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Regular Article
MYELOID NEOPLASIA
Inhibiting glutamine uptake represents an attractive new strategy for
treating acute myeloid leukemia
Lise Willems,1-3 Nathalie Jacque,2 Arnaud Jacquel,4 Nathalie Neveux,5 Thiago Trovati Maciel,6 Mireille Lambert,2
Alain Schmitt,7 Laury Poulain,2 Alexa S. Green,2 Madalina Uzunov,8 Olivier Kosmider,2,3,9 Isabelle Radford-Weiss,10
Ivan Cruz Moura,6 Patrick Auberger,4 Norbert Ifrah,11 Valérie Bardet,2,3,9 Nicolas Chapuis,2,3,9 Catherine Lacombe,2,3,9
Patrick Mayeux,2,3 Jérôme Tamburini,1-3 and Didier Bouscary1-3
1
Unité Fonctionnelle d’Hématologie, Hôpital Cochin, Assistance Publique Hôpitaux de Paris, Paris, France; 2Institut Cochin, Département d’ImmunoHématologie, Le Centre national de la recherche scientifique UMR8104, Institut National de la Santé et de la Recherche Médicale U1016, Paris, France;
3
Université Paris Descartes, Faculté de Médecine Sorbonne Paris Cité, 4Institut National de la Santé et de la Recherche Médicale U1065, Université NiceSophia Antipolis, Centre Méditerranéen de Médecine Moléculaire, Team 2 “Cell death, differentiation, inflammation and cancer,” Nice, France; 5Service de
Biochimie, Hôpital Cochin, Assistance Publique Hôpitaux de Paris, Paris, France; 6Institut National de la Santé et de la Recherche Médicale U699, Faculté
de Médecine Xavier Bichat, Paris, France; 7Plate-forme de microscopie électronique, Institut Cochin, Paris, France; 8Service d’Hématologie Clinique,
Hôpital Pitié-Salpêtrière, Assistance Publique Hôpitaux de Paris, Paris, France; 9Service d’Hématologie Biologique, Hôpital Cochin, Assistance Publique
Hôpitaux de Paris, Paris, France; 10Service de Cytogénétique, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, Paris, France;
and 11Service des Maladies du Sang, Centre Hospitalier Universitaire d’Angers, Angers, France
Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and
protein synthesis inhibition downstream of mammalian target of rapamycin complex 1
(mTORC1) has shown promise as a possible therapy for acute myeloid leukemia
• Glutamine removal and
(AML). Glutamine contributes to leucine import into cells, which controls the amino
knockdown of the glutamine
acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine
transporter SLC1A5 have
removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the
antileukemic activity in AML.
SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor
• The glutaminase activity
formation in a mouse AML xenotransplantation model. L-asparaginase (L-ase) is an antiof L-asparaginase inhibits
cancer agent also harboring glutaminase activity. We show that L-ases from both Escherichia
mTORC1 and protein
coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that
synthesis and induces a
this inhibition correlates with their glutaminase activity levels and produces a strong apostrong autophagy in AML.
ptotic response in primary AML cells. We further show that L-ases upregulate glutamine
synthase (GS) expression in leukemic cells and that a GS knockdown enhances
L-ase–induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon L-ase treatment. These results suggest
that L-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML. (Blood. 2013;122(20):3521-3532)
Key Points
Introduction
The serine threonine kinase mammalian target of rapamycin (mTOR)
forms 2 functionally and structurally distinct complexes, mTORC1
and mTORC2. MTORC1, comprising mTOR, Raptor, and MLST8,
positively regulates protein translation through the phosphorylation of
its substrates, protein S6 Kinase (P70S6K) and eukaryotic initiation
factor 4E-binding protein 1 (4E-BP1). Cap-dependent translation is
controlled by the translational repressor 4E-BP1. 4E-BP1 phosphorylation at its serine 65 residue is absolutely required to initiate the
formation of the translation initiation complex eukaryotic initiation
factor 4F.1 In most primary acute myeloid leukemia (AML) samples,
the mTORC1 complex is activated by an as-yet–unknown mechanism.2,3 Recently, it was shown in a raptor deficiency mouse model
that mTORC1 inactivation induces apoptosis in differentiated leukemic
cells and maintains immature leukemic cells with leukemia initiation
potential in a dormant state, underlying the critical role of mTORC1 in
leukemia.4 In vitro, in primary AML cells, mTORC1 inhibition with
rapamycin has cytostatic effects but does not induce apoptosis,2-5
mainly because it does not inhibit 4E-BP1 phosphorylation on
ser65.6 In contrast, mTOR kinase inhibitors fully inhibit 4E-BP1
phosphorylation, suppress protein synthesis, and induce apoptosis.7,8
Thus, full inhibition of mTORC1 activity represents a promising
therapy in AML.
Many upstream signals regulate mTORC1 activation, including
growth factors, the energy status of the cell via the LKB1/AMPK
pathway, hypoxemia, and low glucose content, all converging at
the TSC1/TSC2 axis that negatively affects the activity of the
Ras homolog enriched in brain (Rheb) protein toward mTORC1.9
However, a major process controlling mTORC1 activity is the
Submitted March 27, 2013; accepted August 19, 2013. Prepublished online as
Blood First Edition paper, September 6, 2013; DOI 10.1182/blood-2013-03493163.
