A new nonpeptidic inhibitor of 14-3-3 induces apoptotic cell death in chronic myeloid leukemia sensitive or resistant to imatinib

Article abstract of DOI:10.1124/jpet.110.172536

Resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitor imatinib mesylate (IM) is most often due to point mutations in the Bcr-Abl fusion gene. T315I mutation (resulting in substitution of Ile for a Thr residue at the gatekeeper position 315) raises particular concern, because it also provides resistance to second-generation kinase inhibitors already approved for clinical use (nilotinib and dasatinib). Much effort is therefore focused on alternative molecular-based strategies. Previous studies proved that binding to 14-3-3 scaffolding proteins leads to cytoplasmic compartmentalization and suppression of proapoptotic and antiproliferative signals associated with Bcr-Abl protein kinase, hence contributing to leukemic clone expansion. Here we investigated the effect of 14-3-3 inhibition disruption on hematopoietic cells expressing the IM-sensitive wild type Bcr-Abl and the IM-resistant T315I mutation. Using a virtual screening protocol and docking simulations, we identified a nonpeptidic inhibitor of 14-3-3, named BV02, that exhibits a remarkable cytotoxicity against both cell types. c-Abl release from 14-3-3σ, promoting its relocation to nuclear compartment (where it triggers transcription of p73-dependent proapoptotic genes) and to mitochondrial membranes (where it induces the loss of mitochondrial transmembrane potential) combined with c-Abl enhanced association with caspase 9 (a critical step of sequential caspase activation further contributing to c-Abl pro-apoptotic function) has a prominent role in the effect of BV02 on Bcr-Abl-expressing cells. In conclusion, BV02 may be considered as a treatment option for CML and, in particular, for more advanced phases of the disease that developed IM resistance as a consequence of Bcr-Abl point mutations. Copyright

Full text of DOI:10.1124/jpet.110.172536

Supplemental material to this article can be found at:
http://jpet.aspetjournals.org/content/suppl/2010/11/01/jpet.110.172536.DC1.html
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022-3565/11/3363-596–604$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
JPET 336:596–604, 2011
Vol. 336, No. 3
172536/3658113
Printed in U.S.A.
A New Nonpeptidic Inhibitor of 14-3-3 Induces Apoptotic Cell
Death in Chronic Myeloid Leukemia Sensitive or Resistant
□
S
to Imatinib
Manuela Mancini, Valentina Corradi, Sara Petta, Enza Barbieri, Fabrizio Manetti,
Maurizio Botta, and Maria Alessandra Santucci
Dipartimento di Ematologia e Scienze Oncologiche “Lorenzo e Ariosto Ser a` gnoli,” Universit a` di Bologna, Italy (M.M., S.P., E.B.,
M.A.S.); and Dipartimento Farmaco Chimico Tecnologico, Universit a` di Siena, Italy (V.C., F.M., M.B.)
Received July 8, 2010; accepted October 20, 2010
ABSTRACT
Resistance of chronic myeloid leukemia (CML) to tyrosine ki- Using a virtual screening protocol and docking simulations, we
nase inhibitor imatinib mesylate (IM) is most often due to point identified a nonpeptidic inhibitor of 14-3-3, named BV02, that
mutations in the Bcr-Abl fusion gene. T315I mutation (resulting exhibits a remarkable cytotoxicity against both cell types. c-Abl
in substitution of Ile for a Thr residue at the “gatekeeper” release from 14-3-3␴, promoting its relocation to nuclear com-
position 315) raises particular concern, because it also provides partment (where it triggers transcription of p73-dependent pro-
resistance to second-generation kinase inhibitors already ap- apoptotic genes) and to mitochondrial membranes (where it
proved for clinical use (nilotinib and dasatinib). Much effort is induces the loss of mitochondrial transmembrane potential)
therefore focused on alternative molecular-based strategies. combined with c-Abl enhanced association with caspase 9 (a
Previous studies proved that binding to 14-3-3 scaffolding critical step of sequential caspase activation further contribut-
proteins leads to cytoplasmic compartmentalization and sup- ing to c-Abl pro-apoptotic function) has a prominent role in the
pression of proapoptotic and antiproliferative signals associ- effect of BV02 on Bcr-Abl-expressing cells. In conclusion, BV02
ated with Bcr-Abl protein kinase, hence contributing to leuke- may be considered as a treatment option for CML and, in
mic clone expansion. Here we investigated the effect of 14-3-3 particular, for more advanced phases of the disease that de-
inhibition disruption on hematopoietic cells expressing the IM- veloped IM resistance as a consequence of Bcr-Abl point
sensitive wild type Bcr-Abl and the IM-resistant T315I mutation. mutations.
emergence of Bcr-Abl point mutations. At the protein level,
Introduction
Bcr-Abl point mutations may either distort the configuration of
The tyrosine kinase (TK) inhibitor imatinib mesylate (IM)
Abl kinase, rendering it unable to adopt the inactive conforma-
has revolutionized the prognosis of chronic myeloid leukemia
tion to which IM binds, or change the identity of residues that
(
CML), with best complete hematologic and cytogenetic re-
directly contact IM (Weisberg et al., 2007). The greatest thera-
peutic challenge is posed by the substitution of highly conserved
Thr residue at position 315, controlling the access to a hydro-
phobic pocket of the enzymatic active site, by Ile (T315I). In fact,
T315I mediates resistance not only to IM but also to second-
generation Abl kinase inhibitors highly effective in patients
with CML who failed IM therapy (Druker, 2008). The need for
sponse rates of 98 and 87%, respectively, after 5 years of fol-
low-up (Druker et al., 2006). However, there is growing concern
for the development of resistance, most often as a result of the
This study was supported by the University of Bologna [ex60% funds];
Ministero della Pubblica Istruzione [PRIN], BolognaAIL, Fondazione Monte
dei Paschi di Siena; Asinex; Department of Radiation Oncology [Postdoctoral
Grant (to M.M.)]; and Department of Biological Sciences, University of Calgary new strategies to treat IM-resistant CML has stimulated con-
(
Calgary, AB, Canada) [Postdoctoral Grant (to V.C.)].
