University of Southern California Dissertations and Theses

Adipocyte production of glutamine protects leukemia cells from L-asparaginase

(USC Thesis Other)

Adipocyte production of glutamine protects leukemia cells from L-asparaginase

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
i
ADIPOCYTE PRODUCTION OF GLUTAMINE PROTECTS LEUKEMIA CELLS FROM
L-ASPARAGINASE
by
Ehsan Ali Ehsanipour

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHYSIOLOGY AND BIOPHYSICS)

August 2013
Copyright 2013 Ehsan Ali Ehsanipour

ii
Acknowledgements
Committee
Steven D. Mittelman
Harvey Kaslow
Yong-Mi Kim
Vassilios I. Avramis

Members of the Mittelman Laboratory
Xia Sheng
James W. Behan
Rebecca Paszkiewicz

Collaborators
Anna Butturini
Xingchao Wang

iii
Table of Contents

Acknowledgements ......................................................................................................... ii
Table of Contents ........................................................................................................... iii
List of Figures................................................................................................................. iv
List of Tables .................................................................................................................. iv
Abstract ........................................................................................................................... v
Introduction ..................................................................................................................... 1
Obesity and cancer ...................................................................................................... 1
Leukemia and its microenvironment ............................................................................ 2
L-Asparaginase: A front-line leukemia treatment ......................................................... 3
Goals of this study ....................................................................................................... 5
Materials and methods .................................................................................................... 6
Human subjects ........................................................................................................... 6
Cell lines and culture ................................................................................................... 6
In vivo leukemia model ................................................................................................ 7
Coculture experiments ................................................................................................. 8
Amino acid analysis and sample preparation ............................................................... 9
Western blotting ......................................................................................................... 10
Immunohistochemistry ............................................................................................... 10
Statistical analysis ..................................................................................................... 11
Results .......................................................................................................................... 12
Adipocytes in the leukemia microenvironment produce glutamine ............................. 12
Obesity impairs L-Asparaginase efficacy in mice ....................................................... 14
Adipocytes protect leukemia cells from ASNase via GLN production ......................... 17
Glutamine secretion by adipocytes protects from L-Asparaginase ............................. 18
Discussion ..................................................................................................................... 23
Conclusions and Future Studies .................................................................................... 26
References .................................................................................................................... 28

iv
List of Figures

Figure 1. Effect of obesity on plasma and bone marrow asparagine and glutamine in
patients following L-Asparaginase treatment. ................................................................ 13
Figure 2. Diet induced obesity impairs L-Asparaginase treatment in leukemic mice. .... 14
Figure 3. Systemic markers of ASNase efficacy are not different in obese and lean mice
...................................................................................................................................... 15
Figure 4. Changes in Glutamine Synthetase in vivo and in vitro ................................... 16
Figure 5. Adipocytes protect leukemia from ASNase in vitro ......................................... 18
Figure 6. Adipocytes protection depends on glutamine production. .............................. 19
Figure 7. Impaired GLN secretion by adipocytes reverses protection from ASNase ..... 20
Figure 8. Leukemia cells show greater dependence on glutamine than asparagine...... 21
Figure 9. Erwinase is more effective than ASNase in killing leukemia cells. ................. 22

List of Tables
Table 1. ASN and GLN secreted by cells over 72 hours………..…………………………21

v
Abstract
Obesity is a significant risk factor for cancer. A link between obesity and a childhood
cancer has been identified: obese children diagnosed with high-risk acute lymphoblastic
leukemia (ALL) had a 50% greater risk of relapse than their lean counterparts. L-
asparaginase (ASNase) is a first-line therapy for ALL that breaks down asparagine and
glutamine, exploiting the fact that ALL cells are more dependent on these amino acids
than other cells. In the present study, we investigated whether adipocytes, which
produce significant quantities of glutamine, may counteract the effects of ASNase. In
children being treated for high-risk ALL, obesity was not associated with altered plasma
levels of asparagine or glutamine. However, glutamine synthetase was markedly
increased in bone marrow adipocytes after induction chemotherapy. Obesity
substantially impaired ASNase efficacy in mice transplanted with syngeneic ALL cells,
and, like in humans, without affecting plasma asparagine or glutamine levels. In co-
culture, adipocytes inhibited leukemic cell cytotoxicity induced by ASNase, and this
protection was dependent on glutamine secretion. These findings suggest that
adipocytes work in conjunction with other cells of the leukemia microenvironment to
protect leukemia cells during ASNase treatment.

