Pharmacologic inhibition of MNKs in acute myeloid leukemia

Article abstract of DOI:10.1124/mol.115.098012

The Ras/Raf/MAPK and PI3K/Akt/mTOR pathways are key signaling cascades involved in the regulation of cell proliferation and survival, and have been implicated in the pathogenesis of several types of cancers, including acute myeloid leukemia (AML). The onc

Full text of DOI:10.1124/mol.115.098012

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2015/06/04/mol.115.098012.DC1.html
1521-0111/88/2/380–389$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.115.098012
Mol Pharmacol 88:380–389, August 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Pharmacologic Inhibition of MNKs in Acute Myeloid Leukemia s
Theodosia Teo, Frankie Lam, Mingfeng Yu, Yuchao Yang, Sunita K. C. Basnet,
Hugo Albrecht, Matthew J. Sykes, and Shudong Wang
Centre for Drug Discovery and Development, Sansom Institute for Health Research, Centre for Cancer Biology, and School of
Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia
Received January 19, 2015; accepted June 4, 2015
ABSTRACT
The Ras/Raf/MAPK and PI3K/Akt/mTOR pathways are key effects with respect to the selective Mnk1/2 kinase inhibition in
signaling cascades involved in the regulation of cell proliferation AML cells causes G1 cell cycle arrest followed by induction of
and survival, and have been implicated in the pathogenesis of apoptosis. MNKI-19 and MNKI-85 demonstrate similar Mnk2
several types of cancers, including acute myeloid leukemia (AML). kinase activity and cellular antiproliferative activity but exhibit
The oncogenic activity of eIF4E driven by the Mnk kinases is different time-dependent effects on cell cycle progression and
a convergent determinant of the two cascades, suggesting that apoptosis. Collectively, this study shows that pharmacologic
targeting the Mnk/eIF4E axis may provide therapeutic opportunity inhibition of both Mnk1 and Mnk2 can result in a more pro-
for the treatment of cancer. Herein, a potent and selective Mnk2 nounced cellular response than targeting Mnk2 alone. However,
inhibitor (MNKI-85) and a dual-specific Mnk1 and Mnk2 inhibitor MNKI-85, a first-in-class inhibitor of Mnk2, can be used as
(MNKI-19), both derived from a thienopyrimidinyl chemotype, a powerful pharmacologic tool in studying the Mnk2/eIF4E-
were selected to explore their antileukemic properties. MNKI-19 mediated tumorigenic mechanism. In conclusion, this study
and MNKI-85 are effective in inhibiting the growth of AML cells that provides a better understanding of the mechanism underlying
possess an M5 subtype with FLT3-internal tandem duplication the inhibition of AML cell growth by Mnk inhibitors and suggests
mutation. Further mechanistic studies show that the downstream their potential utility as a therapeutic agent for AML.
(Milella et al., 2001; Martelli et al., 2006). Another common
molecular defect, FLT3 mutation, is highly expressed in AML,
accounting for 70–100% of all cases (Gilliland and Griffin, 2002).
Introduction
Acute myeloid leukemia (AML) is a genetically heterogeneous
cancer characterized by a collection of mutations and chromo-
Specifically, the presence of internal tandem duplication (ITDs)
somal translocations in immature myeloid cells that cooperate to
in the juxtamembrane domain of FLT3 was found in nearly one-
interrupt the proliferative or survival pathways (Gilliland et al.,
third of patients with AML and is associated with a poor
2004). The aberrant fusion transcription factors expressed in
prognosis (Meshinchi et al., 2001). Therefore, these signal
myeloid cells can impair hematopoietic differentiation; however,
transduction cascades may represent promising areas to exploit
a single genetic alteration is insufficient to induce leukemogenesis
with therapeutic intent in AML.
in most cases (He et al., 1997; Yuan et al., 2001). Particularly,
Interestingly, the aforementioned cascades consequently
constitutively activated signal transduction pathways derived
converge on the Mnks/eIF4E axis, which plays a significant
from the mutated genes that normally regulate hematopoietic
regulatory role in mRNA translation and malignant trans-
cell growth and homeostasis are additional contributors to the
pathogenesis of AML (Dash and Gilliland, 2001).
The Ras/Raf/MAPK and PI3K/Akt/mTOR signaling pathways
formation. The phosphorylation of eIF4E by Mnk at Ser209 has
been implicated in tumorigenesis and development (Waskiewicz
et al., 1997; Furic et al., 2010). While Mnk activity is not
are two vital cascades that are physiologically relevant to
essential for normal cell growth (Ueda et al., 2004), the oncogenic
diverse cell stimuli involved in differentiation, proliferation, and
activity of Mnk-driven eIF4E has been shown to be vital for the
apoptosis (Lewis et al., 1998; Hanada et al., 2004). Activation of
proliferation and survival of cancer cells (Topisirovic et al., 2004;
these pathways has been associated with most AML cases
Wendel et al., 2007). Therefore, therapeutic targeting of Mnk
offers an opportunity in the treatment of cancer.
This work was supported by the Australian Government National Health
Mnks belong to a serine/threonine kinases family, and were
and Medical Research Council [Grant 1050825], and the Beat Cancer Project
identified as unique interacting proteins for ERK and p38
kinases in the MAPK cascade (Waskiewicz et al., 1997; Roux
and Blenis, 2004). Two Mnk human genes have been identified,
expressing Mnk1 and Mnk2 proteins that are ∼72% identical in
Principal Cancer Research Fellowship by South Australian Health and
Medical Research Institute to S.W.
dx.doi.org/10.1124/mol.115.098012.
s
This article has supplemental material available at molpharm.
aspetjournals.org.
ABBREVIATIONS: AML, acute myeloid leukemia; CGP57380, N3-(4-fluorophenyl)-1H-pyrazolo-[3,4-d]pyrimidine-3,4-diamine; DCM, dichloromethane;
DMSO, dimethylsulfoxide; FA, formic acid; ITD, internal tandem duplications; MNKI-19, 4-((4-fluoro-2-isopropoxyphenyl)amino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid; MNKI-85, 4-((4-fluoro-2-isopropoxyphenyl)amino)-N-(2-methoxyethyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide; PI,
propidium iodide; RP-HPLC, reversed-phase high-performance liquid chromatography; WT, wild-type.