M.M. and V.C. contributed equally to this work.
siderable efforts to develop compounds targeting key functional
Article, publication date, and citation information can be found at motifs distant from the ATP-binding pocket of Bcr-Abl protein
http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.172536.
or, alternatively, kinase downstream effectors.
4-3-3 are a highly conserved family of 28- to 33-kDa acidic
proteins consisting of seven members (␤, ␥, ε, ␴, ␨, ␶, and ␩;
1
□S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: TK, tyrosine kinase; CML, chronic myeloid leukemia; IM, imatinib mesylate; BV02, 2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-ylcarbamoyl)terephthalic acid; wt, wild type; JNK, c-Jun NH2 terminal kinase; MD, molecular dynamics; GA, genetic
algorithms; MIFs, molecular interactions fields; PI, propidium iodide; IP, immunoprecipitation; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonic acid; FLICA, fluorochrome-bound inhibitor of caspase.
596

14-3-3 Inhibitors in Chronic Myeloid Leukemia
597
the phosphorylated forms of ␤ and ␥ were initially described
as ␣ and ␦, respectively) (Aitken, 2006). They associate with
more than 300 partners to regulate critical events such as
cell cycle progression, DNA damage response, apoptosis, pro-
tein trafficking, signal transduction, cytoskeletal rearrange-
Materials and Methods
Structure-based-pharmacophore model generation. Protein
Data Bank entry 1ywt corresponds to crystallographic structure of
4-3-3␴ isoform in complex with a mode I-binding motif phosphopep-
tide (Wilker et al., 2005). This structure was imported in Ligand-
1
ments, metabolism, and transcriptional regulation. Struc- Scout 1.0 software (Inte:Ligand Software-Entwicklungs und Con-
tural studies proved the dimeric nature of 14-3-3 proteins sulting GmbH, Vienna, Austria), and the peptide ligand was selected
that can function as homo- or heterodimers. Each monomer for pharmacophore generation using default parameters supplied by
the software and in agreement with Catalyst 4.10 platform later
employed for database searching (Catalyst 4.10; Accelrys, Inc., San
consists of nine ␣-helices arranged in an antiparallel array to
surround an amphipathic groove, forming a highly conserved
Diego, CA). Excluded volume spheres, corresponding to regions in
binding site for client proteins (Xiao et al., 1995). 14-3-3
which the presence of a ligand is not allowed, were automatically
binding is primarily dependent on ligand phosphorylation.
Two main consensus motifs recognized by all 14-3-3 isotypes
added to the pharmacophoric model. The final model consisted of a
large number of features, illustrated in detail in the Supplemental
have been identified. They correspond to RSX-pS/T-XP (mode material. A MD simulation has been applied to investigate the
I) and RXXX-pS/T-XP (mode II) sequences, where pS/T de- stability of interactions involving hydrogen bonds by means of
GROMACS 3.3 software (http://www.gromacs.org/; see Supple-
notes phospho-Ser or phospho-Thr and X any other amino
mental data for further details). Topology file for the phosphopep-
acid except Cys. Crystal structures of phosphopeptide-
tide ligand was generated using PRODRG server (Sch u¨ ttelkopf
and van Aalten, 2004).
Database Searching. Once simplified by MD simulation, the
pharmacophoric model was used as a search query for screening the
bound 14-3-3 complexes demonstrated that pS/T binding
occurs within amphipathic grooves at either edge of the
central channel of 14-3-3 dimer. An additional mechanism
of 14-3-3 binding is provided by the phosphorylation status
Asinex Gold Collection (Asinex Ltd., Moscow, Russia). Catalyst 4.10
of specific Ser/Thr residues. In particular, Ser58 phosphor- was used to perform the screening, whereas molecular weight and
ylation by Akt regulates 14-3-3 dimerization and ligand Lipinski’s properties were computed by means of Cerius2 (ver. 4.10L;
Accelrys).
stabilization, Thr233 phosphorylation by casein kinase I
Docking Studies. To prepare input structures of selected com-
pounds for docking calculations, a geometry optimization was per-
formed using MacroModel 8.5 with OPLSA_2005 force field, the
negatively regulates c-Raf binding, and Ser184 phosphor-
ylation by JNK promotes the release of forkhead box O 3a
transcription factor and c-Abl TK (all residue numbers
above refer to 14-3-3␨) (Dubois et al., 1997; Powell et al.,
Polak-Ribiere conjugate gradient method, and a convergence of 0.001
Ϫ1
Ϫ1
kJ ꢀ mol
ꢀ Å
(Mohamadi et al., 1990; Kaminski et al., 2001).
2
002; Yoshida et al., 2005). Accordingly, 14-3-3␨ phosphor- Charges were computed by means of MOPAC2007 software (http://
ylation at Ser184 (corresponding to Ser186 of 14-3-3␴) openmopac.net) using the AM1 Hamiltonian. All docking studies
were performed by means of GOLD software, version 3.0.1, with
drives cell decision to die through events encompassing the
ChemScore scoring function (Verdonk et al., 2003). For each ligand,
dissociation of Bad and Bax, which allows their transloca-
5
0 independent GA runs were performed, with a maximum number
tion to mitochondrial membranes and the release of c-Abl,
which promotes its nuclear import and interaction with
nuclear proapoptotic proteins p53 and p73 (Tsuruta et al.,
5
of 10 GA operations on a set of five islands with a population size of
00 individuals. Remaining GA parameters were kept to their de-
2
fault values. The binding site cavity was defined with a radius of 10
Å around the phosphopeptide. The X-Score program (Wang et al.,
2
004; Yoshida et al., 2005). c-Abl targeting to mitochon-
dria, eventually communicated by endoplasmic reticulum 2002) was then used with default parameters to rerank results from
stress, further integrates its pro-apoptotic activity pro- GOLD (Verdonk et al., 2003) for each compound.
