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Blood First Edition Paper, prepublished online September 6, 2013; DOI 10.1182/blood-2013-03-493163
Inhibiting glutamine uptake represents an attractive new strategy for
treating acute myeloid leukemia
Running title: Targeting glutamine uptake in AML
Lise Willems1,2,3*, Nathalie Jacque2*, Arnaud Jacquel4, Nathalie Neveux5, Thiago Trovati
Maciel6, Mireille Lambert2, Alain Schmitt7, Laury Poulain2, Alexa S. Green2, Madalina
Uzunov8, Olivier Kosmider2,3,9, Isabelle Radford-Weiss10, Ivan Cruz Moura6, Patrick
Auberger4, Norbert Ifrah11, Valérie Bardet2,3,9, Nicolas Chapuis2,3,9, Catherine Lacombe2,3,9,
Patrick Mayeux2,3, Jérôme Tamburini1,2,3, Didier Bouscary1,2,3.
1
Unité Fonctionnelle d’Hématologie, Hôpital Cochin, AP-HP. Paris, France.
2
Institut Cochin, Département d’Immuno-Hématologie, CNRS UMR8104, INSERM U1016.
Paris, France.
3
Université Paris Descartes, Faculté de Médecine Sorbonne Paris Cité.
4
INSERM U1065, Université Nice-Sophia Antipolis, Centre Méditerranéen de Médecine
Moléculaire, Team 2 “Cell death, differentiation, inflammation and cancer”, Nice, France.
5
Service de Biochimie, Hôpital Cochin, AP-HP. Paris, France.
6
Inserm U699, Faculté de Médecine Xavier Bichat, Paris, France.
7
Plateforme de microscopie électronique, Institut Cochin, Paris, France.
8
Service d’Hématologie Clinique, Hôpital Pitié-Salpêtrière, AP-HP. Paris, France.
9
Service d’Hématologie Biologique, Hôpital Cochin, AP-HP. Paris, France.
10
Service de Cytogénétique, Hôpital Necker-Enfants Malades, AP-HP. Paris, France
11
Service des Maladies du Sang, CHU Angers, Angers, France
1
Copyright © 2013 American Society of Hematology
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*
LW and NJ equally contributed
Correspondence: Didier Bouscary. MD; PhD. UF d’Hématologie, Hôpital Cochin, AP-HP,
Paris. E-mail : [email protected]
2
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Key points
Glutamine removal and knockdown of the glutamine transporter SLC1A5 have antileukemic
activity in AML.
The glutaminase activity of L-asparaginase inhibits mTORC1 and protein synthesis and
induces a strong autophagy in AML
Abstract
Cancer cells require nutrients and energy to adapt to increased biosynthetic activity and
protein synthesis inhibition downstream of mTORC1 has shown promise as a possible therapy
for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which
controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that
glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of
the SLC1A5 high affinity transporter for glutamine induces apoptosis and inhibits tumor
formation in a mouse AML xenotransplantation model. L-asparaginase (L-ase) is an anticancer agent also harboring glutaminase activity. We show that L-ases from both E. coli and
E. chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition
correlates with their glutaminase activity levels and produces a strong apoptotic 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 anti-cancer activity and glutamine uptake inhibition are
promising new therapeutic strategies for AML.
