., 2004), and preliminary studies with
agents such as BMS354825 suggest that inhibition of Src ki-
nases in the setting of the highly drug-resistant T315I mutant
of Bcr-Abl results in no inhibition at all. The added value of
such agents, and potential for added toxicity, will need to be
studied in clinical trials.
AMN107 is a new and highly potent inhibitor of Abl that has
certain advantages over imatinib. First, AMN107 is 10- to 50-
fold more potent as an inhibitor of Bcr-Abl than imatinib, as
assessed by its ability to block proliferation of Bcr-Abl depen-
dent cell lines derived from CML patients (K562, Ku-812F) and
cell lines (32D or Ba/F3) transfected to express the Bcr-Abl pro-
tein. Similarly, AMN107 is 10- to 20-fold more active than ima-
tinib in reducing Bcr-Abl autophosphorylation (IC50 values
ranging from 20–60 nM). Proliferation of the parental 32D and
Ba/F3 cell lines was unaffected at 100-fold greater concentra-
tions, indicating a lack of general toxicity. Similarly, normal my-
eloid progenitor cells are not inhibited at concentrations of
AMN107 <100 nM, and mice show no evidence of hematopoi-
etic toxicity after exposure to high concentrations of drug for
AMN107 also inhibited the tyrosine kinase activity of the
PDGF and c-Kit receptors, displaying similar efficacy to ima-
tinib, and therefore possessing greater selectivity toward Bcr-
Abl. AMN107 showed no activity against a wide panel of other
protein kinases at concentrations below 3 ?M, including c-Src.
A key feature of AMN107 is the ability to inhibit some Bcr-
Abl point mutants resistant to imatinib. The majority of the 15
mutants tested were sensitive to AMN107 to a variable degree,
with IC50values ranging from <10 nM to approximately 1000
nM (when assessed in proliferation assays in vitro), with 10 mu-
tants <100 nM (Table 1). Mutants G250E, Q252H, Y253H,
E255K/V, T315I, and M351T are the most common mutants in
patients with imatinib resistance (>5% incidence each), while
the others tested are detected in 1% to 5% of patients (Hoch-
haus and La Rosee, 2004). Overall, L248R, Y253H, E255K/V,
and L248R were the least sensitive to AMN107, with IC50val-
ues 100–1000 nM, while T315I was resistant at an IC50value
of >10,000 nM. Since plasma levels of AMN107 in excess of
10,000 nM can be readily achieved in mice, these results sug-
gest that many imatinib-resistant Bcr-Abl mutants might be ef-
fectively targeted by AMN107.
In an effort to explain the differences between imatinib and
AMN107 as inhibitors of Abl, crystallographic structural analy-
sis of an Abl-AMN107 complex was performed. Like imatinib,
AMN107 binds to the inactive conformation of Abl kinase
(Cowan-Jacob et al., 2004). From analysis of crystal structures,
the greater affinity of AMN107 compared to imatinib results
from a better topological fit of AMN107 to the protein, contrast-
ing with the need to desolvate/deprotonate the highly basic
N-methylpiperazine and the slightly larger constraints on the
binding surface for this group in imatinib. The binding affinity
contributed by the pyridinyl and pyrimidinyl groups is therefore
relatively large for imatinib, and small compared to the total
energy for AMN107, where the trifluoromethyl/imidazole substi-
tuted phenyl group (Figure 1A) contributes greatly to the po-
tency. Hence, mutations such as F317L from the hinge region
and E255K/V in the P loop, which contact the pyridinyl and
pyrimidinyl groups, have less effect on the overall affinity of
AMN107 than imatinib. The crystal structure of AblM351Tshows
that residues of the C-terminal lobe lining the imatinib binding
site are only marginally affected by the M351T mutation, and
could explain the small reduction in affinity to AblM351Tcom-
pared to wt-Abl, although energetic differences between the
two states might be of greater importance. If imatinib must in-
duce Abl to adopt a specific conformation for binding, then the
affinity will be greater if that conformational state is of lower
energy. The M351T mutation facilitates other positions of helix
E, increasing the entropy and thus increasing the energy re-
CANCER CELL : FEBRUARY 2005137
A R T I C L E
quired to adopt the imatinib binding mode. This mutation has
little effect on AMN107 affinity, since there is less stringent in-
duced-fit binding. In contrast to imatinib, which makes direc-
tional H bonds to Ile360 and His361, the imidazole moiety has
less critical interactions with the C-terminal lobe. Similar ener-
getic effects would be expected for other mutants, e.g. M244V
and F486S, which are distant from the imatinib binding site and
cause mild resistance to imatinib. The T315I mutant remains
insensitive to binding of AMN107 due to the loss of a hydrogen
bond and introduction of a steric clash, as in the case of ima-
tinib (Schindler et al., 2000). A different inhibitor scaffold would
be required to overcome resistance to this mutant. The re-
duced sensitivity of the G250E mutant toward AMN107 is
probably because the glutamate stabilizes the active confor-
mation of Abl. This is in contrast to other mutations in this re-
gion, such as Y253F/H and E255K/V, which destabilize the in-
active conformation of the P loop (Cowan-Jacob et al., 2004).