1
Introduction
Obesity and cancer
Obesity is responsible for significant mortality and morbidity in humans. Obesity is a
major factor in the development of cardiovascular disease, diabetes, liver disease,
osteoarthritis, and other diseases. In the past 20 years, the percentage of obese adults
in the United States has increased from 12% to 35.9% (1,2), and estimated to be as
much as 51% by the year 2030 (3). At the same time, 32% of children are overweight
and 16% are obese (2), creating a huge burden on the healthcare system (4). It is
estimated that 300,000 deaths per year are attributable to poor diet and inactivity,
second only to tobacco as contributors of mortality (5). As the prevalence of obesity
continues to increase, the economic and social burden is likely to become even more
significant.
Obesity is also associated with an increased risk of developing several types of cancer.
A study performed in 2008 estimated that 4 percent of new cases of cancer in men and
7 percent in women were attributable to obesity (6). For some cancer types, such as
endometrial cancer and esophageal adenocarcinoma, as much as 40% of cases were
attributable to obesity. In addition to incidence, new research has found that obesity
leads to greater mortality in cancer patients. Calle et al. estimated that given the
prevalence of obesity in the U.S., 14% of all cancer deaths in men and 20% in women
are attributable to obesity (7).
In a large retrospective study, Dr. Anna Butturini showed that obese children had a 50%
higher risk of relapsing from acute lymphoblastic leukemia (ALL) than their lean
counterparts (8). Childhood leukemia is the most common type of cancer in children and

2
teenagers, and relapsed leukemia is the fourth most common (9). Caloric intake itself
has been correlated with incidence of lymphoblastic leukemia in adults world-wide (10).
Discovering the mechanism through which obesity impairs ALL treatment is vitally
important to improve patient outcome.

Leukemia and its microenvironment
Leukemia is a cancer of the white blood cells and the disease arises from the
uncontrolled growth of these cells, which overcrowd normal cells in the bone marrow and
spread to other organs. Leukemia is the most common cancer in children, making up
one third of all childhood cancer cases (11). In 2000 alone, there were approximately
256,000 new cases of leukemia and 209,000 deaths worldwide (12). Despite advances
in leukemia treatment over the last 40 years, the outcome for relapsed patients remains
poor. It is believed that minimal residual disease (MRD), the small population of cancer
cells surviving initial treatment, can give rise to a new, more aggressive tumor population
resulting in relapse.
A great deal of cancer research has focused on cell-autonomous mechanisms of drug
resistance. However, the tumor microenvironment has recently become a new target of
treatment. Originally proposed by Paget in 1889 (13) as an explanation for why certain
organs are more prone to metastasis than others, the tumor microenvironment
hypothesis argues that malignant cells are able to manipulate the surrounding extra
cellular matrix and normal cells to provide increased growth signals, nutrients, resistance
to chemotherapies, and angiogenesis (14). In the case of leukemia cells the
microenvironment is often considered the bone marrow, a compartment within bones
made up of several cell types including fibroblasts, osteoblasts, macrophages, and