MIFs Analysis. MIFs were calculated by means of GRID version
ceeding from the loss of mitochondrial transmembrane
potential and release of cytochrome c (Ito et al., 2001; Qi
and Mochly-Rosen, 2008).
Resistance to apoptosis is one major cause of illegitimate
expansion of CML hematopoiesis. It is driven by multiple
2
2 (Molecular Discovery Ltd. Pinner, Middlesex, UK) to analyze how
favorable interaction regions for a specific probe are located in the
4-3-3␴ binding site. Minimum energy points of these MIFs were
then computed and used as a tool to analyze the docking results of
selected compounds. In details, probes DRY and C1 were selected to
1
events, including the “loss of function” of c-Abl protein coded calculate MIFs referring to hydrophobic interactions, N1 and O for H
by the allele not rearranged with Bcr because of its cytoplas- bond donor and acceptor interactions, and PO4 for describing nega-
tive interaction regions. The determination of MIF minimum points
was carried out by means of Minim and Filmap programs (both
implemented in the GRID package) using a threshold value of 0
kcal/mol (see Supplemental Fig. S1).
BV02 Chemistry and Synthesis. BV02 was purchased from
Asinex (Moscow, Russia), which declares a purity higher than 95%.
mic retention bound to 14-3-3␴. Accordingly, apoptosis of
leukemic progenitors induced by IM is conditional, at least in
part, upon c-Abl release from 14-3-3␴ phosphorylated by JNK
at Ser186 and nuclear relocation (Mancini et al., 2009). Tar-
geting 14-3-3 might therefore be considered a therapeutic
option in CML either sensitive or resistant to IM (Dong et al.,
2
It was synthesized according to a previously published procedure
008). However, no modulators of 14-3-3 ligands have been (Bauer and Rademann, 2003). In brief, anhydrous reactions were run
reported yet.
under a positive pressure of dry N
2
. Thin-layer chromatography was
carried out using thin-layer chromatography plates silica gel 60 F254
Here we show that the inhibition of 14-3-3␴ by BV02, a
nonpeptidic compound identified by means of in silico vir-
tual screening techniques, promotes the apoptotic death of
cells expressing either wt Bcr-Abl construct or T315I mu-
tation. BV02-induced apoptosis proceeds from c-Abl re-
1
13
from Merck (Milan, Italy). H and C NMR spectra were recorded at
00 MHz on a DPX400 spectrometer from Bruker Avance (Milan,
4
Italy). Chemical shifts relative to chloroform and tetramethylsilane
are at ␦ 7.24 ppm at ␦ 0.00 ppm, respectively. Melting points were
determined with a Gallenkamp melting point apparatus (Sanyo Gal-
lease from 14-3-3␴ and re-location to nuclear compartment lenkamp, Loughborough, UK) and are uncorrected. 4-Aminoantipy-
and at mitochondrial membranes. rine (compound 1 in Fig. 1A: 158.7 mg, 0.78 mmol, 3.00 eq) was

5
98
Mancini et al.
Fig. 1. BV02 chemical synthesis and graphical representation of the final pharmacophoric model simplified by MD simulations. A, BV02 is obtained
by dissolving 4-aminoantipyrine (1) in anhydrous dimethylformamide and adding trimellitic anhydride (2) and N,N-dimethylamino pyrimidine. BЈ,
superposition of the pharmacophoric model and the peptide used to generate it. Pharmacophoric features are labeled. BЉ, the pharmacophoric model
with eight excluded volumes (represented by gray spheres) codifying for forbidden regions of space already occupied by amino acids of 14-3-3. DMF,
dimethylformamide; DMAP, N,N-dimethylamino pyrimidine.
dissolved in anhydrous dimethylformamide (1 ml). Next, trimellitic to identify Bcr-Abl point mutations (Soverini et al., 2005). BV02
anhydride (compound 2 in Fig. 1A: 50.2 mg, 0.26 mmol, 1.00 eq) and
N,N-dimethylamino pyrimidine (10.0 mg) were added and the result- nique to quantify drug impact on cell reproductive integrity. In brief,
ing mixture was stirred at room temperature overnight (Fig. 1A). we assessed the reduction of colony (aggregates containing Ͼ50 cells
cytotoxicy was assayed in clonogenic assays, the best in vitro tech-
The suspension was diluted with H O (5 ml) and then filtered under generated in 0.9% methylcellulose supplemented with 30% fetal calf
2
ϩ
reduced pressure to obtain a white precipitate that was thoroughly serum) number in Bcr-Abl-expressing Ba/F3 cell lines and CD34 in
washed with H O, hexane and finally dried over P O . Compound the presence of increasing doses of BV02 after 7- or 14-day incuba-
2
2
5
purity was confirmed by high-performance liquid chromatography tion at 37°C in fully humidified atmosphere and 5% CO . Linear and
2
analysis, whereas its structure was confirmed by NMR and IR ex-
periments. Spectroscopic data are as follows: H NMR (400 MHz, Bcr-Abl-expressing Ba/F3 cell lines and CD34 progenitors from
nonlinear regression analyses were used to calculate BV02 LD50 in
1
ϩ
DMSO, Me Si): ␦ (ppm); 13.72 (s, 1H); 8.40 (d, J ϭ 8 Hz, 1H); 8.29 (s, patients with CML, respectively. Apoptosis induction and involved
4
1
H); 8.07 (d, J ϭ 8 Hz, 1H); 7.53 (m, 2H); 7.38 (m, 3H); 3.27 (s, 3H); signals were assayed in Bcr-Abl-expressing Ba/F3 cell lines after
13
and 2.24 (s, 3H). C NMR (100 MHz, DMSO, Me Si): ␦ (ppm) ϭ 24-h exposure to 1 ␮M IM and 5 ␮M BV02 by means of cytofluori-
4
1
1
1
1
66.51, 166.20, 160.95, 154.37, 137.14, 136.10, 135.24, 134.88,
metric analysis of Annexin V (F. Hoffmann-La Roche, Basel, Swit-
32.44, 129.73, 127.60, 125.12, 124.47, 124.03, 99.95, 35.66, and zerland) and PI (Sigma) uptake, Western blot and IP/immunoblot-
Ϫ1
0.85. IR: cm ϭ 2961.9, 2892.4, 2725.8, 1726.9, 1578.6, 1460.7, and ting analyses, according to published methods (Mancini et al., 2009).