380

MAPK-Interacting Kinase Inhibition in AML
381
Synthesis of MNKI-19 and MNKI-85. 1H and 13C NMR spectra
were recorded at 298 K on an AVANCE III HD 500 spectrometer (Bruker,
Faellanden, Switzerland) (1H at 500.16 MHz and 13C at 125.76 MHz),
and were analyzed using the Topspin 3.2 software (Bruker). The 1H
and 13C NMR spectra are referenced to 1H signals of residual
nondeuterated solvents and 13C signals of deuterated solvents,
respectively. The 1H NMR signals are reported with chemical shift
values d (ppm), multiplicity (where s 5 singlet, d 5 doublet, t 5 triplet,
dd 5 doublet of doublets, td 5 triplet of doublets, m 5 multiplet, and
br 5 broad), relative integral, coupling constants J (Hz), and assign-
ments. High-resolution mass spectra were recorded on a TripleTOF
5600 mass spectrometer (AB SCIEX, Concord, Ontario, Canada), and
ionization of all samples was carried out using electrospray ionization.
Melting points were determined using an open capillary on a Stuart
SMP10 (Staffordshire, UK) melting point apparatus and were un-
corrected. The purity of the compounds used for biologic evaluation was
determined by analytic reversed-phase high-performance liquid chro-
matography (RP-HPLC), which was carried out on a Prominence UFLC
System (Shimadzu, Kyoto, Japan) equipped with a CBM-20A commu-
nications bus module, a DGU-20A5R degassing unit, an LC-20AD liquid
chromatograph pump, an SIL-20AHT auto-sampler, an SPD-M20A
photo-diode array detector, a CTO-20A column oven and a Phenomenex
their amino acid sequence (Slentz-Kesler et al., 2000). Alterna-
tive splicing in the two Mnk genes further revealed the
existence of four isoforms: Mnk1a, Mnk1b, Mnk2a, and Mnk2b
(Waskiewicz et al., 1997; Scheper et al., 2003). Bearing a nuclear
localization signal and a polybasic sequence in the common
basic N-terminal stretch, all four isoforms preferentially localize
in the nucleus and phosphorylate eIF4E efficiently.
The difference between the four isoforms arises from the
C termini, in which only the a-isoforms have a MAPK-binding
site for interaction with ERK or p38 kinases (Parra et al., 2005).
Despite this, Mnk1a and Mnk2a differ in basal activity as has
been shown in in vitro studies (Scheper et al., 2001). Activation
of ERK or p38 kinase could result in a marked increase in the
activation of the low basal activity of Mnk1a, but would
negligibly influence the high basal activity of Mnk2a. Both
Mnk1a and Mnk2a bind preferentially to ERK but only Mnk1a
is a substrate for p38. In appreciation of the dissimilarity in the
regulatory features of Mnk1 and Mnk2, these findings may
indicate differences in activity and regulation of Mnks toward
eIF4G to phosphorylate eIF4E.
An increasing body of evidence has demonstrated the Kinetex 5u C18 100A 250 mm  4.60 mm column (Shimadzu). Method A
[gradient 5–95% CH3OH containing 0.1% formic acid (FA) over
7 minutes at a flow rate of 1 ml/min, followed by 95% CH3OH containing
0.1% FA over 13 minutes] and method B (gradient 5–95% CH3CN
containing 0.1% FA over 7 minutes at a flow rate of 1 ml/min, followed by
95% CH3CN containing 0.1% FA over 13 minutes) were used for the
analytic RP-HPLC. Data acquired from the analytic RP-HPLC were
processed using LabSolutions Analysis Data System (Shimadzu).
To a solution of methyl 4-((4-fluoro-2-isopropoxyphenyl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylate (200 mg, 0.533 mmol) in
a mixture of THF/MeOH/H2O (1:1:1, 60 ml), LiOH (382 mg, 16.0 mmol)
potential antileukemic properties of pharmacologic Mnk
inhibitors (Altman et al., 2013; Lim et al., 2013; Diab et al.,
2014b). Cercosporamide, a nonselective Mnk inhibitor (Konicek
et al., 2011), reduces leukemic cell proliferation in MM6, K562,
and U937 cell lines in a dose-dependent manner (Altman et al.,
2013). The antitumor efficacy of cercosporamide in MV4-11
xenografts supports its Mnk-targeted antileukemic effects. The
cell-specific effects, predominantly in leukemia cells, of Mnk
inhibition have been recently confirmed by the recent discovery
of 5-(2-(phenylamino)pyrimidin-4-yl)thiazole-2(3H)-one Mnk was added. The reaction mixture was stirred at room temperature
overnight and washed with dichloromethane (DCM) (20 ml). The
aqueous layer was heated at 50°C for 10 minutes and acidified to pH 3
with 2 M HCl. The precipitate was filtered, washed with H2O (3 Â 25 ml),
and dried under reduced pressure to give MNKI-19 as a white solid
(128 mg, 67%), with RF (DCM:MeOH 5 9:1) 0.28; m.p. . 300°C; 1H NMR
(DMSO-d6): d 1.32 [d, 6H, J 6.0, OCH(CH3)2], 3.07 (s, 3H, thiophene-CH3),
4.75–4.80 [m, 1H, OCH(CH3)2], 6.80 (td, 1H, J 8.5 and 2.5, benzene-H),
7.07 (dd, 1H, J 11.0 and 2.5, benzene-H), 8.47 (s, 1H, NH), 8.54 (s, 1H,
pyrimidine-H), and 8.56 (dd, 1H, J 9.0 and 6.5, benzene-H) (one carboxylic
acid proton signal not observed); 13C NMR (DMSO-d6): d 15.3, 21.7, 71.5,
101.1 (d, JC-F 26.8), 106.1 (d, JC-F 21.6), 117.8, 121.9 (d, JC-F 9.4), 123.9,
125.0 (d, JC-F 2.6), 137.5, 148.3 (d, JC-F 10.6), 155.1, 156.1, 158.6 (d, JC-F
239.0), and 163.9, 166.3 (one carbon signal overlapping or obscured);
high-resolution mass spectrometry (electrospray ionization) m/z
362.1500 [M1H]1; calculated for C17H17FN3O3S1 362.0969 [M1H]1;
and analytic RP-HPLC method A: tR 16.95 minutes, purity . 97% and
method B: tR 12.48 minutes, purity . 97%.
inhibitors (Diab et al., 2014b).