3
adipocytes. It has been shown that co-culture with bone marrow stromal cells is
necessary for the survival of primary human leukemia cells (15). In addition, stromal
cells have been shown to protect leukemia cells from chemotherapies (16–18). Although
the primary home of leukemia cells is the bone marrow and spleen, leukemia cells have
been found to migrate towards several different tissues including the brain, kidney, liver,
and adipose tissue (19).
The mechanism(s) through which obesity impairs leukemia treatment and survival have
not been elucidated. Obesity is associated with alterations in inflammation, immune
function, and endocrine hormones. Adipose tissue itself is now viewed as an endocrine
organ, secreting hormones such as leptin (20), adiponectin (21), and resistin (22) which
have been shown to play a role in cancer. Obesity also influences the systemic
hormones insulin (23) and IGF-1 (24), both of which play a role in cancer proliferation.
Adipocytes also secrete the growth factors VEGF and TGF-β, as well as the chemokine
SDF-1α (25). Results from our laboratory have shown that adipocytes and adipose
tissue attract leukemia cells through secretion of SDF-1α (19), and that viable leukemia
cells can be found within the fat tissue of leukemic mice (26). We showed that both
adipocytes and adipose tissue explants confer protection from a range of
chemotherapies (26), though the mechanism(s) of this protection are still being
investigated.

L-Asparaginase: A front-line leukemia treatment
L-Asparaginase (ASNase) is a cornerstone of childhood ALL treatment (27), with
growing application in adult chemotherapy regimens (28). Research into the use of
ASNase as an anti-cancer drug began when researchers found that treatment with

4
guinea pig serum regressed lymphomas in mice and rats (29). It was later found that
guinea pig serum contained the enzyme l-asparaginase, which was responsible for the
cytotoxic effect (30). ASNase has been part of standard induction chemotherapy for ALL
since the 1980s and sensitivity to the drug in vitro has been shown to predict clinical
response (31). ASNase hydrolyzes the non-essential amino acids asparagine (ASN) and
glutamine (GLN) to aspartic acid and glutamic acid, respectively (32). In the United
States the most commonly utilized form of the enzyme, from E. coli, has a 100 times
greater substrate specificity for ASN compared to GLN (33). The efficacy of the drug
relies on the dependence of leukemia cells (and lymphoid cells in general) on these two
amino acids (32,34). It has been suggested that sensitivity to ASNase is due to low
expression of the asparagine producing enzyme asparagine synthetase (ASNS), making
them dependent on circulating ASN to fuel protein and DNA synthesis. However, ASNS
expression alone has not been consistently shown to be correlated with ASNase efficacy
(35–37), and many researchers have argued that other factors can influence sensitivity
and resistance to the drug. Aslanian et al. showed that when MOLT-4 human leukemia
cells are made resistant to ASNase, they not only upregulate ASNS but also increase
availability of its necessary substrate GLN through upregulation of the enzyme glutamine
synthetase (GS) as well as amino acid transporters (38). In fact, the importance of GLN
as a fuel source for nucleic acid synthesis and cell proliferation has been explored in
many types of cancer and there is ongoing research into targeting this dependence on
GLN metabolism to develop new treatment strategies (39,40).
In 2007, Shotaro Iwamoto demonstrated that mesenchymal stromal cells within the bone
marrow express ASNS and secrete asparagine (41). This production of asparagine was
found to be sufficient to protect both human leukemia cell lines as well as primary
leukemia cells from ASNase cytotoxicity in vitro. The clinical relevance of the

5
microenvironment's secretion of asparagine using an in vivo system has not yet been
explored. Interestingly murine primary adipocytes express both ASNS and GS and
differention of 3T3-L1 fibroblasts into adipocytes results in a ~100 fold increase in GS
protein levels (42). Furthermore, adipose tissue has been shown to contribute to the
whole body GLN pool to a similar degree as skeletal muscle (43,44). As adipose tissue
has been shown to produce glutamine into the interstitial fluid (45), and we have shown
obese youths can have 38% body fat (46), the implications of a role of adipocytes in
ASNase efficacy may be extremely clinically relevant.