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Introduction
The serine threonine kinase mTOR (mammalian target of rapamycin) forms two 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 (eIF4E)-binding
protein 1 (4E-BP1). Cap dependent translation is controlled by the translational repressor 4EBP1. 4E-BP1 phosphorylation at its serine 65 residue is absolutely required to initiate the
formation of the translation initiation complex eiF4F1. In most primary AML samples, the
mTORC1 complex is activated by an as yet unknown mechanism2,3. 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 leukemia4. In vitro, in
primary AML cells, mTORC1 inhibition with rapamycin has cytostatic effects but does not
induce apoptosis2-5, mainly because it does not inhibit 4E-BP1 phosphorylation on ser656. In
contrast, mTOR kinase inhibitors (TORKinhib) fully inhibit 4E-BP1 phosphorylation,
suppress protein synthesis and induce apoptosis7,8. 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 Rheb (for Ras
homolog enriched in brain) protein towards mTORC19. However, a major process controlling
mTORC1 activity is the availability of amino acids (AA), mainly leucine10,11. Indeed,
intracellular leucine is required to activate the Rag (for Ras-related GTPases) proteins, which
enable the localization of the mTORC1 complex at the lysosome surface close to its activator,
Rheb12,13. Leucine is imported into the cell in exchange for glutamine by the bidirectional
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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 SLC7A5/3A214. The cellular uptake and subsequent rapid
efflux of glutamine in the presence of leucine make glutamine availability a limiting step for
mTORC1 activation15.
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 up-regulates glutamine synthase
(GS) protein expression, which may limit the L-ase induced anti-leukemic 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 (BM) or peripheral blood (PB) samples with > 70% blast cell content were
obtained from 27 patients with newly diagnosed AML. Cells were isolated using FicollHypaque density-gradient separation. Cells were collected after obtaining their 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
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Supplemental Table 1. The CD34+ cells from healthy donors were purified using
MIDIMACS immunoaffinity columns (Milteny Biotech, Bergish Badgash, Germany).
Cell Culture
Human leukemic cell lines were purchased from DSMZ (Braunschweig, Germany) for MV411 and from the American Type Culture Collection (ATCC; Molsheim, France) for HL-60,
MOLM-14 and OCI-AML3. Cells were cultured in MEM alpha medium containing 10% FBS
and 4 mM glutamine. To induce amino acid starvation, cells were cultured in MEM alpha
medium without glutamine, asparagine and leucine (PAA) supplemented with 10% dialyzed
FBS in the presence or absence of 4 mM glutamine, 0.4 mM leucine or 0.4 mM L-asparagine
(Sigma, St. Louis, MO). HEK-293T cells were cultured in DMEM medium with 10% FBS
without glutamine and leucine, and then supplemented or not with amino acids.
Reagents
We used from 0.1 to 10 UI/mL of Escherichia coli L-asparaginase (Kidrolase®) or Erwinia
chrysanthemi L-asparaginase (Erwinase®), both purchased from EUSA Pharma Inc (Lyon
France).
Western Blotting
Whole-cell extracts and western blots were performed as previously described6. Antibodies
against phospho-p70S6K T389, 4EBP1, phospho-4EBP1 S65, phospho-AKT S473, AKT,
phospho-NDRG1 T346, 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, California, USA); anti-p70S6K and anti-SQSTM1 from Santa Cruz Biotechnology;
anti-actin and anti-Flag antibodies from Sigma; anti-p85 antibodies were raised in-house.
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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 AA, culture supernatants were deproteinized with a
30% (w/v) sulfosalicylic acid solution (ASS), incubated for 5 min at room temperature and
centrifuged (10000 rpm, 10 min, 4°C). Supernatants were then stored at -80°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 x 106 cells
were sonicated and centrifuged (10000 rpm, 10 min). Homogenized samples were then
deproteinized and again centrifuged. The resulting supernatant was subsequently used for AA
measurements by ion exchange chromatography.
Lentiviral sh/siRNAs and cDNAs
SLC1A5
[TCRN0000043120
(shRNA1)
and
TCRN0000043122
(shRNA2)],
GS
[TCRN0000045628 (shRNA1) and TCRN0000045630 (sRNAh2)] and non-targeted (control)
shRNAs were from Open Biosystems and cloned into a pLKO-Tet-On inducible or pLKO.1
plasmids [plasmid #21915 and #8453 from Addgene (Cambridge, MA, USA)]16. Lentiviral
particles were produced using HEK-293T packaging cells. Supernatants were collected 48 h
after transfection and then stored at -80°C. Leukemic cell lines were plated at 2.5 x 106
cells/ml in serum-free medium to which 10 µl of lentiviral supernatants were added. The
transfection of anti-Atg5 and anti-beclin small interfering RNAs was performed in the OCIAML3 cell line using the Amaxa nucleofector kit (Lonza, Basel, Switzerland). The pLJM1
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Flag Raptor Rheb15 (Addgene plasmid #26634) or control vector were transfected in 293T
cells using 10µl of lentiviral supernatant.