376.8.
Protein Analysis. Western blot and IP/immunoblotting analyses
Cell Lines and Treatments. Parental pro-B murine cell line were performed on proteins obtained from whole cells, nuclear frac-
Ba/F3 and clones expressing the wt and T315I, and E255K-mutated tions, and mitochondrial membranes according to published methods
Bcr-Abl constructs were kindly donated by Michael W. Deininger (Mancini et al., 2005, 2007). In brief, whole-cell lysates were obtained
7
(
Department of Hematology and Clinical Oncology, Health and Sci- from 2 ϫ 10 cells in buffer A [10 mM Tris, pH 8.0, 150 mM NaCl, 10
ence University, Cancer Institute, Portland, OR). They were main- mM iodacetamide, 1% CHAPS, and 0.02% sodium azide supple-
tained in RPMI medium (Invitrogen, Paisley, UK) supplemented mented with protease inhibitors: trypsin and pepsin inhibitors, leu-
with 10% fetal calf serum (Invitrogen), 1% l-glutamine (Sigma, St. peptin, antipain, Na VO , and phenylmethylsulfonyl fluoride (all
3
4
Louis, MO) and 10% conditioned medium from WEHI 3 cells as
source of interleukin-3 when required. Indirect immunomagnetic recovered from 2 ϫ 10 cells kept in buffer B [10 mM NaCl, 5 mM
from Sigma)]). Nuclear lysates were obtained from naked nuclei
7
labeling (mini-MACS; Miltenyi Biotec, Bergish Gladback, Germany) MgCl , 10 mM phosphate buffer, and 0.1% Tergitol-type detergent
2
ϩ
was used to isolate CD34 hematopoietic progenitors from bone NP40 (all from Sigma) supplemented with protease inhibitors] by
marrow samples from three patients with CML in blast crisis who means of three sonication rounds [10-s pulses using a Hight Inten-
developed IM resistance. Patient informed consent to use bone mar- sity Ultrasonic Processor/Sonicator from Cole-Parmer Instruments
row samples for our experimental studies was approved by the Eth- (Vernon Hills, IL) equipped with 2-mm tips]. Mitochondrial mem-
ical Committee of S.Orsola-Malpighi Hospital (Bologna, Italy). The branes were separated from whole-cell lysates by 30-min centrifuga-
ϩ
content of CD34 cells in each sample was measured by cytofluori- tion at 14,000g in 2 ml of buffer C (20 mM HEPES, pH 7.5, 1.5 mM
metric analysis of CD34 expression with a FACScan (BD Biosci- MgCl , 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol,
2
ences, Franklin Lakes, NJ). It was Ͼ95% in all cases. Denaturing 0.1% potassium metabisulfite, and protease inhibitors in 250 mM
high-performance liquid chromatography and sequencing were used sucrose) at 4°C. Proteins or IP products obtained through overnight

14-3-3 Inhibitors in Chronic Myeloid Leukemia
599
incubation with primary antibodies in buffer D (250 mM NaCl, 15 acceptors (no more than 10), H bond donors (no more than
mM MgCl , 40 mM HEPES, 60 mM glycerophosphate supplemented five), molecular mass (lower than 500 Da), and octanol-water
2
with protease inhibitors) were resolved in SDS-polyacrylamide gel
partition coefficient (lower than five) (Lipinski, 2000). Com-
electrophoresis. Gels were then transferred onto nitrocellulose mem-
pounds with no more than one violation of the Lipinski’s rule
branes (Whatman Schleicher and Schuell, Dassel, Germany), labeled
were kept.
with primary and secondary antibodies in Tris-buffered saline with
Docking Studies and MIFs. Compounds selected from
5
% bovine serum albumin (Sigma) and 0.1% Tween 20 (Sigma). The
virtual screening were docked to 14-3-3␴ binding site by
means of GOLD software and then ranked on the basis of
antibodies were purchased from Millipore (Billerica, MA), Cell Sig-
naling Technology (Danvers, MA), and Santa Cruz Biotechnology
(
Santa Cruz, CA). Signals were visualized by the enhanced chemi- both ChemScore and XScore scoring functions (Verdonk et
luminescence detection system from Pierce (Rockford, IL). Signal al., 2003). MIF computations were also performed when best
intensities in single blots obtained from three individual experi-
docked conformations did not perfectly match the pharmaco-
ments were quantified by mean of a dedicated software (VisionWork-
phoric model. In particular, points of minimum of MIFs were
sLS; UVP, LLC, Upland, CA). Such software attributes a numerical
calculated, thus identifying the regions of most favorable
value to signals of chemiluminescent substrates transferred on
interaction between the five probes and protein (see Supple-
highly sensitive radiographic films (Pierce), thereby allowing a com-
mental Figs. S1 and S2). Finally, results of docking studies
for selected compounds were compared with distribution of
parative analysis of protein levels in untreated and drug-exposed
samples. The statistical significance of differences in signal intensi-
ties relative to cell treatments was assessed by mean of paired these minima into the groove. Fourteen compounds in which
Student’s t test; p Ͻ 0.05 was kept as limit for statistical significance. the docking pose fit at least three features of pharmacophoric
Cyofluorimetric Analysis of Cell Cycle Distribution and model and that were in agreement with localization of the
6
Caspase 8 Activation. Cell cycle distribution was performed on 10
minima have been selected and evaluated for biological ef-
cells fixed overnight in 70% ethanol and treated with 1 ␮g/␮l PI and
fects. BV02, purchased from Asinex, was the only compound
RNase (Sigma) at 37°C for 30 min. PI uptake was measured by mean
among the 14 selected exhibiting a remarkable in vitro cyto-
of a FACScan flow cytometer set at Ͼ580 nm and dedicated software
toxicity on Bcr-Abl-expressing cells (Fig. 1A and data not
(
both from BD Biosciences). Caspase-8 activity was detected with a
shown).