We have recently identified thieno[2,3-d]pyrimidine deriv-
atives as highly potent and selective Mnk inhibitors. Herein,
a potent dual-specific Mnk1/2 inhibitor (MNKI-19, 4-((4-
fluoro-2-isopropoxyphenyl)amino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid) and a selective Mnk2 inhibitor
(MNKI-85, 4-((4-fluoro-2-isopropoxyphenyl)amino)-N-(2-
methoxyethyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide)
were used to provide mechanistic insights into the effects of
Mnk isoforms on leukemia cells. Both MNKI-19 and MNK-85
are able to reduce the level of phosphorylated eIF4E and
subsequently cause G1 phase cell cycle arrest and apoptosis in
FLT3-ITD–expressed AML cells. The current report also
demonstrates that a combined inhibition of Mnk1 and Mnk2
results in a more pronounced cellular response than targeting
Mnk2 alone, supporting the claim that Mnk inhibitors can be
developed as potential anticancer agents.
To a solution of 4-((4-fluoro-2-isopropoxyphenyl)amino)-5-methyl-
thieno[2,3-d]pyrimidine-6-carboxylic acid (200 mg, 0.533 mmol) in
N,N-dimethylformamide (3 ml), N,N-diisopropylethylamine (193 ml,
1.11 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N9,N9-tetramethy-
luronium hexafluorophosphate (316 mg, 0.830 mmol) were added.
The reaction mixture was stirred in an ice bath for 30 minutes. Then,
2-methoxypropylamine (54.0 ml, 0.609 mmol) was added, and the
reaction mixture was stirred at room temperature overnight and
concentrated under reduced pressure. The residue was dissolved in
DCM (20 ml), washed with saturated NaHCO3 (3 Â 20 ml), 10% citric
acid (3 Â 20 ml), H2O (3 Â 20 ml), and brine (3 Â 20 ml), and
concentrated under reduced pressure. The residue was purified by
Materials and Methods
Inhibitor Compounds and Proteins. Syntheses of MNKI-19
and MNKI-85 are described subsequently. The Mnk inhibitor N3-(4-
fluorophenyl)-1H-pyrazolo-[3,4-d]pyrimidine-3,4-diamine (CGP57380)
was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia), and
used as a positive control. All compounds used were dissolved in
dimethylsulfoxide (DMSO) at a stock concentration of 10 mM, and
stored at 220°C in small aliquots. The eIF4E-derived peptides TAMRA-
DTATKSGSTTKNR, TAMRA-DTATKSG(phospho)STTKNR, and Biotage (Charlotte, NC) FlashMaster Personal1 flash chromatography
DTATKSGSTTKNR were custom synthesized by Mimotopes Pty. Ltd.
(Melbourne, VIC, Australia). Proteins Mnk1 and Mnk2 were
(silica gel, DCM ramping to DCM:EtOAc 5 4:1) to give MNKI-85 as
a white solid (118 mg, 51%), with RF (DCM:EtOAc 5 7:3) 0.54; m.p.
purchased from Life Technologies (Grand Island, NY) and Merck 186–187°C; 1H NMR (CDCl3): d 1.43 [d, 6H, J 6.0, OCH(CH3)2], 3.06
Millipore (Billerica, MA), respectively.
(s, 3H, thiophene-CH3), 3.41 (s, 3H, OCH3), 3.56–3.60 (m, 2H, CH2CH2O),

382
Teo et al.
3.63–3.67 (m, 2H, CH2CH2O), 4.64–4.69 [m, 1H, OCH(CH3)2], 6.34 (br s,
1H, CONH), 6.69 (dd, 1H, J 10.0 and 3.0, benzene-H), 6.73 (td, 1H, J 8.5 &
2.5, benzene-H), 8.47 (br s, 1H, pyrimidine-NH-benzene), 8.61 (s, 1H,
Cell Viability Assays. Resazurin (Sigma-Aldrich) and MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Life
Technologies, Mulgrave, VIC, Australia) assays were performed on all
pyrimidine-H), and 8.77 (dd, 1H, J 9.0 and 6.5, benzene-H); 13C NMR leukemia cell lines and normal lung fibroblast, respectively, as
previously reported (Diab et al., 2014b). Compound concentrations
required to inhibit 50% of cell growth (GI50) were calculated using
nonlinear regression analysis.
Caspase-3/7 Assay. The activity of caspase-3/7 was measured
using the Apo-ONE Homogeneous Caspase-3/7 kit (Promega),
according to the manufacturer’s instructions, and analyzed using
the EnVision Multilabel Plate Reader (PerkinElmer).
(CDCl3): d 16.1, 22.2, 40.1, 59.1, 70.3, 71.9, 100.5 (d, JC-F 27.0), 106.8
(d, JC-F 21.6), 118.3, 121.7 (d, JC-F 9.0), 124.9, 127.3, 132.9, 147.7 (d, JC-F
9.5), 154.2, 156.2, 159.1 (d, JC-F 241.5), and 162.9 (two carbon signals
overlapping or obscured); high-resolution mass spectrometry (electro-
spray ionization) m/z 419.1394 [M1H]1; calculated for C20H24FN4O3S1
419.1548 [M1H]1; and analytic RP-HPLC method A: tR 16.01 minutes,
purity . 96% and method B: tR 11.98 minutes, purity . 96%.
Cell Cycle and Detection of Apoptosis. MOLM-13 and MV4-11
(1 Â 105) cells were seeded and incubated overnight at 37°C, 5% CO2.
Cells were centrifuged at 300g for 5 minutes upon treatment with
each compound. Cell pellets were collected and fixed with 70% ethanol
on ice for 15 minutes, followed by centrifugation at 300g for 5 minutes.
The collected pellets were incubated with staining solution [50 mg/ml
propidium iodide (PI), 0.1 mg/ml ribonuclease A, and 0.05% Triton
X-100] at 37°C for 1 hour and analyzed with a Gallios flow cytometer
(Beckman Coulter, Brea, CA). Then, 1 Â 105 of the remaining cells
were used for the apoptotic assay with the Annexin V-fluorescein
Isothiocyanate Apoptosis Detection Kit (Becton Dickenson, Franklin
Lakes, NJ), following the manufacturer’s instructions. The samples
were analyzed by fluorescence-activated cell sorting with the flow
cytometer within 1 hour of staining. Data were analyzed using Kaluza
v1.2 (Beckman Coulter).