Having demonstrated the potent in vitro efficacy of AMN107,
we evaluated the compound against Bcr-Abl-induced leukemia
in animal models. AMN107 was well absorbed and displayed
good bioavailability in mice; oral administration of 20 mg/kg of
AMN107 yielded a mean plasma level in the range of 6.0–12.1
?M after 2 hr, which is >100-fold greater than the concentra-
tions required to inhibit either Bcr-Abl autophosphorylation or
hematopoietic cell proliferation in vitro. Following a single 75
mg/kg dose, high plasma and bone marrow concentrations
were maintained out to 24 hr, and the compound was well tol-
erated at oral doses up to 150 mg/kg/day.
The potential of AMN107 for in vivo activity was tested in a
short-term model where nude mice were injected with
32Dp210Bcr/Abl cells additionally expressing the luciferase
gene. Serial imaging indicated that compared to vehicle,
AMN107 dramatically reduced the accumulation of leukemic
cells in the bone marrow, spleen, liver, and lymph node areas,
indicating effective distribution into multiple tissues in vivo. To
determine whether AMN107 could also extend the survival of
mice injected with 32Dp210Bcr-Abl cells, Balb/c mice were
treated with an oral dose of 75 mg/kg q.d. over a 16-day
period, commencing three days after the injection of 32D.p210
Bcr-Abl cells, or vehicle control. Treatment with AMN107 re-
sulted in the survival of 15/20 animals over the 105 days of
planned observation, whereas 19/20 vehicle-treated mice had
progressive disease. Spleen weights of the animals receiving
AMN107 were within the normal range at the end of the experi-
ment. Taken together, these two studies with 32Dp210Bcr-Abl
cell lines indicate the potential of AMN107 to rapidly and pro-
foundly suppress disease development.
To determine if the therapeutic effects of AMN107 on Bcr-
Abl+ cell lines would extend to Bcr-Abl+ primary hematopoietic
cells, mice were transplanted with marrow infected with a Bcr-
Abl retrovirus, followed 8 days later by treatment with AMN107,
imatinib, or vehicle control. In this model, mice develop a highly
reproducible CML-like myeloproliferative syndrome charac-
terized by granulocytosis and splenomegaly. Treatment with
AMN107 reduced morbidity and, at the end of the study,
yielded spleen weights within the normal range, as observed
for the long-term survival experiments.
As noted above, the availability of agents that could be used
to treat imatinib-resistant clones of Bcr-Abl+ leukemias would
have significant therapeutic value. This possibility was as-
sessed using a highly imatinib-resistant mutant, E255V Bcr-
Abl, both in mice injected with 32D.p210-E255V cells and in
mice receiving bone marrow transplants after infection with the
Bcr-Abl mutant, E255V. In both of these models, AMN107, but
not imatinib, increased survival and decreased disease volume.
These results were extended by testing a second imatinib-resi-
tant mutant, M351T. AMN107 significantly prolonged survival
compared to imatinib (p < 0.001, Figure 6E), and also resulted
in a significant reduction in spleen weight and other measures
of disease burden (Figure 6F).