[S35] Methionine incorporation assay
Cells were pre-treated without or with L-ase (E. coli) at 10 UI/mL in DMEM medium without
methionine (Eurobio, Courtaboeuf, France) for six hours and pulse-labeled with 160µCi of
[S35] methionine for additional two hours. The amount of radioactivity incorporated into the
proteins was determined by trichloroacetic acid precipitation.
AML xenografts in nude mice
MOLM-14 infected with either a lentiviral vector containing the doxycycline-inducible
SLC1A5 shRNA or a non-specific shRNA were subcutaneously injected into nude mice as
previously reported17. All mice were treated with doxycycline, given ad libitum five days a
week (n=12). Tumor growth was measured three 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
Cells were fixed for one hour in 3% glutaraldehyde in 0.1 M sodium phosphate buffer, pH
7.4, post-fixed for 1.5 h with 1% osmium tetraoxide, dehydrated by successive ethanol washes
(70%, 90%, 100%, 100%), and impregnated with epoxy resin. After polymerization, 80-90
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.
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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’s t-test. *, ** and *** indicate P<0.05, P<0.01 and P<0.001, respectively. The
experiments performed in AML cell lines were performed in independent triplicates.
Results
1/ 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 (Figures 1A and S1A). However, glutamine or leucine starvation
did not inhibit the phosphorylation of AKT on S473 (figure S1B). Glutamine removal for 24
hours induced significant apoptosis in all cell lines with the most dramatic effects seen in
MV4-11 and OCI-AML3, as assessed by Annexin V binding and caspase-3 and PARP
cleavage assays (Figures 1B and 1C). We then tested the hypothesis that AML cells have a
higher level of glutamine dependence than normal CD34+ cells. As shown in Figure 1D,
normal CD34+ cells from three 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.
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2/ Targeting the SLC1A5 glutamine transporter has anti-leukemic effects.
We tested whether inhibiting glutamine uptake via a knockdown of the glutamine transporter
SLC1A5 exerts anti-leukemic activity. The SLC1A5 protein is expressed in all leukemic cell
lines tested (Figure 2A). We engineered two different doxycycline-inducible shRNAs that
were transduced in AML cells using a lentivirus construct and which 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 without or with doxycycline were starved of leucine and glutamine for six hours to
inhibit mTORC1 and then stimulated for 15 and 30 minutes with these 2 AAs. MTORC1
activation was strongly re-induced in the doxycycline untreated cells but not when SLC1A5
was knocked down (Figure 2C).
SLC1A5 knockdown induced significant apoptosis, attested 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 glutaminemediated mTORC1 activation in this cell line (Figure S2). The anti-leukemic effects of
SLC1A5 inhibition were tested in vivo. Two groups of nude mice were injected
subcutaneously 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 mice (P=0.0011) (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 the
SLC1A5 glutamine transporter has anti-leukemic activity both in vitro and in vivo.
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3/ The glutaminase activity of L-asparaginase inhibits mTORC1 and protein translation
in AML cells.
The L-ases have glutaminase activity and transform extra-cellular 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 Lase 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 four-fold 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 three other AML cell lines (Figures S3A and
S3B). 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 Lase-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 (Figure S3C). Moreover, the E.coli L-ase
dramatically inhibited mTORC1 activity in several primary AML samples (Figures 3D). As
glutamine starvation, L-ase over-activated mTORC2 signaling, attested by the increased
phosphorylations of AKT on S473 and NDRG1 on T346 (Figure S3D).