Goals of this study
As adipose tissue is a major contributor to the whole body GLN pool (47), obesity may
impair the ability of ASNase to deplete plasma GLN. Moreover, it has been proposed
that non-malignant cells might support leukemia cells during ASNase treatment through
local secretion of amino acids (48), an idea that has been further explored more recently
(41,49–51). The focus of this dissertation is to study whether obesity and adipocytes
impair the efficacy of ASNase, and if so what the mechanism of this protection is and the
impact of these findings on current cancer treatment protocols.

6
Materials and methods
Human subjects
Bone marrow biopsy and blood samples were obtained from 19 patients, 10-18 years old
before and during treatment for high-risk leukemia. Obesity was defined as a BMI
greater than or equal to the 95
th
percentile per CDC guidelines. All patients were treated
per high risk CCG/COG protocol, involving a four-drug induction regimen including 4
weeks of steroids and PEG ASNase (25,000 IU/m
2
, single dose either intramuscularly or
IV). Samples were obtained after written informed consent and assent were obtained,
under a protocol approved by the CHLA Committee on Clinical Investigation (Institutional
Review Board).

Cell lines and culture
3T3-L1 cells (ATCC, Manassas, VA) were differentiated into adipocytes as previously
described (26), and used for experiments between days +7 and +14 of differentiation.
Undifferentiated 3T3-L1 fibroblasts were irradiated and plated at confluence. The bone
marrow derived mesenchymal cell line, OP9, was differentiated into adipocytes in a
similar manner.
Murine pre-B ALL cells were previously isolated from a BCR/ABL transgenic mouse
(“8093 cells”) (52). Human leukemia cell lines were obtained from ATCC and the
German Collection of Cell Lines (DSMZ), and included BV173 (Pre B Ph+ ALL), K562
(chronic myelogenous leukemia), Molt-4 (T cell leukemia), Nalm-6 (B cell precursor
leukemia), RCH-ACV (pre-B ALL with an E2A-PBX1 fusion protein), RS4;11 (pre-B

7
t(4;11) ALL), SD-1 (pre-B Ph+ ALL), SEM (B cell precursor), and SupB15 (B cell
precursor).
Primary human leukemia cells were passaged in NOD.Cg-Prkdc
scid
Il2rg
tm1Wjll
/SzJ mice
(Jackson Laboratories) and harvested from the spleens of these mice and cultured on
irradiated OP9 feeder layers for all experiments. These cells were kindly provided by
Markus Müschen, Yong-Mi Kim, and Nora Heisterkamp (53). These cells are hereafter
referred to as human leukemia cells. US7 and US7R were from a Ph-negative patient
before after the patient developed relapse. TXL-2 and BLQ-1 ALL cells were Ph-positive
and taken from patients at diagnosis.
Asparagine/Glutamine-free (AGF) media was prepared with DMEM and 10% dialyzed
FBS. FBS was dialyzed against 100 volumes of PBS three times, using 3kDa SnakeSkin
dialysis tubing (Thermo Fisher Scientific). HPLC analysis confirmed removal of all amino
acids.
To determine ASN or GLN dependence, cells were plated onto 96-well plates in AGF
media alone, with 2mM GLN, with 400uM ASN, or with both amino acids. After 72 hours,
cell growth was measured using resazurin (R&D Systems, MN). Experiments with
human leukemia cells were performed over OP9 feeder layers and cells counted by a
blinded observer using trypan blue after vigorous trituration to remove cells within and
below the feeder layers.
In vivo leukemia model
10,000 8093 cells were injected retro-orbitally into 46 diet-induced obese (DIO; raised on
60% of calories from fat diet) and 42 nonobese (raised on 10% of calories from fat)
C57Bl6/j mice (Jackson Laboratories). Seven to ten days after implantation, mice were
treated with ASNase or vehicle, proportional to body weight (3,000 IU/kg/day, 5