BV02 Effects on Bcr-Abl-Expressing Cells. We first
commercial kit (Carboxyfluorescein FLICA Apoptosis Detection kits;
Immunochemistry Technologies LLC, Bloomington, MN) according
to the manufacturer’s instructions. In brief, cells labeled with fluo- confirmed the differences in response to IM of pro-B murine
rochrome-bound inhibitor of caspase (FLICA: 300 ␮l/well at 37°C for cell line Ba/F3 expressing either the wt or the T315I-mutated
1
h in 5% CO , fully humidified atmosphere) were quantified by
2
Bcr-Abl construct. To this purpose, we evaluated drug impact
on cell reproductive integrity [i.e., the ability of generating
colonies (aggregates containing Ͼ50 cells) in semisolid cul-
ture media (0.9% methylcellulose)]. Such a method allowed
the plotting of dose-response curves and of IM LD50 (0.41 Ϯ
means of cytofluorimetric analysis at an excitation range from 488 to
92 nm and an emission range from 515 to 535 nm. Apoptosis was
4
quantified as the level of fluorescence emitted from FLICA probes
bound to caspases. Nonapoptotic cells appeared unstained, whereas
cells undergoing apoptosis were brightly fluorescent. Caspase-8 ac-
tivity was quantified by mean of dedicated software (DIVA; BD
Biosciences).
0.01 ␮M for Ba/F3 cells expressing the wt Bcr-Abl and Ͼ10
␮M for Ba/F3 cells expressing T315I mutation) (Fig. 2A, top
and bottom left). Apoptosis induction in response to IM (1 ␮M
for 24 h) was apparent in wt Bcr-Abl-expressing Ba/F3 cells,
but not in Ba/F3 cells expressing T315I-mutation (Fig. 2B,
first and third bars). BV02 induced a significant dose-depen-
dent reduction of reproductive integrity of Ba/F3 cells ex-
pressing the wt and T315I-mutated Bcr-Abl with LD50 of
Results
Structure-Based Pharmacophore Model Generation.
Nonpeptidic compounds able to affect interactions between
1
4-3-3 and their client proteins are not yet known. The nine-
1.04 Ϯ 0.09 and 1.47 Ϯ 0.12 ␮M, respectively (Fig. 2A, top
amino acid, mode I-phosphopeptide MARSH-pS-YPA in com-
plex with 14-3-3␴ (Protein Data Bank entry 1ywt) has been
selected as starting point to generate a structure-based phar-
macophoric model, in turn used as a search query for a
virtual screening protocol aimed at identifying small mole-
cules targeting the 14-3-3 binding site (Wilker et al., 2005). A
preliminary model accounting for the large number of inter-
actions found in the complex was then simplified as described
in the Supplemental data, thus resulting in the final model
consisting of two hydrogen bond acceptor features (HBA1
and HBA2), one hydrogen bond donor feature (HBD4), one
negative ionizable feature (NEG.ION.), two hydrophobic fea-
and bottom right). Moreover, BV02 (5 ␮M for 24 h) recruited
both cell types into the sub-G1 phase of cell cycles and in-
duced their apoptotic death (Fig. 2, B and C, second and
fourth bars). Apoptosis induction in response to BV02 was
further confirmed by caspase 8 activation, similar to that
elicited by IM in wt Bcr-Abl-expressing Ba/F3 cells (Fig. 2D,
first, second, and fourth bars). BV02 antiproliferative and
proapoptotic effects were also seen in Ba/F3 cells expressing
another Bcr-Abl point mutation associated with in vivo IM
resistance [i.e., a Glu-to-Lys substitution at position 255 of
Abl ATP-binding loop (E255K)] (Barthe et al., 2002). Results
tures (HYD1 and HYD2), and eight excluded volumes (shown concerning BV02 effects on reproductive integrity, survival,
as gray spheres in Fig. 1B).
and sub-G recruitment of Ba/F3 cells expressing the E255K
1
Database Searching. The final pharmacophoric model mutation are shown in Supplemental Fig. S3, A–C). BV02
ϩ
was then used as a three-dimensional query for screening a effects were then investigated in CD34 cells from patients
database of approximately 2 ϫ 10 commercially available with CML who were in blast crisis and developed in vivo IM
5
compounds. The 99 compounds selected were further reduced resistance associated with the outcome of T315I Bcr-Abl mu-
ϩ
to 87 entries by application of the Lipinski’s rule of five that tation. BV02 elicited an antiproliferative response in CD34
allows the prediction of the drug likeness of a compound on cells from all three patients, with LD50 values of 0.5, 2.5, and
the basis of several molecular properties, such as H bond 0.7 ␮M (Fig. 2E).

6
00
Mancini et al.
Fig. 2. BV02 effects on Bcr-Abl-expressing cells. A, BV02 impact on reproductive integrity of Ba/F3 cells expressing the wild-type Bcr-Abl sensitive
to IM (top right) or the T315I mutation resistant to IM (bottom right). IM response of both cell types was preliminarily confirmed (top and bottom left).