L-ase may inhibit leucine entry into the cells by depleting intracellular glutamine, thereby
preventing mTORC1 activation by Rheb at the lysosomal surface12,13. To investigate this
mechanism further, 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 activation18. In HEK-293T cells expressing the Raptor-Rheb15 protein, glutamine
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removal only partially inhibited p70S6K T389 phosphorylation. Interestingly, mTORC1
activation was maintained in Raptor-Rheb15 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 [S35] methionine
incorporation into newly synthesized proteins, indicative of global protein synthesis inhibition
(Figure 3F). Moreover, in AML cell lines and in a representative primary AML sample, L-ase
inhibited c-Myc and Mcl-1 expression, whose translation is cap-dependent, in a dosedependent 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.
4/ L-asparaginase induce apoptosis in AML cells through its glutaminase activity.
The functional effects of L-ase were tested in leukemic cell lines and in 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 depletion obtained with E.
chrysanthemi L-ase (Figure 3C). Similar results were observed in the MOLM-14, MV4-11
and HL-60 cell lines (Figure S4A). 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 these samples
(Figure 4D). In AML cell lines and patient samples analyzed, L-ase induced caspase-3
cleavage (Figure 4E).
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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 (Figure S4B). Overall, these
results indicate that L-ase has significant anti-leukemic activity in AML, and that this is
mainly related to its glutaminase activity.
5/ L-asparaginase increase glutamine synthase (GS) expression in AML.
The glutamine synthase (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 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 (Figures 5B and S5A). 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 (Figures 5C and S5B).
These results indicate that in some leukemic cells, GS expression may represent a mechanism
of resistance to the anti-leukemic activity of L-ase.
6/ L-asparaginase induces a strong autophagy in AML cells.
As mTORC1 controls the activity of key proteins of the autophagic process, such as
Atg1/ULK and ATG1319, 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
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and time-dependent 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 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 small interfering RNAs (siRNAs) to knockdown 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 than
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 emerge 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 cells20,21,22. Glutamine is a
core substrate for protein, glucosamine and nucleotide synthesis, and metabolized by
glutaminases into glutamate which enter the TCA cycle to produce ATP after conversion to
α-ketoglutarate23. 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
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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 CD34+
cells.
We then focused our attention on the SLC1A5 transporter whose loss of function has been
shown to inhibit cell growth and activate autophagy15. 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 reported24. 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 anti-leukemic 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 knockdown25,26.
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 (ALL) and induces a 60% of
complete remission (CR) rate as a monotherapy27,28,29. 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 treatment30,31,32. Repeated injections of E. chrysanthemi L-ase (25000 UI/m2
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every 48 hours) resulted in very low levels of asparagine and glutamine in the serum of the
patients33.
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 term 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 over-activation, as we previously reported in AML
cells using the mTORC1 inhibitor RAD0012. 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 mTORC134,35. In ALL cell lines,
L-ase was found previously to inhibit mTORC1 activity, whereas other kinases, including
Akt, were unaffected34. Moreover, the L-ase-induced inhibition of S6K phosphorylation was
also previously found to be diminished in a Jurkatt clone resistant to this drug34. 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 mice35. 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 surface18, is sufficient to render the
mTORC1 pathway partially resistant to L-ase and glutamine starvation.
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We further show from our present analyses that L-ase has anti-leukemic potential and potently
induces apoptosis in AML cells. In the years from 1970-1980, L-ase was tested in AML with
CR rates of around 10% as a monotherapy36,37. 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 protocols39. 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 ALL40,41,42. 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 (ASNS) gene is
transcriptionally increased by L-ase43,44. Similarly, L-ase increased the expression of the
glutamine synthase (GS) protein in the ALL MOLT-4 cell line which correlated with L-ase
sensitivity45. In T-ALL, rat fibrosarcoma or human hepatocarcinoma cells, GS inhibition by
the L-methionine-sulfoximine (MSO) restores the sensitivity to L-ase in resistant cell
lines45,46,47. 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-aseinduced 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 Lase in certain cases. Similar results were observed only in MV4-11 cells when glutamine was
removed.
17
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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 treatments48,49. 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.