Overall, the data presented here suggest that AMN107 is
highly cytotoxic to both cell lines and primary hematopoietic
cells expressing Bcr-Abl, and that it could have certain advan-
tages over imatinib in terms of higher potency and the ability
to inhibit some imatinib-resistant mutants. However, since the
IC50value of AMN107 for some imatinib-resistant mutants is
higher than for wild-type Bcr-Abl, it may be necessary to
achieve substantially higher plasma concentrations of AMN107
in such patients to achieve responses.
If human clinical trials validate the effectiveness of AMN107
demonstrated in the preclinical studies reported here, it may
be possible to either use AMN107 in selected patients with
imatinib resistance, or to use both agents together, simulta-
neously or sequentially. Highly potent inhibitors of Bcr-Abl
should reduce the number of residual Bcr-Abl+ cells capable of
undergoing mutation. Consequently, monotherapy with highly
potent Abl inhibitors, or combinations of Abl inhibitors with dif-
ferent mechanisms of action, might prevent or delay the emer-
gence of some types of drug-resistant mutants of Bcr-Abl. In
support of simultaneous administration, AMN107 was shown
to be synergistic when combined with imatinib against cells
expressing p210Bcr-Abl or p190Bcr-Abl, despite the fact that
both inhibitors bind to the same site.
While we anticipate that new Bcr-Abl point mutations could
eventually emerge to confer resistance to AMN107, it may be
possible to cycle or combine agents to suppress or delay the
emergence of resistant clones. Thus, the availability of novel,
high-potency, Abl tyrosine kinase inhibitors will usher in a new
generation of clinical studies that will hopefully result in addi-
tional major advances in the therapy of CML and Ph+ ALL.
Systemic 32D Bcr-Abl leukemia model
32D.p210 and 32D-E255V cells free of Mycoplasma and viral contamination
were washed once with Hank’s Balanced Salt Solution (HBSS; Mediatech,
Inc.,VA), and resuspended in HBSS prior to administration to mice. Solu-
tions of AMN107 were prepared just prior to administration, by dissolving
75 mg in 1.0 ml of NMP to give a clear solution and diluting with 9.0 ml
PEG300. Female BALB/c mice (weighing 15–18 g and 6–7 weeks of age at
delivery; Taconic, NY) were administered suspensions containing 32D.p210
or 32D-E255V cells by tail vein injection (1 × 10532D.p210 cells/mouse;
7 × 10532D-E255V cells/mouse; day 0). 32D.p210-injected mice were
treated via gavage with either vehicle (10% NMP-90% PEG300) or AMN107
(75 mg/kg/day) on days 3, 4, 7, 8, 9, 10, 11, 15, 16, 17, and 18 (eleven
doses total), and monitored for signs of leukemia. 32D-E255V-injected mice
were treated via gavage with either vehicle or AMN107 (100 mg/kg/day)
once daily for 21 days. Mice were sacrificed if they became morbid, accord-
ing to institute protocols. At the planned end of each study, any remaining
mice were sacrificed, body and spleen weights were recorded, and tissues
preserved in 10% formalin for histopathological analysis.
Survival was measured as time from cell injection to death or sacrifice.
All starting animals were included in the statistical analysis. Survival analysis
was performed using the method of Kaplan and Meier with statistical signifi-
cance assessed using the log rank test.
CANCER CELL : FEBRUARY 2005
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Azam, M., Latek, R.R., and Daley, G.Q. (2003). Mechanisms of autoinhibition
and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell
Bioluminescent Bcr-Abl model of CML
Cells were transduced with a retrovirus encoding firefly luciferase (MSCV-
Luc), and selected with puromycin at 2 ?g/ml to produce 32D.p210-Luc+
and 32D-E255V-Luc+ cell lines. Female NOD-SCID mice (8–10 weeks of
age; Jackson Laboratory) were sublethally irradiated with a single fraction
of 300 rads, and 3–6 hr later, a total of 1 × 106cells were administered by tail
vein injection. Mice were imaged and total body luminescence quantified as
previously described (Armstrong et al., 2003). Baseline imaging 5–7 days
after tumor cell inoculation was used to establish treatment cohorts with
matched tumor burden. Cohorts of mice were treated with oral administra-
tion of vehicle (formulated as above), 75 mg/kg imatinib (twice daily), or 100
mg/kg/day AMN107 (formulated as above). Repeat imaging was performed
every 3–4 days.