Dose-response curves were analyzed by means of linear regression analyses to calculate LD50 [i.e., the compound dose required to reduce to 50% the
cell plating efficiency (aggregates containing Ͼ50 cells: percentage values versus untreated controls) in 0.9% methylcellulose]. Results shown here
represent the mean values of three individual experiments. S.D. did not exceed 10% (data not shown). B, cytofluorimetric analysis of Annexin V and
PI uptake was used to measure apoptosis induction in response to IM (1 ␮M for 24 h) and BV02 (5 ␮M for 24 h) in Ba/F3 cells expressing the wild-type
Bcr-Abl protein (first and second bar) or the T315 mutation (third and fourth bar). Results shown here represent the mean values Ϯ S.D. of three
individual experiments. Apoptotic cell fractions in untreated controls was Ͻ5% (data not shown). IM has a statistically significant effect (ء) on survival
of Ba/F3 cells expressing wt Bcr-Abl construct (p Ͻ 0.001), but not on survival of Ba/F3 cells expressing T315I mutation (p Ͻ 0.5). BV02 has a
statistically significant effect (ء) on survival of both cell types (p Ͻ 0.0001). C, the percentage of cells recruited in the sub-G phase of cell cycle by IM
1
and BV02 was assessed by mean of cytofluorimetric analysis of PI uptake. In untreated controls, cells in sub-G1 phase were Ͻ10% (data not shown).
Sub-G size increment was statistically significant (ء) in wild-type Bcr-Abl-expressing cells exposed to IM and BV02 (p Ͻ 0.001) (first and second bar)
1
and in cells expressing T315I exposed to BV02 (p Ͻ 0.05) (fourth bar) but remained steady in the latter cell type exposed to IM (p Ͻ 0.5). D, caspase
8
activity was analyzed by mean of cytofluorimetric analysis of FLICA labeling. It was totally absent in untreated cells (data not shown), significantly
raised (ء) by IM and BV02 in wild type Bcr-Abl-expressing cells (p Ͻ 0.001 and Ͻ0.0001, respectively) (first and second bar) and by BV02 in cells
ϩ
expressing T315I (p Ͻ 0.001) (fourth bar). E, BV02 survival curves of CD34 cells isolated from bone marrow samples of three patients with CML who
were in blast crisis who developed in vivo IM resistance associated with the outcome of T315I mutation. Nonlinear regression analysis was used to
calculate BV02 LD50
.
BV02 Effects on c-Abl Interaction with 14-3-3␴. Reac- Here we investigated BV02 effects on subcellular distribu-
tivation of proapoptotic or antiproliferative signals after tion of c-Abl, the proapoptotic function of which is precluded
their release from the 14-3-3 binding site is the most likely by persistent 14-3-3 ligand associated with p210 Bcr-Abl TK
cause of BV02 cytotoxic effects on Bcr-Abl-expressing cells. (Mancini et al., 2009). In wt Bcr-Abl-expressing Ba/F3 cells,

14-3-3 Inhibitors in Chronic Myeloid Leukemia
601
Fig. 3. BV02 effects on c-Abl subcellular relocation in wt Bcr-Abl-expressing Ba/F3 cells. Protein expression and interactions in cytoplasmic (A) and
nuclear (B) compartments and at mitochondrial membranes (C) of wild-type Bcr-Abl-expressing Ba/F3 cells either untreated or after 24 h exposure
to 1 ␮M IM or 5 ␮M BV02 were analyzed by mean of Western blot or IP/immunoblotting. ␤-Actin, histone H1, and cytochrome c (Cyt C) levels served
as controls for protein loading and to exclude cross-contamination among three subcellular compartments. Results identical to those shown here were
obtained in two additional experiments. Signal intensities in single blots were measured by a chemoluminescence detection system and quantified by
means of dedicated software (see Materials and Methods for details). ء, statistically significant differences relative to treatments (p Յ 0.05).
Phosphorylation of p210 Bcr-Abl at Tyr245 was not significantly affected by BV02 (p Ͻ 0.1) but completely revoked by IM. c-Abl phosphorylation at
Tyr245 was not affected by either BV02 or IM (p Ͻ 0.1). The levels of 14-3-3␴ were left steady by BV02 (p Ͻ 0.1) and reduced by IM (p Ͻ 0.005). In
the cytoplasm, the reduction of c-Abl (145 kDa) level and interaction with 14-3-3␴ and the increase of c-Abl interaction with caspase 9, caspase 9 (p35),
and caspase 8 (p18) activated fragments in response to BV02 (as well as to IM) were statistically significant (p Յ 0.05). In the nuclear compartment,
Kip1
c-Abl (145 and 120 kDa) and p27
were significantly increased by BV02 and IM (p Յ 0.01). At mitochondrial membranes, c-Abl (145 and 60 kDa),
caspase 9 (p35), caspase 8 (p18), and t-Bid (p15) were significantly raised by BV02 and IM (p Յ 0.01).
BV02 (5 ␮M for 24 h) promoted c-Abl release from 14-3-3␴, ated with c-Abl enhanced interaction with caspase 9 and a
abolished c-Abl expression in the cytoplasm (as IM did), but remarkable increase of caspase 9 p35 cleaved subunit both in
had no influence on 14-3-3␴ levels (Fig. 3A). It is noteworthy cytoplasmic compartment and at mitochondrial membranes
that the p145 c-Abl form was the only one present in the (Fig. 3, A and C).