18
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Acknowledgements
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 Escherichia coli L-asparaginase
(Kidrolase®) and the Erwinia chrysanthemi L-asparaginase (Erwinase®) for in vitro
experiments. This work was supported by grants from the Association de la Recherche contre
le Cancer (ARC; contrat libre). We also thank the Institut National du Cancer (INCa) and the
Fond d’Etude et de Recherche du Corps Médical des hôpitaux de Paris (FERCM) for the
funding of N Jacque.
Authorship Contributions
LW and NC analyzed data, performed the experiments and wrote the manuscript with equal
contribution. AJ and PA performed experiments concerning autophagy. NN performed
experiments concerning measurement of glutamine and L-asparagine levels. TT and ICM
performed in vivo experiments. ML helped designing the molecular constructions. AS
performed the electron microscopy experiments. LP and ASG performed in vitro experiments
on primary AML cells. MU and NI helped recruiting and analyzing AML samples. OK
performed molecular analysis on primary AML samples. IRW performed cytogenetic analysis
on primary AML samples. VB and NC performed the cytological and flow cytometry analysis
on primary AML samples. CL and PM wrote the manuscript. JT analyzed data and wrote the
manuscript. DB designed the research plan, analyzed the data and wrote the manuscript.
Disclosure
The authors declare no competing financial interests.
19
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References
1. Gingras AC, Raught B, Gygi SP, et al. Hierarchical phosphorylation of the translation
inhibitor 4E-BP1. Genes Dev. 2001;15(21):2852–2864.
2. Tamburini J, Chapuis N, Bardet V, et al. Mammalian target of rapamycin (mTOR)
inhibition activates phosphatidylinositol 3-kinase/Akt by up-regulating insulin-like
growth factor-1 receptor signaling in acute myeloid leukemia: rationale for therapeutic
inhibition of both pathways. Blood. 2008;111(1):379–382.
3. Dos Santos C, Demur C, Bardet V, et al. A critical role for Lyn in acute myeloid
leukemia. Blood. 2008;111(4):2269–2279.
4. Hoshii T, Tadokoro Y, Naka K, et al. mTORC1 is essential for leukemia propagation but
not stem cell self-renewal. J. Clin. Invest. 2012;
5. Xu Q, Thompson JE, Carroll M. mTOR regulates cell survival after etoposide treatment
in primary AML cells. Blood. 2005;106(13):4261–4268.
6. Tamburini J, Green AS, Bardet V, et al. Protein synthesis is resistant to rapamycin and
constitutes a promising therapeutic target in acute myeloid leukemia. Blood.
2009;114(8):1618–1627.
7. Willems L, Chapuis N, Puissant A, et al. The dual mTORC1 and mTORC2 inhibitor
AZD8055 has anti-tumor activity in acute myeloid leukemia. Leuk. Off. J. Leuk. Soc. Am.
Leuk. Res. Fund Uk. 2011;
8. Chapuis N, Tamburini J, Green AS, et al. Dual inhibition of PI3K and mTORC1/2
signaling by NVP-BEZ235 as a new therapeutic strategy for acute myeloid leukemia.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2010;16(22):5424–5435.
9. Laplante M, Sabatini DM. mTOR Signaling in Growth Control and Disease. Cell.
2012;149(2):274–293.
10. Hara K, Yonezawa K, Weng QP, et al. Amino acid sufficiency and mTOR regulate p70
S6 kinase and eIF-4E BP1 through a common effector mechanism. J. Biol. Chem.
1998;273(23):14484–14494.
11. Avruch J, Long X, Ortiz-Vega S, et al. Amino acid regulation of TOR complex 1. Am. J.
Physiol. Endocrinol. Metab. 2009;296(4):E592–602.
12. Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan K-L. Regulation of TORC1 by Rag
GTPases in nutrient response. Nat. Cell Biol. 2008;10(8):935–945.
13. Sancak Y, Peterson TR, Shaul YD, et al. The Rag GTPases bind raptor and mediate
amino acid signaling to mTORC1. Science. 2008;320(5882):1496–1501.