There is an Inside Blood commentary on this article in this issue.
L.W. and N.J. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2013 by The American Society of Hematology
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
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WILLEMS et al
availability of amino acids (AAs), mainly leucine.10,11 Indeed, intracellular leucine is required to activate the Ras-related GTPases
proteins, which enable the localization of the mTORC1 complex at
the lysosome surface close to its activator, Rheb.12,13 Leucine is
imported into the cell in exchange for glutamine by the bidirectional
transporter SLC7A5/3A2. Thus, the level of leucine depends on the
intracellular glutamine concentrations. Glutamine uptake is essentially mediated by the high-affinity SLC1A5 transporter, which cooperates with SLC7A5/3A2.14 The cellular uptake and subsequent
rapid efflux of glutamine in the presence of leucine make glutamine
availability a limiting step for mTORC1 activation.15
We herein tested the effects of glutamine depletion in AML cells.
We show from our experiments that leukemic cells are sensitive to
glutamine removal, leading to mTORC1 inhibition and apoptosis.
Knockdown of the SLC1A5 transporter induces apoptosis and inhibits tumor formation in an AML mouse xenotransplantation model.
Moreover, we find that L-ase inhibits mTORC1 activity, suppresses
protein synthesis, and induces apoptosis in AML through its
glutaminase activity. However, L-ase also upregulates glutamine
synthase (GS) protein expression, which may limit the L-ase–induced
antileukemic effects in AML in some cases. Finally, we demonstrate
that L-ase strongly triggers an autophagic process. Taken together, these
results indicate that AML cells absolutely require glutamine and that
targeting glutamine uptake represents a promising therapeutic approach.
Material and methods
Patients
Bone marrow or peripheral blood samples with .70% blast cell content were
obtained from 27 patients with newly diagnosed AML. Cells were isolated
using Ficoll-Hypaque density-gradient separation. Cells were collected after
obtaining patients’ written informed consent in accordance with the Declaration of Helsinki and approval obtained by the Cochin Hospital Institutional
ethic committee. Patient characteristics are listed in supplemental Table 1. The
CD341 cells from healthy donors were purified using MIDIMACS immunoaffinity columns (Milteny Biotech, Bergish Badgash, Germany).
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
Santa Cruz Biotechnology; anti-actin and anti-Flag antibodies from Sigma; and
anti-p85 antibodies were raised in house.
Flow cytometry
Apoptosis was quantified by Annexin V-PE (Becton Dickinson, Le Pont De
Claix, France) and 7-aminoactinomycin D (Sigma) staining.
Measurement of extracellular and intracellular AA levels
For the quantification of extracellular AAs, culture supernatants were deproteinized with a 30% (w/v) sulfosalicylic acid solution, incubated for 5 minutes
at room temperature, and centrifuged (10 000 rpm, 10 minutes, 4°C). Supernatants were then stored at 280°C until analysis. AA levels were measured by
ion exchange chromatography using an AA auto analyzer (model Aminotac;
Jeol, Tokyo, Japan). For quantification of AA in cell extracts, 5 3 106 cells
were sonicated and centrifuged (10 000 rpm, 10 minutes). Homogenized samples
were then deproteinized and again centrifuged. The resulting supernatant was
subsequently used for AA measurements by ion exchange chromatography.
Lentiviral constuctions expressing sh/siRNAS
SLC1A5 (TCRN0000043120 [short hairpin RNA (shRNA)1] and
TCRN0000043122 [shRNA2]), GS (TCRN0000045628 [shRNA1] and
TCRN0000045630 [shRNA2]), and nontargeted (control) shRNAs were
from Open Biosystems and cloned into a pLKO-Tet-On inducible or pLKO.1
plasmids (plasmid numbers 21915 and 8453 from Addgene, Cambridge, MA).16
Lentiviral particles were produced using HEK-293T packaging cells. Supernatants were collected 48 hours after transfection and then stored at 280°C.
Leukemic cell lines were plated at 2.5 3 106 cells/mL in serum-free medium
to which 10 mL of lentiviral supernatants were added. The transfection of antiAtg5 and anti-beclin small interfering RNAs (siRNAs) was performed in
the OCI-AML3 cell line using the Amaxa nucleofector kit (Lonza, Basel,
Switzerland). The pLJM1 Flag Raptor Rheb15 (Addgene plasmid 26634) or control vector were transfected in 293T cells using 10 mL of lentiviral supernatant.
[35S] Methionine incorporation assay
Cells were pretreated without or with L-ase (E coli) at 10 UI/mL in Dulbecco’s
modified Eagle medium without methionine (Eurobio, Courtaboeuf, France)
for 6 hours and pulse-labeled with 160 mCi of [35S] methionine for an additional 2 hours. The amount of radioactivity incorporated into the proteins was
determined by trichloroacetic acid precipitation.