cytoplasm, whereas the p120-kDa fragment generated by
Caspase 9 is the apical member of intrinsic apoptotic path-
caspase-dependent cleavage was completely absent either in way: it triggers sequential activation of caspases 3, 7, and 6,
control or treated cells (Fig. 3A) (Baril a` et al., 2003). As leading in turn to the activation of caspase 8, the apical
expected, BV02 had no influence on phosphorylation of c-Abl member of extrinsic pathway (Inoue et al., 2009). Accord-
and p210 Bcr-Abl at Tyr245 (the residue located at inter- ingly, caspase 9 activation in response to BV02 and IM was
linker region and required for both protein enzymatic activ- followed by caspase 8 cleavage into the p18 active fragment
ity) (Fig. 3A). c-Abl release from 14-3-3␴ in response to BV02 both in the cytoplasmic compartment and at mitochondrial
induced its translocation into the nuclear compartment sim- membranes (Fig. 3, A and C). It is noteworthy that caspase 8
ilar to that elicited by IM (Fig. 3B). In the nuclear compart- activation in response to either IM or BV02 evoked the cleav-
ment c-Abl was seen as 145 kDa full-length protein and 120 age and mitochondrial targeting of p15 fragment of the BH3-
kDa fragment (Fig. 3B). The concomitant increment of cyclin- interacting death agonist Bid (Fig. 3C) (Billen et al., 2008).
Kip1
dependent kinase inhibitor p27
in response to BV02 sup- Such an event may further contribute to the outer mitochon-
ports the idea that the release from 14-3-3 is a common drial membrane permeabilization. BV02-induced 1) c-Abl re-
mechanism for nuclear import of proteins whose 14-3-3 bind- lease from 14-3-3␴, 2) nuclear import, 3) mitochondrial mem-
ing causes cytoplasmic compartmentalization and “loss of brane targeting associated with the activating cleavage of
Kip1
function” associated with p210 Bcr-Abl TK (Fig. 3B) (Fujita caspases 9 and 8, 4) nuclear translocation of p27
et al., 2002).
, 5)
cleavage, and 6) mitochondrial accumulation of Bid were also
Moreover, BV02 promoted c-Abl targeting to mitochondrial seen in IM-resistant Ba/F3 cells expressing T315I and E255K
membranes in the full-length (145-kDa) form and cleaved Bcr-Abl mutations (Fig. 4, A–C, and Supplemental Fig. S4,
fragments, in particular the 60-kDa form resulting from A–C). As in wt Bcr-Abl-expressing Ba/F3 cells, the exposure
caspase-induced cleavage (Fig. 3C). A previous study proved to BV02 reduced p145-kDa c-Abl but did not change 14-3-3␴
that c-Abl association with caspase 9 promotes the caspase 9 expression (Fig. 4A). All these events contribute to drive the
phosphorylation-dependent auto-cleavage, a critical step of apoptotic death of IM-resistant Ba/F3 cells expressing T315I
DNA damage-induced auto-processing of caspase 9 leading to and E255K Bcr-Abl mutations in response to BV02 (Fig. 2B
the activation of caspase 3 and apoptosis in response to and Supplemental Fig. S3C). As in the IM-sensitive cell con-
genotoxic stress (Raina et al., 2005). Here we saw that c-Abl text, c-Abl dissociation from 14-3-3␴ was not contingent upon
release from 14-3-3␴ in response to BV02 and IM is associ- Tyr245 dephosphorylation (Fig. 4A and Supplemental Fig.

6
02
Mancini et al.
Fig. 4. BV02 effects on c-Abl subcellular
relocation in Ba/F3 cells expressing
T315I Bcr-Abl mutation. Protein expres-
sion and interactions in cytoplasmic (A)
and nuclear (B) compartments and at mi-
tochondrial membranes (C) of wild-type
Bcr-Abl-expressing Ba/F3 cells either un-
treated or after 24 h exposure to 1 ␮M IM
or 5 ␮M BV02 were analyzed by mean of
Western blot or IP/immunoblotting. ␤-Ac-
tin, histone H1, and cytochrome c (Cyt C)
levels served as controls for protein load-
ing and to exclude cross-contamination
among three subcellular compartments.
Results identical to those shown here
were obtained in two additional experi-
ments. Signal intensities in single blots
were measured by a chemoluminescence
detection system and quantified by
means of dedicated software (see Materi-
als and Methods for details). A slight in-
crease (p Ͻ 0.05) of Tyr245 phosphoryla-
tion of normal and Bcr-rearraged c-Abl
was apparent in Ba/F3 cells expressing
the T315I mutation after exposure to
BV02.
S4A). On the contrary, a slight increase (p Ͻ 0.05) of Tyr245
phosphorylation of normal and Bcr-rearranged c-Abl was
apparent in Ba/F3 cells expressing the T315I mutation after
exposure to BV02 (Fig. 4A). This increase may be ascribed to
the release of unknown signal(s) that integrate phospho-Tyr
signaling and/or protein stability. Further studies are re-
quired to elucidate the matter.
Despite the critical role of 14-3-3 ligand in the balance
between life and death, no 14-3-3 antagonists with pharma-
cological applications have been developed so far. The only
known compound capable of blocking 14-3-3 interaction with
most or all of its client proteins is R18 (otherwise named
difopein), a high-affinity peptide ligand binding with the
amphipathic groove of all 14-3-3 isoforms (Wang et al., 1999).