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ATP-binding site of BCR-ABL in a patient with chronic myeloid leukaemia
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alternative to bone marrow cytogenetics. Br. J. Haematol. 107, 587–599.
Bone marrow transplant Bcr-Abl model of CML
The murine BMT assays and drug treatment were performed as described
previously (Liu et al., 2000; Cools et al., 2003; Kelly et al., 2002). In brief,
1 × 106bone marrow cells transduced with distinct retroviral constructs
(either native p210-BCR-ABL or BCR-ABL E255V mutant both in MSCV-
IRES-GFP) were injected into the lateral tail veins of lethally irradiated
(450cGy X2) syngeneic Balb/c recipient mice. After 8 days, mice were ad-
ministered either vehicle (10% NMP-90% PEG300), 75 mg/kg/day AMN107
(formulated in 10% NMP-90% PEG300), or 125 mg/kg/day imatinib (two
divided doses formulated in 0.05% methylcellulose) via oral gavage. Dis-
eased animals, identified by splenomegaly or moribund appearance, were
sacrificed. Spleen and liver weights were recorded and histopathological
analyses were performed for each animal; single-cell suspensions of bone
marrow, spleen, and peripheral blood were analyzed by flow cytometry as
described previously (Schwaller et al., 1998). Survival analysis was per-
formed using the method of Kaplan and Meier with statistical significance
assessed using the log rank test. Tumor burden as assessed by white cell
counts (WCC) and spleen and liver weights were compared by the Mann-
Whitney U test for each treatment category.
Branford, S., Rudzki, Z., Walsh, S., Grigg, A., Arthur, C., Taylor, K., Herr-
mann, R., Lynch, K.P., and Hughes, T.P. (2002). High frequency of point
mutations clustered within the adenosine triphosphate-binding region of
BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute
lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood
Buchdunger, E., Zimmermann, J., Mett, H., Meyer, T., Muller, M., Druker,
B.J., and Lydon, N.B. (1996). Inhibition of the Abl protein-tyrosine kinase in
vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res. 56,
Buchdunger, E., Cioffi, C.L., Law, N., Stover, D., Ohno-Jones, S., Druker,
B.J., and Lydon, N.B. (2000). Abl protein-tyrosine kinase inhibitor STI571
inhibits in vitro signal transduction mediated by c-kit and platelet-derived
growth factor receptors. J. Pharmacol. Exp. Ther. 295, 139–145.
Buchdunger, E., Matter, A., and Druker, B.J. (2001). Bcr-Abl inhibition as a
modality of CML therapeutics. Biochim. Biophys. Acta 1551, M11–M18.
Information on cell lines and cell culture, chemical compounds and biologic
reagents, antibodies, cell proliferation studies, apoptosis assays, immuno-
precipitation and immunoblotting, effects of imatinib and AMN107 on phos-
phorylation status of target kinases in cells, determination of AMN107 bind-
ing sites in Abl (including preparation of the c-AblM351T-AMN107 complex,
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structure determination and refinement), and synergy studies can be found
in the Supplemental Data at http://www.cancercell.org/cgi/content/full/7/2/
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J.D.G. and D.G.G. are supported by NIH grant CA66996, and a Specialized
Center of Research Award from the Leukemia and Lymphoma Society.
J.D.G. is also supported by NIH grants CA36167 and DK50654. G.Q.D. is
supported by grants from the National Cancer Institute (CA86991), the NIH
Director’s Pioneer Award (DP1-OD000256), and the Burroughs Wellcome
Fund. P.W.M., W.B., J.B., S.W.C.-J., D.F., G.F., and J.M. are employees of
Novartis Pharma AG, Basel, Switzerland. J.D.G has a financial interest with
Novartis Pharma AG. B.H. is a senior clinical fellow of the LRF (UK).
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