Cell culture
Human leukemic cell lines were purchased from DSMZ (Braunschweig,
Germany) for MV4-11 and from the American Type Culture Collection
(Molsheim, France) for HL-60, MOLM-14, and OCI-AML3. Cells were
cultured in minimum essential medium (MEM) a medium containing 10%
fetal bovine serum (FBS) and 4 mM glutamine. To induce AA starvation,
cells were cultured in MEM a medium without glutamine, asparagine, and
leucine supplemented with 10% dialyzed FBS in the presence or absence of
4 mM glutamine, 0.4 mM leucine, or 0.4 mM L-ase (Sigma, St. Louis, MO).
HEK-293T cells were cultured in Dulbecco’s modified Eagle medium with 10%
FBS without glutamine and leucine and then supplemented or not with AAs.
AML xenografts in nude mice
MOLM-14 infected with either a lentiviral vector containing the doxycyclineinducible SLC1A5 shRNA or a nonspecific shRNA were subcutaneously
injected into nude mice as previously reported.17 All mice were treated with
doxycycline given ad libitum 5 days per week (n 5 12). Tumor growth was
measured 3 times per week. All experiments were conducted in accordance
with the guidelines of the Association for Assessment and Accreditation of
Laboratory Animal Care International.
Electron microscopy
Reagents
We used from 0.1 to 10 UI/mL of Escherichia coli L-asparaginase (L-ase;
Kidrolase) or Erwinia chrysanthemi L-ase (Erwinase), both purchased from
EUSA Pharma Inc (Lyon, France).
Western blotting
Whole-cell extracts and western blots were performed as previously described.6 Antibodies against phospho-p70S6K T389, 4EBP1, phospho-4EBP1
S65, phospho-AKT S473, AKT, phospho-NDRG1 T346, poly ADP-ribose
polymerase (PARP), caspase 3, SLC1A5, c-Myc, Mcl-1, LC3b, ATG5, and
beclin were purchased from Cell Signaling Technology; anti-GS antibodies
from BD Bioscience (San Jose, CA); anti-p70S6K and anti-SQSTM1 from
Cells were fixed for 1 hour in 3% glutaraldehyde in 0.1 M sodium phosphate
buffer, pH 7.4, post-fixed for 1.5 hours with 1% osmium tetraoxide, dehydrated
by successive ethanol washes (70%, 90%, 100%, 100%), and impregnated with
epoxy resin. After polymerization, 80- to 90-nm sections were prepared using
a Reichert Ultracut S ultramicrotome, stained with 2% uranyl acetate plus
Reynold’s lead citrate, and visualized under a JEOL 1011 transmission electron
microscope with a GATAN Erlangshen CCD camera.
Statistical analysis
Data from this study are expressed as mean values with standard deviations.
Statistical significance in the differences between experimental groups was
determined using the Student t test. *, ** and *** indicate P , .05, P , .01,
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BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
and P , .001, respectively. The experiments performed in AML cell lines
were performed in independent triplicates.
Results
Glutamine removal inhibits mTORC1 and induces apoptosis in
AML cells
We tested the effects of either leucine or glutamine deprivation on
mTORC1 activity in AML cells. Leucine- or glutamine-starved cells
exhibited, in a time-dependent manner, a decrease of p70S6K T389
and 4E-BP1 S65 phosphorylation in all AML cell lines tested as well
as in a representative AML sample (Figure 1A; supplemental
Figure 1A). However, glutamine or leucine starvation did not inhibit
the phosphorylation of AKT on S473 (supplemental Figure 1B).
Glutamine removal for 24 hours induced significant apoptosis in all
cell lines, with the most dramatic effects seen in MV4-11 and OCIAML3, as assessed by Annexin V binding and caspase-3 and PARP
cleavage assays (Figure 1B-C). We then tested the hypothesis that
AML cells have a higher level of glutamine dependence than normal
CD341 cells. As shown in Figure 1D, normal CD341 cells from 3
donors were resistant to apoptosis induction following glutamine removal. In contrast, an analysis of 8 primary AML samples indicated
that some samples are glutamine dependent, whereas others are not.
These results indicate that glutamine deprivation induces mTORC1
inhibition and apoptosis in AML cells.
Targeting the SLC1A5 glutamine transporter has
antileukemic effects
We tested whether inhibiting glutamine uptake via a knockdown
of the glutamine transporter SLC1A5 exerts antileukemic activity. The SLC1A5 protein is expressed in all leukemic cell lines
tested (Figure 2A). We engineered 2 different doxycycline-inducible
shRNAs that were transduced in AML cells using a lentivirus
construct and inhibited SLC1A5 expression with similar efficiency (results are reported for shRNA1). SLC1A5 knockdown in
MOLM-14 cells was maximal from day 2 (Figure 2B). We investigated the role of this transporter in the control of mTORC1 activation.
SLC1A5 shRNA-infected MOLM-14 cells cultured with or without
doxycycline were starved of leucine and glutamine for 6 hours to
inhibit mTORC1 and then stimulated for 15 and 30 minutes with
these 2 AAs. MTORC1 activation was strongly reinduced in the
doxycycline-untreated cells but not when SLC1A5 was knocked
down (Figure 2C).