Cell Culture. All leukemia cell lines including HL-60, K562,
MOLM-13, MV4-11, PL-21, THP-1, and U937 cells were kindly
provided by Richard D’Andrea (University of South Australia, Adelaide,
SA, Australia) and were maintained in Roswell Park Memorial
Institute-1640 medium with 10% fetal bovine serum. The normal lung
fibroblast cell line, WI-38, was obtained from the cell bank at the Centre
for Drug Discovery and Development, University of South Australia,
and was maintained in minimum essential media with 10% fetal bovine
serum, 2 mM L-glutamine, and 1% nonessential amino acid. All cell
lines were cultured in a humidified 37°C, 5% CO2 incubator.
Kinase Assays. Inhibition of CDK2/cyclin A1, CDK9/cyclin T1,
CDK4/cyclin D1, FLT3, Pim-1, B-Raf, MAPK1 (ERK2), MAPK2,
PKBa (Akt1), PI3K (p110b/p85a), SAPK2a (p38a), and mTOR was
measured by radiometric assays using the Millipore KinaseProfiler
services with ATP concentrations within 15 mM of apparent Km(app)
for each kinase. For CDK2/cyclin A1, CDK9/cyclin T1, CDK4/cyclin
D1, FLT3, and Pim-1 kinases, the IC50 values were calculated from
10-point dose-response curves and the apparent inhibition constants
(Ki) were calculated from the IC50 values and appropriate ATP values
using the Cheng and Prusoff (1973) equation. For the kinase panel
screen, the kinase activities were reported as remaining activities
that were calculated as percentage relative to the averages of
multiplying the 0 and 100% controls, at a compound concentration
of 5 mM.
Immobilized Metal Ion Affinity Particle Time-Resolved
Fluorescence Energy Transfer Progressive Binding System.
Each compound (1 and 10 mM in 0.5% DMSO) was added to the kinase
reaction containing 1Â immobilized metal ion affinity particle
reaction buffer with Tween-20 dithiothreitol, distilled H2O,
TAMRA-labeled eIF4E peptide substrate, and Mnk kinase in a total
assay volume of 20 ml. The kinase reaction was initiated by addition of
ATP, incubated at 30°C for 90 minutes, and stopped with 60 ml
progressive binding solution [30% binding buffer A, 70% binding
buffer B, progressive binding reagent (1:600) and terbium donor
(1:400)], followed by incubation in the dark at room temperature for
5 hours. After the incubation, the plate was read in an EnVision
Multilabel Plate Reader (PerkinElmer, Beaconsfield, UK). The
excitation wavelength for the terbium donor was set at 330 nm,
while emissions were measured at 545 nm (terbium emission) and
572 nm (time-resolved fluorescence energy transfer emission) in
a time-resolved manner with a delay of 200 microseconds. Positive
and negative controls were performed in 0.5% DMSO in the presence
and absence of Mnk kinases, respectively.
ADP-Glo Kinase Assay. The ADP-Glo assay kits were purchased
from Promega (Madison, WI). Serial dilution of compounds with
a dilution factor of 1:3 was performed to give eight concentrations
ranging from 10 to 4.5 nM with 0.5% DMSO. Each compound was
added to the kinase reaction containing 1Â kinase reaction buffer,
0.1 mg/ml bovine serum albumin, distilled H2O, eIF4E peptide substrate,
and Mnk kinase in a total assay volume of 15 ml. The kinase reaction was
initiated by addition of ATP, incubated at 30°C for 45 minutes, and
stopped with 15 ml ADP Glo reagent, followed by incubation in the dark
at room temperature for 40 minutes, and then 30 ml kinase detection
reagent was added. The mixture was further incubated for 40 minutes
before reading in the EnVision Multilabel Plate Reader (PerkinElmer).
Positive and negative controls were performed with 0.5% DMSO in the
presence and absence of Mnk kinases, respectively.
Western Blots. Western blotting was performed as described
previously (Wang et al., 2004). The antibodies used were as follows:
Mnk2 and phosphorylated Rb at Thr821 (p-RbT821) (Abcam, Cambridge,
UK), b-actin, caspase-3, CDK4, CDK6, cyclin D3, eIF4E, phosphor-
ylated eIF4E at Ser209 (p-eIF4ES209), 4E-BP1, phosphorylated
4E-BP1 at Thr37/46 (p-4E-BP1T37/46
)
and Thr70 (p-4E-BP1T70),
phosphorylated p42/p44 at Thr202/Tyr204 (p-ERKT202/Y204), Mcl-1,
PARP, cleaved PARP, Mnk1, p38, phosphorylated p38 at Thr180/
Tyr182 (p-p38T180/Y182) (Cell Signaling Technologies, Danvers, MA),
Bcl-2 (Dako, Kingsgrove, NSW, Australia), p-RbT826 (Sigma-Aldrich),
and p42/44 (Protein Simple, San Jose, CA). Both anti-mouse and anti-
rabbit immunoglobulin G horseradish peroxidase–conjugated anti-
bodies were obtained from Dako. Enhanced chemiluminescence
reagents were obtained from GE Healthcare Life Sciences (Rydalmere,
NSW, Australia).
Statistical Analyses. All experiments were performed in tripli-
cate and repeated at least three times; representative experiments
were selected for figures. The statistical significance of the differences
observed between the experimental groups was determined using
one-way analysis of variance with a minimal level of significance at
P , 0.05.
Results
MNKI-19 and MNKI-85 Are Potent and Selective Mnk
Inhibitors. The thienopyrimidinyl derivative MNKI-19
inhibits Mnk1 and Mnk2 potently with Ki values of 0.186
and 0.068 mM, respectively, by biochemical assays (Table 1).
MNKI-85 exhibits ∼2-fold increased inhibitory activity
against Mnk2 (Ki 5 0.031 mM) when compared with MNKI-
19, but is completely inactive against Mnk1. No inhibitory
activities against CDK2A, CDK9T, and CDK4D were detected
with both compounds (Ki . 10 mM). To further profile kinase
selectivity, MNKI-19 and MNKI-85 were tested against
a panel of upstream activating kinases of Mnks, including
B-Raf, MAPK1, MAPK2, Akt1, PI3K, p38a, and mTOR, at
5 mM (Fig. 1). FLT3 protein is an upstream affector of both the
Ras/Raf/MAPK and PI3K/Akt/mTOR pathways (Wander
et al., 2014). As one of the downstream effectors of FLT3,
Pim-1 was inhibited potently by cercosporamide (Konicek

MAPK-Interacting Kinase Inhibition in AML
383
TABLE 1
Chemical structure and kinase inhibitory activity of Mnk inhibitors
The ATP concentrations used in these assays were within 15 mM of Km. Apparent inhibition constants (Ki) were calculated from IC50 values and the appropriate Km (ATP)
values for each kinase using the Cheng-Prusoff equation. Data shown are the mean values derived from two replicates 6 S.D.