8
days/week via intraperitoneal injection x 3 weeks, Elspar, Ovation Pharmaceuticals).
Additional experiments were performed with pegylated ASNase (3,000 IU/kg/week x 3
weeks, Enzon Pharmaceuticals, NJ). Animals were euthanized upon development of
progressive leukemia (weight loss >10%, paralysis, hunched posture, or palpable mass
> 1cm). Additional transplanted and treated mice underwent cardiac perfusion with
heparinized saline under ketamine/xylazine anesthesia for analysis of tissue ASNS and
GS levels. Fat pads were collected, weighed, snap frozen in liquid nitrogen, and stored
at -80°C. All animal studies were approved by the Institutional Animal Care and Use
Committee.
Coculture experiments
Leukemia cells were cultured with fibroblasts, adipocytes, or no feeder layer. In
experiments of drug resistance, ASNase was added to achieve an approximate IC
50
.
After 72 hours, cells were counted as above. In additional experiments, 8093 cells were
cultured in 0.4 µm pore size TransWells (Corning, Inc., Lowell, MA) separated from the
feeder layers. To inhibit glutamine synthetase (GS), adipocytes were treated overnight
with 1.5 mM methionine sulfoximine (MSO), and washed three times before
experiments. Complete GS inhibition was confirmed by HPLC measurement of GLN
secretion. Erwinase investigational drug was kindly provided for experimental
evaluations by Dr. Paul Plourde (Jazz Pharmaceuticals, Langhorne, PA), and used at a
dose with equivalent asparagine-deamination activity, as determined by Nessler’s
reaction (54). 8093 cells in TransWells were analyzed for cell cycle and apoptosis after
48 hours in culture by BrdU incorporation (BD Biosciences, San Jose, CA) on a
FACScan (BD Biosciences, CellQuest software).

9
Amino acid analysis and sample preparation
To measure amino acid secretion, feeder layers were cultured in 24 well plates as
above, washed with PBS twice, then cultured in 1 mL per well of AGF media. Media was
collected, filtered through 0.45 μm syringe filters, and snap frozen. All samples were
stored at -80°C until assay.
Tissue explant amino acid production was measured using fat pads from perfused mice.
Fat was cut into approximately 50 mg pieces, washed thoroughly with PBS, and placed
in 24-well culture plates with 1mL AGF media for conditioning.
Blood was sampled from the submandibular plexus of unanesthetized mice into BD
EDTA-coated Microtainer tubes, cooled to 4°C to prevent ex vivo deamination, spun at
13,000 g, and then plasma was snap frozen.
Murine plasma and conditioned media amino acid measurements were performed as
previously described (55) with slight modifications. Samples were deproteinized using
20% 5-sulfosalicylic acid containing 1.0 mM L-Norleucine (internal standard, Sigma).
Samples were dried in a speedvac, resuspended with a derivatization reagent
(Methanol, TEA, H
2
0, and PITC at 7:1:1:1 ratios) and dried again. Samples were
measured using a Waters 1525 Binary HPLC pump and absorbance detected at 254nm.
Clinical plasma amino acid samples were measured in the clinical laboratory. Briefly,
samples were deproteinized with 5-sulfosalicylic acid followed by addition of N
G
-
Methylarginine. On-line derivatization was carried out using mixture solution of OPA (o-
phthaladehyde) and MPA (3-mercaptopropionic acid). After derivatization and
neutralization, 5 µl was injected to HPLC. Separation was performed on a Synergi 4U
Fusion RP80A C18 column (110 x 4.6 mM) with guard column (2 Fusion-RP 4.0 X 3.0

10
mm) (both from Phenomenex, Torrance, CA, USA) using a fluorescence detector by
their native fluorescence at λ
EX
: 340 nm, λ
EM
: 455 nm