R18 was widely used to probe 14-3-3 participation in various
signaling pathways. Here we used R18 to confirm that c-Abl
release from 14-3-3 after the inhibition of the 14-3-3 binding
site triggers the chain of events leading to apoptotic death of
Bcr-Abl-expressing cells. In Ba/F3 cells expressing the wt
Bcr-Abl, R18 (25 ␮M for 24 h) promoted c-Abl release from Fig. 5. R18 effects on c-Abl subcellular relocation. c-Abl release from
1
4-3-3␴, nuclear import and translocation to mitochondrial membranes
1
4-3-3␴ (Fig. 5). Moreover, it induced c-Abl nuclear import
in response to IM (1 ␮M for 24 h) and R18 (25 ␮M for 24 h) were assayed
and translocation to mitochondrial membranes and was as-
by means of Western Blot and IP/immunoblotting analyses in wild type
sociated with the increase of caspase 8 cleaved fragments Bcr-Abl-expressing Ba/F3 cells. Protein expression and interactions in
cytoplasmic and nuclear compartments and at mitochondrial membranes
(
Fig. 5). It is noteworthy that c-Abl nuclear translocation was
of wild-type Bcr-Abl-expressing Ba/F3 cells either untreated or after 24 h
significantly enhanced by R18 association with IM, suggest-
exposure to 1 ␮M IM or 5 ␮M BV02 were analyzed by mean of Western
ing that the two compounds may have additive effects on blot or IP/immunoblotting. ␤-Actin, histone H1, and cytochrome c (Cyt C)
1
4-3-3 interaction(s) with client proteins (Fig. 5).
levels served as controls for protein loading and to exclude cross-contam-
ination among three subcellular compartments. Results identical to those
shown here were obtained in two additional experiments. Signal inten-
sities in single blots were measured by a chemiluminescence detection
system and quantified by means of dedicated software (see Materials and
Methods for details). Cross-contamination among subcellular compart-
ments was excluded by simultaneous labeling of single blots with ␤-actin,
histone H1 and cytochrome c (data not shown).
Discussion
TK of the Abl moiety of the p210 Bcr-Abl fusion protein
constitutively activated by the coiled-coil domain at the N
terminus of BCR is the causative event of CML (McWhirter
et al., 1993). Accordingly, IM, a 2-phenylaminopyrimidine
derivative having a specific inhibitory activity against c-Abl driven by Bcr-Abl point mutations that directly impair drug
and Bcr-Abl TK, has revolutionized the disease prognosis binding to the Abl TK domain (T315I, F317L, and F359V) or
(
Druker, 2008). However, secondary resistance (defined as preclude the fusion protein conformational changes required
the loss of IM response during treatment) emerged as a major for drug binding either in the P-loop (M244V, G250E,
problem with IM therapy in CML, particularly in more ad- Q252H/R, and Y253F/H) or near the activation loop (E255K)
vanced phases of the disease. IM resistance is most often (Weisberg et al., 2007). The development of other therapeutic

14-3-3 Inhibitors in Chronic Myeloid Leukemia
603
options is therefore crucial to overcome drug resistance in (Reuther et al., 1994). Such interactions occur at the N-ter-
CML. minal Ser/Thr kinase domain encoded by the first exon of Bcr
Previous studies demonstrated that p210 Bcr-Abl TK in- (Braselmann and McCormick, 1995). Nevertheless, in our
duces an overexpression of 14-3-3, which therefore binds experience, p210 Bcr-Abl was neither bound to 14-3-3␴ nor
substrates with proapoptotic and antiproliferative effects. released from 14-3-3␴ and allowed to translocate into the
This event causes their “loss of function” through cytoplasmic nuclear compartment in response to IM (Mancini et al.,
compartmentalization, thereby contributing to the illegiti- 2009). Indeed, in the Bcr-Abl fusion protein, Bcr interaction
mate expansion of CML hematopoiesis. Accordingly, the de- with 14-3-3 is abrogated by Tyr autophosphorylation at the
phosphorylation of 14-3-3-binding motifs and the phosphor- Bcr first exon (Liu et al., 1993; Peters and Smithgall, 1999).
ylation of 14-3-3 at critical binding residues restore the Moreover, Bcr phosphorylates 14-3-3 at Thr233, but not at
Kip1
functions of Bad, p27
, and forkhead box O 3a, and of Bax Ser184, the JNK substrate involved in c-Abl interaction
and c-Abl, respectively, through events encompassing their (Clokie et al., 2005). However, further investigation is re-
release from 14-3-3 (Tsuruta et al., 2004; Yoshida et al., 2005; quired to elucidate whether 14-3-3 phosphorylation at
Dong et al., 2008; Mancini et al., 2009). Targeting 14-3-3 Thr233 has a role in 14-3-3 binding with Bcr-Abl proteins.
binding sites may be therefore considered a useful strategy to
To conclude, our study supports the idea that the inhibition
induce apoptotic death and growth arrest of leukemic pro- of 14-3-3 binding site may be considered for the treatment of
genitors. We applied a virtual screening protocol to a data- CML and, in particular, to overcome the outcome of drug
base of approximately 200,000 commercially available non- resistance associated with the disease progression (Dong et
peptidic compounds. We found that among 14 compounds al., 2008). To our knowledge, BV02 represents the first non-
selected in agreement with the filters applied during the peptidic inhibitor of 14-3-3 proteins potentially useful for
virtual screening and docking studies, the one named BV02 clinical purposes. Further work for the improvement of BV02
exhibits a remarkable cytotoxicity against Brc-Abl-express- for clinical purpose is currently in progress.
ing cells either sensitive or resistant to IM (Figs. 1 and 2, A
and C, and Supplemental Fig. S3A). c-Abl repartitioning
among subcellular compartments is one component of BV02
cytotoxicity. First, the inhibition of 14-3-3␴ binding site in
response to BV02 promotes c-Abl release in the Tyr245 phos-
phorylated isoform, confirming that the two protein interac-
tion is independent from c-Abl activating phosphorylation (Yo-
Authorship Contributions
Participated in research design: Mancini, Corradi, Petta, Barbieri,
Manetti, Botta, and Santucci.
Conducted experiments: Mancini, Petta, Barbieri, and Santucci.
Performed data analysis: Corradi, Manetti, and Botta.
Wrote or contributed to the writing of the manuscript: Mancini,
shida et al., 2005; Mancini et al., 2009). Once released from Corradi, Petta, Barbieri, Manetti, Botta, and Santucci.
1
4-3-3␴, c-Abl relocates to the nuclear compartment and mito-
chondrial membranes, where it evokes apoptotic death signals
Figs. 3 and 4, Supplemental Fig. S4). Previous studies proved
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