Ki (mM)
Compound
Mnk1
Mnk2
CDK2A
CDK9T1
CDK4D1
FLT3
ND
Pim-1
ND
1.010 6 0.098
0.877 6 0.212
.10
.10
.10
CGP57380
MNKI-19
0.186 6 0.011
0.068 6 0.009
0.031 6 0.012
.10
.10
.10
.10
.10
.10
.10
.10
2.35
MNKI-85
.10
.10
ND, not determined.
et al., 2011), thus it is of great interest to evaluate the inhibitory insensitive to both MNKI-19 and MNKI-85 with GI50 values of
activities of the compounds against FLT3 and Pim-1. The results 98.63 and 43.40 mM, respectively (Table 2).
obtained from in-house immobilized metal ion affinity particle
MNKI-19 and MNKI-85 Inhibits the Phosphorylation
assays for Mnk1 and Mnk2 at 1 mM of the tested compounds were of eIF4E and 4E-BP1. We next examined cellular Mnk
included for comparison. Both compounds are inactive against inhibition of MNKI-19 and MNKI-85 by Western blot analysis
most of the upstream kinases, with the exception that MNKI-19 (Fig. 2). MOLM-13 cells were treated with CGP57380, MNKI-
displays moderate activity against Pim-1 (Ki 5 2.35 mM) 19, or MNKI-85 at 5 and/or 10 and 20 mM for a period of 1 hour.
(Table 1). However, it is 12- and 35-fold less active when As shown in Fig. 2A, both MNKI-19 and MNK-85 completely
compared with Mnk1 and Mnk2, respectively. Collectively, depleted the phosphorylation of eIF4E at Ser209 (p-eIF4ES209
)
these data suggest the high specificity of MNKI-19 and at a lower concentration (i.e., 5 mM), whereas CGP57380
MNKI-85 for Mnks.
moderately suppressed p-eIF4ES209 within the same concen-
MNKI-19 and MNKI-85 Exhibit Antileukemic Activity. tration. These results confirm the cellular inhibition of Mnk
The antiproliferative activity of both MNKI-19 and MNKI-85 and the relative potency of MNKI-19 and MNKI-85 against
was assessed on a panel of six AML cell lines and one chronic Mnks over CGP57380. The treatment caused no changes in the
myeloid leukemia cell line (Table 2) using 72-hour resazurin
assays. CGP57380 was included as a control. All compounds
exhibited the greatest antiproliferative effects on MOLM-13
and MV4-11, both of which expressed FLT3-ITD (Quentmeier
et al., 2003), with GI50 values ranging between 5.65 and 8.81 mM.
In contrast, at least 4-fold higher concentrations of com-
pounds MNKI-19 and MNKI-85 were required to suppress the
proliferation of HL-60, K562, THP-1, and U937 [wild-type
(WT) FLT3 expression]. Surprisingly, PL-21 cells, expressing
both WT and mutated alleles, showed less sensitivity in
response to the same treatments. Different antiproliferative
effects on these cells have also been observed with CGP57380,
which modestly suppressed cell proliferation with GI50 values
ranging from 6.89 to 38.97 mM, suggesting an alternative
mechanism underlying the growth inhibitory effects for
CGP57380. Taken together, these data suggest that cellular
Fig. 1. Selectivity of MNKI-19 and MNKI-85 against a panel of kinases.
The remaining percentages of kinase activity upon treatment with each
compound at a concentration 5 mM are shown, unless otherwise specified.
effects of the Mnk inhibitors are more pronounced against
AML cells with FLT3-ITD expression than the nonmutated
FLT3 cells. It is worth noting that these thienopyrimidinyl
derivatives also display cell-specific effects between cancerous
and normal cells. The normal lung fibroblast WI-38 cells were
Both Mnk1 and Mnk2 are tested by the in-house immobilized metal ion
affinity particle assays at 1 mM of compound inhibitors; 15 mM within Km
of ATP for each kinase is used in the assays. Data represent one repeat,
and kinases that show ,50% in kinase activity upon compound treatment
are further evaluated with dose-response analysis to obtain their apparent
inhibition constants (Ki) as shown in Table 1.

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Teo et al.
TABLE 2
Antiproliferative activity of CGP57380, MNKI-19, and MNKI-85 in leukemia cell lines with various FLT3 mutational statuses
and normal fibroblast cells by 72-hour resazurin and MTT assays, respectively
72-hour GI50 (mM)a 6 S.D.
Cell Line
FLT3 Status
FAB Subtype
CGP57380
MNKI-19
MNKI-85
MOLM-13
MV4-11
PL-21
ITD
ITD
M5a
M5
8.81 6 0.80
6.89 6 0.16
9.39 6 0.11
16.83 6 1.98
38.97 6 2.98
9.40 6 0.60
34.13 6 1.82
62.37 6 0.24
5.65 6 0.84
7.30 6 0.75
57.17 6 6.74
69.18 6 6.15
.100
33.59 6 4.21
93.16 6 9.68
98.63 6 1.54
7.19 6 0.08
7.44 6 0.77
.100
ITD/WT
M3
HL-60
THP-1
U937
WT
M2
M5
M4/5
(CML)
.100
WT
94.06 6 8.40
.100
.100
43.40 6 5.05
WT
K562
WT
WI-38b
Normal fibroblast cells
Normal fibroblast cells
CML, chronic myeloid leukemia; FAB, French-American-British classification system.
aData shown are the mean values derived from at least two replicates 6 S.D.
bThe GI50 values were determined by 72-hour MTT assay.
expression of total eIF4E, Mnk1, and Mnk2 proteins. To assess point (i.e., 1 hour) (see also Supplemental Fig. 2). However,
the Mnk-targeting specificity, the effects of MNKI-19 or MNKI- 24-hour incubation of MOLM-13 cells with Mnk inhibitors
85 on the upstream activating kinases, including p38 MAPK, resulted in the suppression of cyclin D3 expression in a dose-
ERK, eIF4E-binding protein 1 (4E-BP1), and their phosphory- dependent manner (Fig. 3D). CDK4 was also downregulated by
lated forms, were analyzed in MOLM-13 cells. Incubation with Mnk inhibitors at low concentrations.