14. Fuchs BC, Bode BP. Amino acid transporters ASCT2 and LAT1 in cancer: partners in
crime? Semin. Cancer Biol. 2005;15(4):254–266.
15. Nicklin P, Bergman P, Zhang B, et al. Bidirectional transport of amino acids regulates
mTOR and autophagy. Cell. 2009;136(3):521–534.
16. Wiederschain D, Wee S, Chen L, et al. Single-vector inducible lentiviral RNAi system for
oncology target validation. Cell Cycle Georget. Tex. 2009;8(3):498–504.
17. Callens C, Coulon S, Naudin J, et al. Targeting iron homeostasis induces cellular
differentiation and synergizes with differentiating agents in acute myeloid leukemia. J.
Exp. Med. 2010;207(4):731–750.
18. Sancak Y, Bar-Peled L, Zoncu R, et al. Ragulator-Rag complex targets mTORC1 to the
lysosomal surface and is necessary for its activation by amino acids. Cell.
2010;141(2):290–303.
19. Puissant A, Robert G, Auberger P. Targeting autophagy to fight hematopoietic
malignancies. Cell Cycle Georget. Tex. 2010;9(17):3470–3478.
20. Levine AJ, Puzio-Kuter AM. The control of the metabolic switch in cancers by oncogenes
and tumor suppressor genes. Science. 2010;330(6009):1340–1344.
20
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
21. Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even warburg did
not anticipate. Cancer Cell. 2012;21(3):297–308.
22. Vander Heiden MG, Locasale JW, Swanson KD, et al. Evidence for an alternative
glycolytic pathway in rapidly proliferating cells. Science. 2010;329(5998):1492–1499.
23. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in cancer. Trends
Biochem. Sci. 2010;35(8):427–433.
24. Ochiai H, Higa K, Hishiyama N, Hisamatsu S, Fujise H. Characterization of several
amino acid transports and glutamine metabolism in MOLT4 human T4 leukemia cells.
Clin. Lab. Haematol. 2006;28(6):399–404.
25. Evans K, Nasim Z, Brown J, et al. Acidosis-sensing glutamine pump SNAT2 determines
amino acid levels and mammalian target of rapamycin signalling to protein synthesis in
L6 muscle cells. J. Am. Soc. Nephrol. Jasn. 2007;18(5):1426–1436.
26. Pinilla J, Aledo JC, Cwiklinski E, et al. SNAT2 transceptor signalling via mTOR: a role
in cell growth and proliferation? Front. Biosci. Elite Ed. 2011;3:1289–1299.
27. Capizzi RL, Bertino JR, Skeel RT, et al. L-asparaginase: clinical, biochemical,
pharmacological, and immunological studies. Ann. Intern. Med. 1971;74(6):893–901.
28. Sallan SE, Hitchcock-Bryan S, Gelber R, et al. Influence of intensive asparaginase in the
treatment of childhood non-T-cell acute lymphoblastic leukemia. Cancer Res.
1983;43(11):5601–5607.
29. Pui C-H, Evans WE. Treatment of acute lymphoblastic leukemia. N. Engl. J. Med.
2006;354(2):166–178.
30. Miller HK, Salser JS, Balis ME. Amino acid levels following L-asparagine
amidohydrolase (EC.3.5.1.1) therapy. Cancer Res. 1969;29(1):183–187.
31. Müller HJ, Boos J. Use of L-asparaginase in childhood ALL. Crit. Rev. Oncol. Hematol.
1998;28(2):97–113.
32. Avramis VI, Sencer S, Periclou AP, et al. A randomized comparison of native Escherichia
coli asparaginase and polyethylene glycol conjugated asparaginase for treatment of
children with newly diagnosed standard-risk acute lymphoblastic leukemia: a Children’s
Cancer Group study. Blood. 2002;99(6):1986–1994.