SLC1A5 knockdown induced significant apoptosis, attested
to by caspase-3 and PARP cleavage assays and by Annexin V
fixation (Figure 2D). In contrast, no apoptosis was detected in the
HL-60 cell line when SLC1A5 expression was fully inhibited
(data not shown). This result was further supported by our finding
that in SLC1A5 knockdown HL-60 cells, mTORC1 was reactivated by AA stimulation, suggesting that other glutamine transporters control glutamine-mediated mTORC1 activation in this
cell line (supplemental Figure 2). The antileukemic effects of
SLC1A5 inhibition were tested in vivo. Two groups of nude mice
were subcutaneously injected with MOLM-14 cells transduced with
SLC1A5 or control shRNAs and then treated with doxycycline.
SLC1A5 ablation prevented tumor development (Figure 2E) and
statistically improved overall survival of the mice (P 5 .0011)
(Figure 2F). Western blotting analysis of tumor samples confirmed
this inducible SLC1A5 inhibition in mice injected with SLC1A5
shRNA-MOLM-14 cells (Figure 2G). These results reveal that
TARGETING GLUTAMINE UPTAKE IN AML
3523
targeting the SLC1A5 glutamine transporter has antileukemic
activity both in vitro and in vivo.
The glutaminase activity of L-ase inhibits mTORC1 and protein
translation in AML cells
The L-ases have glutaminase activity and transform extracellular
glutamine into glutamate. In OCI-AML3 cells, L-ase drastically
reduces the intracellular glutamine levels (Figure 3A). Similar results
were observed in MOLM-14 and MV4-11 cell lines (not shown). We
tested L-ase effects on mTORC1 activity in AML cells. We compared
the effects of L-ase from E coli with those of the L-ase from
E chrysanthemi, whose glutaminase activity is fourfold higher despite
the similar levels of asparaginase activity. In OCI-AML3 cells, both
L-ases induced the dose- and time-dependent inhibition of p70S6K
T389 and 4E-BP1 S65 phosphorylations (Figure 3B). Similar results
were obtained in 3 other AML cell lines (supplemental Figure 3A-B).
E chrysanthemi L-ase was found to induce a maximal inhibition
of mTORC1 at lower concentrations than E coli L-ase (Figure 3B).
Moreover, mTORC1 inhibition by L-ase correlated with extracellular glutamine depletion (Figure 3C). These results suggest that
L-ase–induced mTORC1 inhibition is due to extracellular glutamine
degradation. Indeed, asparagine starvation did not inhibit mTORC1
in all AML cell lines tested in contrast to the findings for leucine or
glutamine privation (supplemental Figure 3C). Moreover, the E coli
L-ase dramatically inhibited mTORC1 activity in several primary AML
samples (Figures 3D). Glutamine starvation and L-ase overactivated
mTORC2 signaling, attested by the increased phosphorylations of
AKT on S473 and NDRG1 on T346 (supplemental Figure 3D).
L-ase may inhibit leucine entry into the cells by depleting intracellular glutamine, thereby preventing mTORC1 activation by Rheb
at the lysosomal surface.12,13 To further investigate this mechanism, we
used a chimeric Raptor-Rheb15 protein in which the mTORC1 partner protein Raptor was fused to the 15-AA Rheb sequence. This
chimeric protein produces the constitutive localization of mTORC1
at the lysosomal surface, leading to AA-independent mTORC1
activation.18 In HEK-293T cells expressing the Raptor-Rheb15 protein,
glutamine removal only partially inhibited p70S6K T389 phosphorylation. Interestingly, mTORC1 activation was maintained in RaptorRheb15–expressing cells treated with L-ase, which strongly suggests
that L-ase inhibits mTORC1 in an AA-dependent manner (Figure 3E).
Finally, as L-ase inhibits 4E-BP1 phosphorylation at residue S65,
which is absolutely required to maintain active cap-mRNA translation, we evaluated the effects of L-ase on protein synthesis. In all
leukemic cell lines tested, L-ase significantly inhibited [35S] methionine
incorporation into newly synthesized proteins, indicative of global
protein synthesis inhibition (Figure 3F). Moreover, in AML cell
lines and a representative primary AML sample, L-ase inhibited
c-Myc and Mcl-1 expression, whose translation is cap dependent,
in a dose-dependent manner (Figure 3G).
Overall, these results indicate that the glutaminase activity of
L-ase leads to mTORC1 inhibition in AML cells, resulting in protein
synthesis inhibition, and that this effect is due, at least in part, to an
inhibition of the AA/mTORC1-sensing machinery.
L-ase
induces apoptosis in AML cells through its
glutaminase activity
The functional effects of L-ase were tested in leukemic cell lines
and primary AML cells. In OCI-AML3 cells, E coli L-ase induced
both time- and dose-dependent apoptosis (Figure 4A), with higher
potency seen for E chrysanthemi L-ase than E coli L-ase (Figure 4B).