MNKI-19 for 1 hour upregulated an early transient phosphor-
To investigate whether the perturbation of cell cycle pro-
ylation of ERK and p38 in a dose-dependent manner, without gression is a consequence of CDK4 activity, the phosphorylation
affecting their total levels. The expression levels of protein of Rb at both CDK4-specific Thr826 (p-RbT826) sites (Macdonald
4E-BP1 and its phosphorylated forms at Thr37/46 and Thr70 and Dick, 2012) was also measured. A decreased level of
are not affected, which is consistent with the kinase assays p-RbT826 was detected upon treatment of MOLM-13 cells with
showing no inhibitory activity against mTOR with MNKI-19 20 mM of MNKI-19 or MNKI-85 (Fig. 3D; Supplemental Fig. 3),
and MNKI-85 (Fig. 1). Similar effects were seen in CGP57380- indicating the loss of CDK4 activity and restriction of the G1 to
treated cells but not in MNKI-85. However, little to no effect on S transition. This observation is further supported by kinase
the levels of p38 and ERK proteins and their phosphorylated assays showing that both MNKI-19 and MNKI-85 have no
forms was observed after treating MOLM-13 with 20 mM inhibitory effect on CDK4 kinase activity (Table 1), implying
compounds for 24 hours (Fig. 2B). After 24 hours, the complete the decreased CDK4 protein expression is a consequence of the
abolition of p-eIF4ES209 by Mnk inhibitors gave rise to a downregulating Mnk/eIF4E-mediated protein synthesis of
reduction in the phosphorylation of 4E-BP1 at Thr37/46 (Fig. 2C; cyclin D3. In contrast, little effect on CDK6 or CDK6-specific
Supplemental Fig. 1) and Thr70 (Fig. 2C), particularly at the p-RbT821 levels was observed (Fig. 3E; Supplemental Fig. 4),
latter in a dose-dependent manner, in MOLM-13 cells.
MNKI-19 and MNKI-85 Arrest Cancer Cells in the G1 cell cycle progression (Neganova and Lako, 2008).
Phase of the Cell Cycle via Downregulation of CDK4 MNKI-19 and MNKI-85 Induce Apoptosis in FLT3-
supporting the relative importance of CDK4 over CDK6 in the
and Cyclin D3. We next investigated whether the anti- ITD–Expressed AML Cells. The apoptotic mechanism of
proliferative activity of MNKI-19 and MNKI-85 was a conse- action by Mnk inhibitors was further examined using annexin
quence of cell cycle effects. As shown in Fig. 3A, treatment of V/PI staining. Exposure of MOLM-13 or MV4-11 cells to either
MOLM-13 cells with MNKI-19 or MNKI-85 at 5 mM for MNKI-19, MNKI-85, or CGP57380 at a concentration of 10 or
24 hours resulted in the accumulation of G1 cells, which was 20 mM for 24 hours caused a 2- to 4-fold increase in apoptotic
accompanied by a loss in cell population at the G2/M phase. cells (annexin V1/PI2 and annexin V1/PI1) when compared
This is consistent with previous studies (Zhang et al., 2008; with untreated cells (Fig. 4, A and B). Consistent with the cell
Diab et al., 2014b). The cell cycle effect of MNKI-19 was found cycle profile, MNKI-19 induced significant apoptosis in a dose-
to behave in a dose- and time-dependent manner. Consistently, and time-dependent manner. While the proteins involved in the
this was observed with MV4-11 cells after treatment with the apoptosis mechanism, including Mcl-1, Bcl-2, PARP, and
compounds (Fig. 3B). However, in the case of MNKI-85 there caspase-3, were not affected in MOLM-13 cells at the early time
was no significant change in the cell cycle distribution observed point (i.e., 1 hour) (Fig. 4C), Western blot analysis revealed the
with longer treatment time. Time-course Western blot analysis downregulation of the anti-apoptotic protein Mcl-1 and/or an
was conducted to study the levels of key proteins involved in increase of PARP cleavage after exposure to 5 mM of MNKI-19,
the cell cycle after treatment of MOLM-13 cells with compound MNKI-85, or CGP57380 for 24 hours (Fig. 4D), indicating the
inhibitors for 1 and 24 hours. Attempts were made to study the apoptotic effect caused by the inhibition of Mnks. The levels of
expression level of cyclin D1 in MOLM-13 cells but the protein Bcl-2 remain unchanged (Fig. 4E), which is in agreement with
was too weak to be detected (unpublished data), hence cyclin our previous results obtained using MV4-11 (Diab et al., 2014b).
D3 was pursued. In human somatic cells, the G1 to S phase
In conjunction with no aberration in full-length caspase-3
transition is tightly regulated by the cyclin-D/CDK4/6 proteins, protein levels (Fig. 4E) and no detectable caspase-3/7 activity
along with their downstream target, the Rb protein (Neganova (Fig. 4F), the data indicate that Mnk inhibitor-induced apoptosis
and Lako, 2008). As shown in Fig. 3C, no significant changes in MOLM-13 and MV4-11 cells may be independent of caspases-
were detected in these cell cycle regulators at the early time 3 and -7.

MAPK-Interacting Kinase Inhibition in AML
385
sensitivity between the cell lines could be attributed to the
different subtypes, which are classified based on the morphol-
ogy and immunohistochemistry of myeloid neoplasms (Bennett
et al., 1985). Both MOLM-13 and MV4-11 are acute mono-
blastic leukemia cells (M5/M5a) (Quentmeier et al., 2003) that
carry the respective t(9,11) and t(4,11) chromosomal trans-
locations, resulting in the fusion proteins MLL-AF9 and MLL-
AF4, respectively (Matsuo et al., 1997; Andersson et al., 2005).
Of note, these subtypes of AML are correlated with aberrant
upregulation of eIF4E (Topisirovic et al., 2003), which is absent in
the less mature promyelocytic cells (M3; PL-21 cells) (Quentmeier
et al., 2003). As a comparator, THP-1 cells with the t(9,11)
chromosomal translocation and no detectable FLT3-ITD expres-
sion exhibited negligible sensitivity to MNKI-19 and MNKI-85
(.12-fold). Similarly, HL-60, U937, and K562, which do not
express ITD mutations, have no cytotoxic effects when treated
with the compounds for up to 3 days. These results are in
agreement with a previous study, in which inhibiting 50% of cell
growth in K562, U937, and MM6 cell lines after 5-day treatment
also required .10 mM of cercosporamide (Altman et al., 2013). On
the other hand, CGP57380 is not particularly selective against
the ITD-positive cells, indicating an alternative mechanism
underlying the growth inhibitory effects. When considered
together, concurrently activated upstream signaling pathways
of Mnks with overexpressed eIF4E levels, as characterized by the
combined FLT3-ITD mutations and M5 subtypes, may serve as
the right platform for investigation of the antileukemic effects of
Mnk-specific inhibitors.