Western blotting
Protein was extracted from leukemia cells, cultured adipocytes, and fat tissue from
perfused mice as previously described(26) Cell lysates were separated on Novex® Tris-
Glycine precast gels (Invitrogen) and transferred to 0.2 μm nitrocellulose membranes
(Invitrogen). Membranes were then incubated with a mouse anti-GS monoclonal
antibody (Abcam), a rabbit anti-ASNS polyclonal antibody (Abcam), or rabbit anti-
GAPDH antibody (Cell Signaling Technologies), with appropriate horseradish
peroxidase-conjugated secondary antibody from Cell Signaling Technologies.
Membranes were developed using the HyGLO HRP detection kit (Denville). To allow
inter-gel comparison of fat pad western blots, K562 cell lysates (positive for ASNS, GS,
and GAPDH) were run on all gels and used to correct for exposure time and run
variances. Band intensity was quantified using ImageJ software
(http://rsb.info.nih.gov/ij/).
Immunohistochemistry
Paraformaldehyde fixed bone marrow samples were embedded with paraffin, sliced, and
mounted by the CHLA Pathology Core. Sections were subjected to antigen retrieval with
Tris-EDTA, pH 8.0, steam for 30 min. Endogenous peroxidases were inactivated with
3% H
2
O
2
. Non-specific staining was blocked with 2.5% normal goat serum before
staining with rabbit anti-mouse GS or ASNS (Abcam, Cambridge, MA), and detected
with the ImmPRESS reagent (Vector Laboratories Inc., Burlingame, CA) containing

11
polymerized peroxidase labeled goat anti-rabbit immunoglobulin (mouse adsorbed). The
reaction was detected with ImmPACT DAB (Vector Laboratories Inc.) and
counterstained with Mayer’s hematoxylin. Images were acquired on a Zeiss Axioplan
Microscope (40x/1.3) with a SPOT QE Color Digital Camera.

Statistical analysis
Body weights were compared with unpaired, two-sided t tests. Survival curves were
generated by Kaplan Meier Life Tables, and compared using Cox Proportional Hazards.
Each coculture experiment was performed on different days or using different cell thaws,
and the averages of three triplicate wells for each condition in each experiment were
calculated. Paired t tests were used to compare number of viable leukemia cells over the
various feeder layers. A p value of less than 0.05 was considered significant.

12
Results
Adipocytes in the leukemia microenvironment produce
glutamine
We and others have previously found that obesity worsens treatment outcome in
adolescents with high-risk ALL (8,56). To test whether obesity might impair ASNase
efficacy, we measured plasma levels of amino acids in adolescents before and after
induction chemotherapy for high-risk ALL, which included a single dose of PEG-ASNase.
There were no significant differences in amino acid levels between obese and lean
subjects, with ASN being fully suppressed by ASNase, and GLN largely unaffected in
both groups (Figure 1A).
Since plasma amino acid levels might not reflect conditions in the leukemia
microenvironment, we examined bone marrow biopsy specimens from four obese and
four lean adolescent leukemia patients for expression of ASN synthetase (ASNS) and
GLN synthetase (GS), the rate limiting steps for ASN and GLN production. Cells positive
for ASNS were found throughout the marrow, and expression appeared unaltered after
treatment (Figure 1B). Prior to treatment, GS expression was low and appeared to be
localized in scattered adipocytes. After treatment, there was a large increase in the area
occupied by adipocytes, as has been previously shown (57), together with an apparent
increase of GS in these cells.

13

Figure 1. Effect of obesity on plasma and bone marrow asparagine and glutamine in patients
following L-Asparaginase treatment.
(A) Plasma amino acid measurements of ASN (left) and GLN (right) in lean and obese patients during
induction chemotherapy for newly diagnosed high-risk ALL, treated on CCG1961, including a single
dose of 2,500 IU/m2 of pegylated L-asparaginase. (B) ASN synthetase (ASNS, left) and GLN
synthetase (GS, right) staining of bone marrow taken from four lean (Pt1-4) and four obese (Pt5-8)
children before and after induction chemotherapy. Images were acquired on a Zeiss Axioplan
Microscope (40x/1.3) with a SPOT QE Color Digital Camera. Calibration bar (upper right image) is
50µm