33. Avramis VI, Martin-Aragon S, Avramis EV, Asselin BL. Pharmacoanalytical assays of
Erwinia asparaginase (erwinase) and pharmacokinetic results in high-risk acute
lymphoblastic leukemia (HR ALL) patients: simulations of erwinase population PK-PD
models. Anticancer Res. 2007;27(4C):2561–2572.
34. Iiboshi Y, Papst PJ, Hunger SP, Terada N. L-Asparaginase inhibits the rapamycintargeted signaling pathway. Biochem. Biophys. Res. Commun. 1999;260(2):534–539.
35. Reinert RB, Oberle LM, Wek SA, et al. Role of glutamine depletion in directing tissuespecific nutrient stress responses to L-asparaginase. J. Biol. Chem. 2006;281(42):31222–
31233.
36. Clarkson B, Krakoff I, Burchenal J, et al. Clinical results of treatment with E. coli Lasparaginase in adults with leukemia, lymphoma, and solid tumors. Cancer.
1970;25(2):279–305.
37. Ohnuma T, Holland JF, Freeman A, Sinks LF. Biochemical and pharmacological studies
with asparaginase in man. Cancer Res. 1970;30(9):2297–2305.
38. Capizzi RL, Davis R, Powell B, et al. Synergy between high-dose cytarabine and
asparaginase in the treatment of adults with refractory and relapsed acute myelogenous
leukemia--a Cancer and Leukemia Group B Study. J. Clin. Oncol. Off. J. Am. Soc. Clin.
Oncol. 1988;6(3):499–508.
39. Perel Y, Auvrignon A, Leblanc T, et al. Treatment of childhood acute myeloblastic
leukemia: dose intensification improves outcome and maintenance therapy is of no
benefit--multicenter studies of the French LAME (Leucémie Aiguë Myéloblastique
21
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
Enfant) Cooperative Group. Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund Uk.
2005;19(12):2082–2089.
Zwaan CM, Kaspers GJ, Pieters R, et al. Cellular drug resistance profiles in childhood
acute myeloid leukemia: differences between FAB types and comparison with acute
lymphoblastic leukemia. Blood. 2000;96(8):2879–2886.
Yamada S, Hongo T, Okada S, et al. Clinical relevance of in vitro chemoresistance in
childhood acute myeloid leukemia. Leuk. Off. J. Leuk. Soc. Am. Leuk. Res. Fund Uk.
2001;15(12):1892–1897.
Okada S, Hongo T, Yamada S, et al. In vitro efficacy of l-asparaginase in childhood acute
myeloid leukaemia. Br. J. Haematol. 2003;123(5):802–809.
Hutson RG, Kitoh T, Moraga Amador DA, et al. Amino acid control of asparagine
synthetase: relation to asparaginase resistance in human leukemia cells. Am. J. Physiol.
1997;272(5 Pt 1):C1691–1699.
Appel IM, den Boer ML, Meijerink JPP, et al. Up-regulation of asparagine synthetase
expression is not linked to the clinical response L-asparaginase in pediatric acute
lymphoblastic leukemia. Blood. 2006;107(11):4244–4249.
Aslanian AM, Kilberg MS. Multiple adaptive mechanisms affect asparagine synthetase
substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells. Biochem.
J. 2001;358(Pt 1):59–67.
Rotoli BM, Uggeri J, Dall’Asta V, et al. Inhibition of glutamine synthetase triggers
apoptosis in asparaginase-resistant cells. Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol.
Biochem. Pharmacol. 2005;15(6):281–292.
Tardito S, Chiu M, Uggeri J, et al. L-Asparaginase and inhibitors of glutamine synthetase
disclose glutamine addiction of β-catenin-mutated human hepatocellular carcinoma cells.
Curr. Cancer Drug Targets. 2011;11(8):929–943.
Lamoureux F, Thomas C, Crafter C, et al. Blocked Autophagy Using Lysosomotropic
Agents Sensitizes Resistant Prostate Tumor Cells to the Novel Akt Inhibitor AZD5363.
Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2013;
Han W, Sun J, Feng L, et al. Autophagy inhibition enhances daunorubicin-induced
apoptosis in K562 cells. Plos One. 2011;6(12):e28491.
22
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Figure legends
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 (WB)
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 WB. (D) Normal CD34+ cell were obtained
from three healthy allogeneic transplant donors after positive sorting and primary AML cell
from eight AML patients were also tested. Cells were cultured 48 hours with or without
glutamine (4mM) and the apoptotic responses were quantified by flow cytometry analysis of
Annexin-V binding.
Figure 2. Targeting the SLC1A5 glutamine transporter has pro-apoptotic effects and in
vivo anti leukemic activity. (A) SLC1A5 expression was studied by WB in the four 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 without or with doxycycline for the indicated times. The inhibition of SLC1A5
expression with doxycycline was assessed by WB. (C) MOLM-14/SLC1A5 shRNA cells and
MOLM-14/CTR shRNA cells were starved or not for six hours of glutamine and leucine to
inhibit mTORC1, as detected by the inhibition of p70S6K T389 phosphorylation. Previously
23
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starved cells were then stimulated for 15 or 30 minutes with leucine + 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
WB 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=8 in each group). (F) Kaplan-Meier survival
curves of nude mice treated with doxycycline. (G) SLC1A5 expression was detected by WB
analysis of tumor extracts from MOLM-14/SLC1A5 shRNA or MOLM-14/CTR shRNA mice
treated with doxycycline (day 22).
Figure 3. The glutaminase activity of L-asparaginase 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. WB was
performed with anti-phospho-p70S6K T389, anti-phospho-4E-BP1 S65, anti-p70S6K, anti4E-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 (A) and the same
analysis was assessed by WB. The intensities of phospho-p70S6K T389, phospho-4E-BP1
S65 and actin signals were quantified in different AML samples. Ratios of phospho-p70S6K
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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 (10UI/mL). WB was then performed with anti-flag, anti-actin or
35
anti-phospho-p70S6K T389 antibodies. (F) [S ] 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 10UI/ml during a 6 hours period. (G) OCI-AML3,
MOLM-14, MV4-11, 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
WB.
Figure 4. L-asparaginase 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 +/- 7AAD binding using flow cytometry (A–D) or by WB using an anti-caspase 3
antibody (E).
Figure 5. L-asparaginase increases glutamine synthase (GS) expression in AML, which
may represent a mechanism of resistance to L-asparaginase. (A) MOLM-14, OCI-AML3,
and primary AML blast cells from three patients were cultured over a 24 hours period with or
without 0.1, 1 or 10 UI/ml E.coli L-ase. GS expression was determined by WB. (B) OCIAML3 and MV4-11 cells were stably infected with a lentiviral vector expressing either a
control (CTR) or a GS shRNA and then cultured +/- 10 UI/mL E.coli L-ase and +/- glutamine
at 4 mM for 24 hours. WB 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
25
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apoptosis induction after 24 hours in the presence or not of E.coli L-ase at 10 UI/ml and with
glutamine starvation or not.
Figure 6. L-asparaginase induces strong protective autophagy in AML cells. (A, B) OCIAML3 cells were cultured for 6 or 24 hours in 10% FCS MEM +/- 0.1, 1 and 10 UI/ml of E.
coli L-ase and +/- Gln as indicated. Autophagy was detected by WB using anti-LC3b and
p62/SQSTM1 antibodies. (C) Representative electron micrographs of OCI-AML3 cells
collected after 24 hours of culture in MEM +/- E. coli L-ase (1 UI/ml) and without glutamine.
Arrows indicate autophagic vesicles. Upper and lower panels show two different scales. (D)
OCI-AML3 cells were transfected with anti-ATG5 or anti-beclin small interfering RNAs
(siRNAs) using the Amaxa nucleofector kit. Two days after transfection, cells were cultured
for 24 hours +/- E. coli L-ase at 10 UI/ml and WB 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
three independent experiments.
26
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Prepublished online September 6, 2013;
doi:10.1182/blood-2013-03-493163
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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