Western blot analysis further confirmed the role of MNKI-19
and MNKI-85 as potent Mnk inhibitors, as evidenced by the
blockage of eIF4ES209 phosphorylation. Because Mnks are
important convergence nodes of the MAPK pathways (Faivre
et al., 2006; Chappell et al., 2011), the expression of the
upstream effectors, p38 and ERK, was also investigated.
Interestingly, an early transient phosphorylation of ERK and
p38 was found to be upregulated upon incubation with MNKI-
19 for 1 hour, without affecting their total levels. Similar effects
were seen in CGP57380-treated cells but not in MNKI-85. This
could be attributed to the induction of a negative-feedback
mechanism upon Mnk1 inhibition since both ERK and p38 are
substrates for Mnk1 (Waskiewicz et al., 1997). Such an
observation is consistent with a study done by Zhang et al.
(2008), who reported that CGP57380-mediated ERK and Mnk
activation was determined at an early time point (i.e.,
30 minutes) and followed by a second late phosphorylation
starting at 24 hours in the Bcr-Abl–dependent cells. Little to no
effect on the levels of ERK and p38 proteins and their
phosphorylated forms was observed after treating MOLM-13
with 20 mM compounds for 24 hours, supporting the observation
that the sustained phosphorylation at a late time point is
dependent on the presence of Bcr-Abl and signifying the specific
effect of our Mnk inhibitors on Mnk/eIF4E. The compounds
were also evaluated against a panel of upstream activating
Fig. 2. Western blot analysis of key proteins involved in the Ras/Raf/
MAPK and PI3K/Akt/mTOR pathways in MOLM-13 cells treated with
CGP57380, MNKI-19, and MNKI-85 for (A) 1 hour and (B and C) 24 hours.
In (A) and (C) compounds are tested at 5 and/or 10 and 20 mM, whereas in
(B) compounds are used at 20 mM. Antibodies used and the protein
molecular weight marker are indicated on the left and right sides of each
panel, respectively. DMSO diluent is used as a control and b-actin is used
as an internal loading control. A representative blot is selected from at
least two independent repeats.
Discussion
Screening against a panel of seven leukemia cell lines with kinases, including B-Raf, ERK, p38a, PI3K, Akt1, and mTOR,
various FLT3 mutational statuses revealed that the selective using biochemical assays, and showed no significant kinase
Mnk inhibitors MNKI-19 and MNKI-85 are particularly inhibitory activity, supporting selectivity of the compounds.
effective in suppressing the growth of FLT3/ITD-expressing
Interestingly, downregulation of the levels of phosphory-
AML cells, including MOLM-13 and MV4-11. PL-21 cells, in lated 4E-BP1 at Thr37/46 and Thr70 (the two most priming
contrast, expressing both WT and mutated alleles (Quentmeier phosphorylation events for the upstream affector mTOR)
et al., 2003), were less sensitive to these compounds. However, (Gingras et al., 2001) were detected in MOLM-13 cells treated
it remains debatable that PL-21 cells could only express the WT with either MNKI-19 or MNKI-85. Binding of eIF4E to
allele (Yokota et al., 1997). Nevertheless, the differences in the 4E-BP1 is regulated by hierarchical multisite phosphorylation,

386
Teo et al.
Fig. 3. Cell cycle analysis of (A) MOLM-13 and (B) MV4-11 cells after treatment with 5, 10, or 20 mM of MNKI-19, MNKI-85, or CGP57380 for 24 and 48 hours. Data
represent the mean 6 S.D. of three independent repeats. *P , 0.05 when compared with the control. Western blot analysis of key proteins involved in the cell cycle
upon treatment with Mnk inhibitors in MOLM-13 cells for (C) 1 hour and (D and E) 24 hours. In (C) and (D) compounds are tested at 5 and/or 10 and 20 mM, whereas in
(E) compounds are used at 20 mM. Antibodies used and the protein molecular weight marker are indicated on the left and right sides of each panel, respectively. DMSO
diluent is used as a control and b-actin is used as an internal loading control. A representative blot is selected from at least two independent repeats.
and the role of the phosphorylation site at Thr37/46 on eIF4E the compound-treated cells. Translation processes involve the
binding remains controversial: several studies claimed that the assembly of several eIF4 translational factors, including the
phosphorylation of Thr37/46 has little effect on the binding of eIF4E and activated Mnks to form the eIF4F complex (Hou et al.,
eIF4E (Gingras et al., 1999; Karim et al., 2001), while the other 2012). Inhibition of Mnks, especially Mnk2, may increase the
group showed that a significant reduction in eIF4E-binding levels of eIF4E in the cells since the formation of eIF4F
affinity was induced by such a phosphorylation event (Burnett complex has been disrupted. Because the Akt/mTOR pathway
et al., 1998). However, mutations of these residues and/or Thr70 is overly activated in FLT3-ITD MOLM-13 cells (Hayakawa
to alanine have significantly led to the dissociation of eIF4E from et al., 2000), it is suspected that the upregulated eIF4E levels
4E-BP1 (Gingras et al., 2001), confirming the important role of have initiated the negative-feedback mechanism of Akt/mTOR
these phosphorylation sites on the eIF4E/4E-BP1 equilibrium.
to dephosphorylate 4E-BP1. Upon hypo-phosphorylation, 4E-
In light of these findings, it is speculated that a negative- BP1 interacts strongly with eIF4E (Khaleghpour et al., 1999).
feedback regulatory mechanism involving the equilibrium While our compounds were inactive to the upstream kinases in
shift toward the eIF4E/4E-BP1 complex could be triggered in the Akt/mTOR pathway, particularly mTOR, it is believed that

MAPK-Interacting Kinase Inhibition in AML
387
Fig. 4. Induction of apoptosis by MNKI-19, MNKI-85, or CGP57380 in (A) MOLM-13 and (B) MV4-11 cells. Cells are exposed at 5, 10, and 20 mM of each
compound for 24 and 48 hours, and examined with Annexin V/PI staining. The percentage of cells undergoing apoptosis is represented by the sum of the
early and late apoptosis. Data represent the mean 6 S.D. of three independent repeats. *P , 0.05 when compared with the control. Western blot analysis
of key proteins involved in apoptosis upon treatment with Mnk inhibitors in MOLM-13 cells for (C) 1 hour and (D and E) 24 hours. In (C) and (D)
compounds are tested at 5 and/or 10 and 20 mM, whereas in (E) compounds are used at 20 mM. Antibodies used and the protein molecular weight marker
are indicated on the left and right sides of each panel, respectively. DMSO diluent is used as a control and b-actin is used as an internal loading control.