14
Obesity impairs L-Asparaginase efficacy in mice
To test whether obesity per se can cause ASNase resistance, we implanted diet-induced
obese (DIO, 41.5±4.4 g) and non-obese (30.4±2.0 g, p<0.001) male mice with syngeneic
leukemia cells at 20±2 weeks of age (26). There was no difference in survival between
vehicle treated non-obese and obese mice (28±4.5 vs 26±4.2, Figure 2A). ASNase,
administered proportional to body weight, prolonged survival in non-obese mice over
vehicle (33.4±12.0 vs 26.6±5.6 days, p<0.01), but yielded no detectible survival benefit
to obese mice (26.4±7.5 days, p<0.0001 vs. non-obese, p=n.s. vs. vehicle, Figure 2B).
Obesity similarly decreased survival after treatment with the more stable pegylated form
of ASNase (p<0.05 obese vs. non-obese, Figure 2C).
0 20 40 60
0%
50%
100%
0 20 40 60 80
0%
50%
100%
B
Days Post Transplant
% Survival
Obese
Nonobese
Vehicle
Native Pegylated
A
C
Vehicle
0 20 40 60
0%
50%
100%

Figure 2. Diet induced obesity impairs L-Asparaginase treatment in leukemic mice.
(A) Survival of mice transplanted with 8093 leukemia cells and treated with saline. Solid line, obese
mice (n=13); dashed black line, nonobese mice (n=6). (B) Survival of transplanted mice treated with
ASNase. Solid line, obese mice (n=28); dashed black line, nonobese mice (n=31), gray dotted line,
vehicle-treated mice (n=10). Gray bar shows treatment period. p<0.001 obese vs. nonobese. (B)
Survival of transplanted mice treated with pegylated L-Asparaginase (n=5), p<0.01 obese vs.
nonobese

Plasma amino acid levels showed a similar pattern to that of humans, with no
differences between diet groups (Figure 3A). Nor was there any significant difference
between plasma ASNase activity following a single dose of E. coli ASNase between diet
groups, though obese mice tended to have higher levels than nonobese mice (Figure

15
3B). Thus, similar to humans, obese mice exhibited impaired leukemia outcome with no
significant differences in plasma ASN or GLN.

Pre .25 .5 1 5 7
0
20
40
60
80
100
A
uM Amino A cid
Asparagine
Glutamine
Pre .25 .5 1 5 7
0
400
800
1,200
Nonobese
Obese
0 2 4 6
0
20
40
60
80
100
Asparaginase Activity
(IU/mL)
Timepoint (hours)
B
Days after treatment
Obese
Non-obese

Figure 3. Systemic markers of ASNase efficacy are not different in obese and lean mice
(A) Plasma ASN and GLN concentrations in leukemic obese or nonobese mice prior to and after
treatment with ASNase at above dose. (B) Plasma asparaginase activity in leukemic obese and
control mice following a single dose of E. coli L-asparagianse at 3,000 IU/kg

Unlike in humans, we did not observe a change in bone marrow GS expression in mice
treated with ASNase (Figure 4A). Likewise, although GS was dramatically higher in 3T3-
L1 adipocytes than in undifferentiated 3T3-L1 cells, as has been previously shown (42),
expression of ASNS and GS appeared to decrease following 72 hours of culture in ASN
and GLN depleted (AGF) media (Figure 4B). We therefore considered whether the
increase in GS found in human samples could be caused by another chemotherapy
given during induction. Indeed, dexamethasone increased 3T3-L1 adipocyte GS levels
~2 fold, as has been shown in other studies (58).
Since we have shown that ALL cells infiltrate adipose tissue during treatment (26), we
next investigated GS expression in adipose tissue. Mouse adipose tissue expressed
detectible GS, but not ASNS, by western blot analysis (Figure 4C). Furthermore, fat
tissue explants from wild-type C57 mice secreted GLN (105.69±53.00 nmol/100 mg

16
tissue/24 hours) but not ASN, into the media (Table 1). Dosing obese mice with ASNase
daily for five days resulted in a ~50% increase in GS expression in subcutaneous fat
(Figure 4C), but no overall effect in other fat pads (Figure 4D). We observed no
significant differences between GS expression in fat pads between obese and lean mice
(Figure 4D). ASNase dosing also did not lead to detectible ASNS protein expression in
fat pads (not shown).