This difference is probably due to the higher rate of glutamine
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WILLEMS et al
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
Figure 1. Glutamine removal inhibits mTORC1 activity and induces apoptosis in AML cells. (A) The MOLM-14 and OCI-AML3 cell lines, and primary AML cells from
a representative patient, were cultured for 6 or 24 hours with or without 4 mM glutamine (Gln) and/or 0.4 mM leucine (Leu). Cells were lysed in Laemmli buffer and western
blotting was performed to analyze the p70S6K T389 and 4E-BP1 S65 phosphorylation status. (B) MOLM-14, OCI-AML3, MV4-11, and HL-60 cell lines were cultured with or
without Gln for 24 hours and apoptosis was quantified by flow cytometry analysis of Annexin-V binding. (C) The indicated cell lines were cultured for 24 hours with or without
glutamine and then assessed for caspase-3 and PARP cleavage by western blotting. (D) Normal CD341 cell were obtained from 3 healthy allogeneic transplant donors after
positive sorting and primary AML cells from 8 AML patients were also tested. Cells were cultured 48 hours with or without glutamine (4 mM), and the apoptotic responses were
quantified by flow cytometry analysis of Annexin-V binding.
depletion obtained with E chrysanthemi L-ase (Figure 3C). Similar
results were observed in the MOLM-14, MV4-11, and HL-60 cell
lines (supplemental Figure 4A). Significant apoptosis was also
induced by E coli L-ase in almost all primary AML samples
examined (Figure 4C). E chrysanthemi L-ase–induced apoptosis
was statistically superior to E coli L-ase at the 0.1 UI/mL dose in
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BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
TARGETING GLUTAMINE UPTAKE IN AML
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Figure 2. Targeting the SLC1A5 glutamine transporter has
proapoptotic effects and in vivo antileukemic activity. (A)
SLC1A5 expression was studied by western blotting in the 4
indicated AML cell lines. (B-F) MOLM-14 cells were infected
with a lentiviral vector expressing either an inducible SLC1A5
or a control (CTR) shRNA. Stably infected cell lines were
established after selection in puromycin. (B) MOLM-14 cells
infected with SLC1A5 or CTR shRNA were cultured with or
without doxycycline for the indicated times. The inhibition of
SLC1A5 expression with doxycycline was assessed by western
blotting. (C) MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR
shRNA cells were starved or not for 6 hours of glutamine and
leucine to inhibit mTORC1, as detected by the inhibition of
p70S6K T389 phosphorylation. Previously starved cells were
then stimulated for 15 or 30 minutes with leucine 1 glutamine in
the presence or absence of doxycycline. The reactivation of
mTORC1 was then determined. (D) Apoptosis was evaluated in
MOLM-14/SLC1A5 shRNA cells and MOLM-14/CTR shRNA
cells in the presence of doxycycline by flow cytometry analysis
of Annexin-V fixation or by western blotting analysis of PARP
and caspase-3 cleaved fragments. Analysis was performed
every day from day 2 to day 6. (E) MOLM-14/SLC1A5shRNA
cells (red) or MOLM-14/CTR shRNA cells (black) were injected
into nude mice. The mice were then treated with doxycycline by
oral gavage, and the tumor volumes were determined at the
indicated times. The means of the individual tumor sizes were
then plotted (n 5 8 in each group). (F) Kaplan-Meier survival
curves of nude mice treated with doxycycline. (G) SLC1A5
expression was detected by western blotting analysis of tumor
extracts from MOLM-14/SLC1A5 shRNA or MOLM-14/CTR
shRNA mice treated with doxycycline (day 22).
these samples (Figure 4D). In the AML cell lines and patient samples
analyzed, L-ase induced caspase-3 cleavage (Figure 4E).
To determine the sensitivity of AML cells to asparagine starvation, we starved leukemic cell lines from this AA. Asparagine
removal induced significant apoptosis only in MV4-11 cells, whereas
other leukemic cell lines were resistant to this condition (supplemental
Figure 4B). Overall, these results indicate that L-ase has significant
antileukemic activity in AML and that this is mainly related to its
glutaminase activity.
L-ase
increases GS expression in AML
The GS enzyme produces glutamine from glutamate. We studied the
basal expression of this enzyme and after 24 hours of E coli L-ase
exposure. Basal levels of GS expression were rather low in OCI-AML3
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WILLEMS et al
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Figure 3. The glutaminase activity of L-ase inhibits mTORC1 and protein translation in AML cells. (A) OCI-AML3 cells were cultured for 24 hours with the indicated
doses of E coli L-ase, and the levels of glutamine and asparagine in the intracellular compartment were quantified by ion exchange chromatography. (B) The OCI-AML3 cell
line was cultured for 6 or 24 hours with or without E coli or E chrysanthemi L-ases at an equal level of asparaginase activity (0.1-10 UI/mL) and then lysed in Laemmli buffer.
Western blotting was performed with anti-phospho-p70S6K T389, anti-phospho-4E-BP1 S65, anti-p70S6K, anti-4E-BP1, and anti-actin antibodies. (C) Cells were treated as
indicated in B, and the level of Gln in the extracellular medium was quantified by ion exchange chromatography. (D) Primary bone marrow leukemic cells from AML patients
were treated as in A, and the same analysis was assessed by western blotting. The intensities of phospho-p70S6K T389, phospho-4E-BP1 S65, and actin signals were
quantified in different AML samples. Ratios of phospho-p70S6K T389 or phospho-4E-BP1 S65 to actin were calculated and assigned a reference value of 1 in the control
culture. (E) HEK-293T cells were transfected with a Flag-Raptor-Rheb15 expression vector or a control vector and then cultured for 24 hours with or without glutamine at 4 mM
or E coli L-ase (10 UI/mL). Western blotting was then performed with anti-flag, anti-actin, or anti-phospho-p70S6K T389 antibodies. (F) [S35] methionine pulses were
performed in MOLM-14, OCI-AML3, MV4-11, and HL-60 cells to evaluate the global protein synthesis profile with or without E coli L-ase at 10 UI/mL during a 6-hour period. (G)
OCI-AML3, MOLM-14, MV4-11, and HL-60 cell lines and a representative AML sample were cultured for 24 hours with or without E coli L-ase. The expression of Mcl-1 and
c-Myc was then analyzed by western blotting.