A representative blot is selected from at least two independent repeats. (F) Quantification of caspase-3/7 activity in MOLM-13 (left) and MV4-11 (right)
cells upon treatment with 5, 10, and 20 mM of MNKI-19, MNKI-85, or CGP57380 for 48 hours.
the dephosphorylation events were induced upon Mnk complex was decreased when treated with the compound
inhibition.
(Li et al., 2010). This implies that inhibiting Mnks may reduce
Complementary to this, a recent study used CGP57380 to the eIF4F assembly. Another recent study also suggested that
investigate the effect of eIF4E phosphorylation on translation the dissociation of 4E-BP1 is highly dependent on the Mnk1-
initiation, showing that the amount of eIF4G in the eIF4F mediated eIF4E phosphorylation (Das et al., 2013). The

388
Teo et al.
dephosphorylation of 4E-BP1 by MNKI-19 could be also due to of Mcl-1 levels were observed. Lack of procaspase-3 processing
the Pim-1 inhibition as shown in the kinase assay. 4E-BP1 and caspase-3/7 activation suggest that the apoptotic cell
has recently been identified as a target of Pim-1 in the FLT3- death by Mnk inhibitors may be via a caspase-independent
ITD–driven activation of STAT-5. Pim-1 inhibitors have mechanism. In contrast, a recent study demonstrated a
significantly reduced p-4E-BP1T37/46 in FLT3-ITD AML cells caspase-dependent apoptosis was induced with Mnk degrad-
(Chen et al., 2011). However, more studies on the binding ing agents in breast cancer cell lines (Ramalingam et al.,
affinity of compounds MNKI-19 and MNKI-85 for the relevant 2014). Perhaps modes of action are contingent on compounds
proteins, such as eIF4G and 4E-BP1, are required to unveil treated and cell lines tested. However, it is noteworthy that
the underlying mechanism.
a combined use of Mnk1 and Mnk2, as represented by MNKI-
The oncogenic role of eIF4E has been reported in the cell 19, gives rise to a more pronounced apoptosis than inhibiting
cycle progression (Diab et al., 2014a). D-type cyclins, as G1 Mnk2 alone (MNKI-85).
progression factors, play a vital role in enhancing the G1 to
Acknowledgments
S transition through binding to CDK4/6 (Sherr, 1995). Elevated
eIF4E levels have been shown to enhance the translational
levels of a wide array of malignancy-related mRNAs involved in
the regulation of cell senescence, proliferation, and apoptosis,
including cyclin D1 and Mcl-1 (Zimmer et al., 2000), whereas
the oncogenic functions of eIF4E are closely linked to its
phosphorylation by Mnks (Ueda et al., 2010; Diab et al., 2014a).
Consistent with previous studies (Zhang et al., 2008; Diab
et al., 2014b; Teo et al., 2015), Mnk inhibitors in the current
study triggered a cell cycle arrest in both MOLM-13 and MV4-11
cells by disrupting the transition of the G1 phase to the S phase.
Such an effect is likely to be the result of decreased expression of
the key cell cycle regulators, including CDK4 and cyclin D3,
which further hampered the phosphorylation of Rb at the
CDK4-specific Thr826 site (Macdonald and Dick, 2012). This
was further supported when kinase assays showed both MNKI-
19 and MNKI-85 have no effect on CDK4 kinase activity (Ki . 10
mM). Previous studies also demonstrated the importance of
cyclin D3 at the translational level over other D-type cyclins
(Prabhu et al., 2007; Zhang et al., 2008).
Collectively, these results indicate that the effect of eIF4E-
mediated cell cycle arrest is through phosphorylation by Mnks.
While MNKI-19 increased the G1 cell accumulation in a time-
dependent manner, MNKI-85 failed to show a similar cell cycle
effect, suggesting that targeting a combination of Mnk1 and
Mnk2 may be more effective than one of the isoforms alone in
preventing cell cycle progression. Our observation agrees with
a recent study on a series of Mnk degrading agents, which
showed they induce G1/G0 cell cycle arrest, along with the
downregulation of cyclin D3 and CDK4 in MDA-MB-231 cells
(Ramalingam et al., 2014).
The apoptotic effect of Mnk inhibitors is likely mediated
through the 4E-BP1/eIF4E axis. Several lines of evidence have
indicated the positive correlation between the suppression of
eIF4E by binding to 4E-BP1 and induction of apoptosis
(Clemens, 2001; Yellen et al., 2013). To date, Mnk1 and Mnk2
are the only identified kinases that phosphorylate eIF4E, and
phosphorylation of eIF4E modulates cap-dependent trans-
lation. In addition, eIF4E phosphorylation can be downregu-
lated by 4E-BP1 binding (Li et al., 2010); therefore, this has
raised the possibility that inhibiting Mnks triggers the de-
phosphorylation of 4E-BP1 and results in the reduction of cap-
dependent protein synthesis, including Mcl-1. In contrast, the
significant induction of G1 arrest and apoptosis by MNKI-19
could also be an additive effect of Pim-1 inhibition. A previous
study has shown that Pim-1 inhibitors reduced Mcl-1 and
induced apoptosis in FLT3-ITD AML cells (Chen et al., 2011).
We have shown that inhibition of eIF4E phosphorylation
with Mnk inhibitors is associated with an increase in cell
death. An increase cleavage of PARP and a marked suppression
The authors thank Professor Richard D’Andrea (University of
South Australia) for the generous supply of the AML cell lines.
Authorship Contributions
Participated in research design: Wang, Teo.
Conducted experiments: Teo, Basnet.
Contributed new reagents or analytic tools: Yang, Yu.
Performed data analysis: Teo, Lam, Basnet, Albrecht, Wang.
Wrote or contributed to the writing of the manuscript: Teo, Wang,
Yu, Sykes.
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Address correspondence to: Shudong Wang, Centre for Drug Discovery and
Development, School of Pharmacy and Medical Sciences, University of South
Australia, Adelaide, South Australia 5001, Australia. E-mail: shudong.wang@
unisa.edu.au
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