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BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
TARGETING GLUTAMINE UPTAKE IN AML
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Figure 4. L-ase induces apoptosis in leukemic cells. OCI-AML3 cells and primary AML samples were cultured for 24 or 48 hours in 10% FBS MEM with or without 0.1, 1, or
10 UI/mL of E coli or E chrysanthemi L-ase. Apoptosis was determined by Annexin V 1/2 7AAD binding using flow cytometry (A-D) or by western blotting using an anti-caspase
3 antibody (E).
and HL-60 cells but more prominent in MOLM-14 and MV4-11
cells, and L-ase increased GS expression in all of these leukemic cell
lines (Figure 5A upper panel). In primary AML samples, L-ase also
increased GS expression in a dose-dependent manner (Figure 5A
lower panel). All cell lines were stably infected with a lentiviral
vector expressing a GS shRNA (shRNA1 shown; similar results with
shRNA2) that produced a persistent inhibition of GS expression
even when cells were treated with E coli L-ase or cultured without
glutamine (Figure 5B; supplemental Figure 5A). GS inhibition had
no effect on glutamine starvation or L-ase–induced apoptosis in
OCI-AML3, MOLM-14, or HL-60 cells but significantly increased
the level of apoptosis in MV4-11 cells (Figure 5C; supplemental
Figure 5B). These results indicate that in some leukemic cells, GS
expression may represent a mechanism of resistance to the antileukemic activity of L-ase.
L-ase
induces a strong autophagy in AML cells
As mTORC1 controls the activity of key proteins of the autophagic
process, such as Atg1/ULK and ATG13,19 we addressed the question
of autophagy induction by L-ase or glutamine removal in AML cells.
In OCI-AML3 cells, L-ase significantly induced, in a dose- and timedependent manner, an accumulation of both the LC3-II form and the
p62/SQSTM1 protein, reflecting autophagy induction (Figure 6A).
Similar results were observed in the MOLM-14 cell line (data not
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WILLEMS et al
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Figure 5. L-ase increases GS expression in AML, which may represent a mechanism of resistance to L-ase. (A) MOLM-14, OCI-AML3, and primary AML blast cells
from 3 patients were cultured over a 24-hour period with or without 0.1, 1, or 10 UI/mL E coli L-ase. GS expression was determined by western blotting. (B) OCI-AML3 and
MV4-11 cells were stably infected with a lentiviral vector expressing either a CTR or a GS shRNA and then cultured with or without 10 UI/mL E coli L-ase and with or without
glutamine at 4 mM for 24 hours. Western blotting was then performed to assess the GS expression level. (C) The same cell lines were tested by flow cytometry analysis of
Annexin V fixation for apoptosis induction after 24 hours in the presence or absence of E coli L-ase at 10 UI/mL and with or without glutamine starvation.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
TARGETING GLUTAMINE UPTAKE IN AML
3529
Figure 6. L-ase induces strong protective autophagy in AML cells. (A-B) OCI-AML3 cells were cultured for 6 or 24 hours in 10% FCS MEM with or without 0.1, 1, and 10%
FCS MEM with or without 0.1, 1, or 10 UI/mL of E coli L-ase and with or without Gln as indicated. Autophagy was detected by western blotting using anti-LC3b and
p62/SQSTM1 antibodies. (C) Representative electron micrographs of OCI-AML3 cells collected after 24 hours of culture in MEM with or without E coli L-ase (1 UI/mL) and
without glutamine. Arrows indicate autophagic vesicles. Upper and lower panels show 2 different scales. (D) OCI-AML3 cells were transfected with anti-ATG5 or anti-beclin
siRNAs using the Amaxa nucleofector kit. Two days after transfection, cells were cultured for 24 hours with or without E coli L-ase at 10 UI/mL and western blotting was
performed to verify ATG5 and Beclin knockdowns, respectively. (E) Viability was measured in OCI-AML3 cells (transfected or not with anti-ATG5 or anti-Beclin siRNA and
treated or not with E coli L-ase) by flow cytometry analysis of Annexin V fixation. The histograms shown represent the mean values of 3 independent experiments.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3530
WILLEMS et al
shown). Moreover, glutamine starvation increased LC3-II and
p62/SQSTM1 expression (Figure 6B). We then examined the morphology of OCI-AML3 cells by transmission electron microscopy and
observed the appearance of numerous electron-dense inclusions in
the cytosol after L-ase exposure, indicative of autophagy (Figure 6C).
To determine the impact of L-ase–induced autophagy on AML cell
death, we blocked autophagy in OCI-AML3 cells using siRNAs
to knock down ATG5 or Beclin; the inhibition of ATG5 or Beclin
expression decreased L-ase–induced autophagy, attested by lowered
LC3-II band accumulation (Figure 6D). Apoptosis was then
quantified using Annexin V fixation in OCI-AML3 cells expressing
these same siRNAs, and we observed that an autophagy blockade
significantly increased L-ase–induced apoptosis in OCI-AML3 cells
that had undergone an ATG5 or beclin knockdown (Figure 6E).
We conclude from these results that L-ase induces strong autophagy
that could partially protect AML cells from apoptosis.
Discussion
The targeting of metabolic processes has emerged as a very promising
approach to cancer treatment, which is due to the increased metabolic
inputs, including glucose and glutamine, that are required for optimal
proliferation and survival of cancer cells.20-22 Glutamine is a core
substrate for protein, glucosamine, and nucleotide synthesis and is
metabolized by glutaminases into glutamate, which then enters the
TCA cycle to produce ATP after conversion to a-ketoglutarate.23 In
addition to its role in cellular metabolism, glutamine appears to be
essential for mTORC1 activation by leucine. Hence, we analyzed in
this study the dependence of AML cells on glutamine for survival as
well as different ways to target glutamine uptake by leukemic cells.
We show from our experiments that leucine or glutamine removal
inhibits mTORC1 activation in different AML cell lines and in
primary AML samples, further inducing apoptosis, whereas glutamine starvation seems to have no effect on normal CD341 cells.
We then focused our attention on the SLC1A5 transporter whose
loss of function has been shown to inhibit cell growth and activate
autophagy.15 SLC1A5 is necessary for glutamine uptake by epithelial
cells and by some cancer cells in culture, but few data concerning
glutamine transport in hematopoietic cells have been reported.24 The
SLC1A5 transporter is expressed in all leukemic cell lines we tested.
In MOLM-14 cells, shRNA-mediated inhibition of SLC1A5 expression prevented p70S6K T389 phosphorylation induced by AA
stimulation. Moreover, inhibition of SLC1A5 expression showed
antileukemic activity, triggered apoptosis, and markedly reduced
the growth of MOLM-14 tumors in a xenograft mouse model.
However, full inhibition of SLC1A5 expression in HL-60 cells did
not induce apoptosis. This may be due to the expression of other
glutamine transporters in accordance with previous observations
that mTORC1 can be reactivated by AA stimulation in HL-60 cells
after SLC1A5 knockdown.25,26
Because AML cells depend on glutamine for survival, we tested
the effects of L-ase. This drug is essential in the treatment of acute
lymphoid leukemias (ALLs) and induces a 60% of complete
remission (CR) rate as a monotherapy.27-29 L-ases from E coli and
E chrysanthemi were tested in our experiments. Each of these
species of L-ase has glutaminase activity corresponding to 3% and
10% of the total asparaginase activity, respectively. In vivo, a
strong correlation between the serum L-ase activity and the rates of
AA depletion have been observed under treatment.30-32 Repeated
injections of E chrysanthemi L-ase (25 000 UI/m2 every 48 hours)
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
resulted in very low levels of asparagine and glutamine in the
serum of the patients.33
We observed that L-ase induces the rapid and complete depletion
of intracellular glutamine in leukemic cells. We also observed that,
in contrast to ALL cells, asparagine is not essential for AML cell
survival. Indeed, only MV4-11 cells are moderately sensitive to
asparagine removal in terms of apoptosis induction. Moreover,
glutamine, but not asparagine, starvation inhibits mTORC1 activity.
We then observed that both the E coli and E chrysanthemi L-ases
inhibit mTORC1 activity through their glutaminase activity. Similar
results were observed in primary AML samples treated with E coli
L-ase; mTORC1 inhibition was achieved in all patients tested at
24 hours using a dose of 1 UI/mL. We observed that mTORC1 inhibition by glutamine depletion results in mTORC2 overactivation, as
we previously reported in AML cells using the mTORC1 inhibitor
RAD001.2 Moreover, by inhibiting 4E-BP1 phosphorylation downstream of mTORC1, L-ase inhibited global protein synthesis. A few
reports have previously established a link between L-ase and
mTORC1.34,35 In ALL cell lines, L-ase was previously found to
inhibit mTORC1 activity, whereas other kinases, including Akt,
were unaffected.34 Moreover, the L-ase–induced inhibition of S6K
phosphorylation was also previously found to be diminished in a
Jurkatt clone resistant to this drug.34 MTORC1 inhibition has also
been observed in vivo in the liver and pancreas of mice treated with
L-ase. In contrast, L-ase without glutaminase activity isolated from
Wolinella succinogenes does not suppress mTORC1 activity in mice.35
The mechanism by which L-ase inhibits mTORC1 is probably interference with AA-mediated mTORC1 activation. Indeed, we showed in
our present experiments that expression of a Raptor-Rheb15 protein in
HEK293 cells, which forces the localization of mTORC1 to the
lysosome surface,18 is sufficient to render the mTORC1 pathway
partially resistant to L-ase and glutamine starvation.
We further show from our present analyses that L-ase has antileukemic potential and potently induces apoptosis in AML cells. In
the years from 1970 to 1980, L-ase was tested in AML with CR rates
of ;10% as a monotherapy.36,37 Subsequently, a randomized study
compared the effects of a high dose of cytarabine with or without
E coli L-ase in refractory and relapsed AML in adults and the association revealed a significantly increased CR rate (40% vs 24%).38
The high-dose cytarabine plus L-ase regimen is currently used in
pediatric AML treatment protocols.39 In children, all studies that have
compared the sensitivity of primary blast cells from ALL and AML to
L-ase treatment concluded that AML blast cells are less sensitive to
L-ase. However, blast cells from the M1, M4, and M5 FAB subtypes
previously displayed an in vitro sensitivity to L-ase that was very close
to that of ALL.40-42 Although our current study testing the effects of
L-ase in a cohort of 21 patients does not allow us to draw definitive
conclusions, we speculate that some AML subtypes may exhibit a
particular sensitivity to L-ase.
A further question we addressed was the characterization of acquired or intrinsic mechanisms of resistance to L-ase. In ALL, expression of the asparagine synthase gene is transcriptionally increased
by L-ase.43,44 Similarly, L-ase increased the expression of the GS
protein in the ALL MOLT-4 cell line, which correlated with L-ase
sensitivity.45 In T-cell–ALL, rat fibrosarcoma, or human hepatocarcinoma cells, GS inhibition by the L-methionine-sulfoximine
restores the sensitivity to L-ase in resistant cell lines.45-47 We observed
that L-ase increased GS expression in all of the AML cell lines and
primary AML cells tested. However, shRNA-mediated GS knockdown
increased L-ase–induced apoptosis only in MV4-11 cells but not in
other AML cell lines in our panel, indicating that L-ase–induced GS
expression may constitute a mechanism of resistance to L-ase in certain
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 14 NOVEMBER 2013 x VOLUME 122, NUMBER 20
cases. Similar results were observed only in MV4-11 cells when
glutamine was removed.
Finally, we observed that autophagy is strongly induced in AML
by either glutamine removal or L-ase treatment. This process may be
partially protective against AML cell death and may represent a
general mechanism of resistance to L-ase in cancers. Autophagy protects some tumor cells from chemotherapy-induced cell death, and
ongoing clinical trials are aimed at combining autophagy inhibitors
with other cancer treatments.48,49 The future testing of a combination
of autophagy inhibitors and L-ase is therefore warranted in AML.
Overall, by using different approaches, we show that targeting the
glutamine uptake into leukemic cells represents a new therapeutic
strategy in AML. It facilitates protein synthesis inhibition downstream
of mTORC1. Our results also suggest that high glutamine uptake may
sustain constitutive mTORC1 activation in AML. We also provide a
rationale for L-ase treatment in AML and suggest that inhibiting
autophagy or GS activity may improve the antileukemic activity of
L-ases in this disease.
Acknowledgments
We thank Dr D. Rabier (Biochimie Métabolique, Hôpital Necker
Enfants Malades, Paris, France) for helpful discussions concerning
glutaminolysis. We also thank the EUSA Pharma laboratory (Lyon,
France) for kindly providing the E coli L-ase (Kidrolase) and the
E chrysanthemi L-ase (Erwinase) for in vitro experiments.
TARGETING GLUTAMINE UPTAKE IN AML
3531
This work was supported by grants from the Association de la
Recherche contre le Cancer (contrat libre). We also thank the Institut
National du Cancer and the Fond d’Etude et de Recherche du Corps
Médical des hôpitaux de Paris for the funding of N.J.
Authorship
Contribution: L.W. and N.J. analyzed data, performed the experiments, and wrote the manuscript with equal contribution; A.J. and
P.A. performed experiments concerning autophagy; N.N. performed
experiments concerning measurement of glutamine and L-ase
levels; T.T.M. and I.C.M. performed in vivo experiments; M.L.
helped designing the molecular constructions; A.S. performed the
electron microscopy experiments; L.P. and A.S.G. performed in
vitro experiments on primary AML cells; M.U. and N.I. helped
with recruiting and analyzing AML samples; O.K. performed
molecular analysis on primary AML samples; I.R.-W. performed
cytogenetic analysis on primary AML samples; V.B. and N.C.
performed the cytological and flow cytometry analysis on primary
AML samples; C.L. and P.M. wrote the manuscript; J.T. analyzed
data and wrote the manuscript; and D.B. designed the research
plan, analyzed the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Didier Bouscary, UF d’Hématologie, Hôpital
Cochin, AP-HP, Paris, France; e-mail: [email protected].
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2013 122: 3521-3532
doi:10.1182/blood-2013-03-493163 originally published
online September 6, 2013
Inhibiting glutamine uptake represents an attractive new strategy for
treating acute myeloid leukemia
Lise Willems, Nathalie Jacque, Arnaud Jacquel, Nathalie Neveux, Thiago Trovati Maciel, Mireille
Lambert, Alain Schmitt, Laury Poulain, Alexa S. Green, Madalina Uzunov, Olivier Kosmider, Isabelle
Radford-Weiss, Ivan Cruz Moura, Patrick Auberger, Norbert Ifrah, Valérie Bardet, Nicolas Chapuis,
Catherine Lacombe, Patrick Mayeux, Jérôme Tamburini and Didier Bouscary
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