Figure 4. Changes in Glutamine Synthetase in vivo and in vitro
(A) Representative images of bone marrow glutamine synthetase (GS) protein in control and obese
leukemic mice prior to treatment and following five days of ASNase treatment. Marrow was stained
with rabbit anti mouse GS, with a secondary HRP-goat anti-rabbit IgG (B) Representative western
blot of irradiated 3T3-L1 fibroblasts (lane 1) and 3T3-L1 adipocytes (lanes 2-4). Adipocytes were
collected without additional treatment (lane 2) or after 72 hours of exposure to ASN/GLN-free media
(lane 3), 1 IU/mL ASNase (lane 4), or 125nM dexamethasone (lane 5). (C) Western blot of ASNS and
GS levels in adipose tissue taken from obese leukemic mice prior to (pre) and 5 days after (post)
treatment with L-asparaginase. (D) Analysis of western blots of mouse subcutaneous, visceral,
omental and epididymal fat pads for glutamine synthetase. Fat was collected from obese and lean
leukemic mice prior to asparaginase treatment (Pre) or following five days of treatment at 3,000IU/kg
bodyweight (Post).

In vitro, 3T3-L1 adipocytes secreted a small amount of ASN. Supplementing the media
with the required substrates for ASN synthesis, aspartic acid and GLN, along with the
GLN precursor glutamic acid, increased ASN secretion by adipocytes (Table 1).

17
Adipocytes secreted a substantial amount of GLN, ~18 fold more than undifferentiated
3T3-L1 cells.

Cell Type ASN, nmol/mL GLN, nmol/mL
3T3-L1 Fibro <0.005 23±27
3T3-L1 Adipo (AGF) 23±13 417±176
3T3-L1 Adipo (+ASP, GLU, GLN)
a
87±15 -
3T3-L1 Adipo (MSO Treated) 1.6±2.6 56±50
Fat Explant (100mg) <0.005 247±43
Table 1. ASN and GLN secreted by cells over 72 hours.
a
400uM aspartic acid, 400uM glutamic acid, and 2000uM glutamine supplemented

Adipocytes protect leukemia cells from ASNase via GLN
production
To determine whether adipocytes could protect ALL cells from ASNase, we cultured
8093 murine ALL cells over irradiated 3T3-L1 fibroblast-like cells or differentiated 3T3-L1
adipocytes, in media with 1 IU/mL ASNase. Neither fibroblast nor adipocyte feeder
layers affected growth of leukemia cells in the absence of drug (Figure 5A). 3T3-L1
adipocytes protected ALL cells from ASNase both with and without direct contact (Figure
5B). The similar pattern was observed with adipocytes differentiated from OP9 bone
marrow mesenchymal cells (Figure 5C). Adipocyte protection was associated with
increased cell cycling during ASNase exposure (Figure 5D).

18

Direct Coculture Transwell Separated
0
2.0×10
5
4.0×10
5
6.0×10
5
*
*
F
A
N
*
*
0
10
20
30
40
50
BrdU incorporation (%)
*
*
* *
No Drug ASNase
A
C
D
Viable Cells
N F A
0
1.0×10
5
2.0×10
5
3.0×10
5
4.0×10
5 *
*
Viable Cells
B
N F A
0
5.0×10
5
1.0×10
6
1.5×10
6

Figure 5. Adipocytes protect leukemia from ASNase in vitro
(A) 8093 leukemia cells were cultured for 72 hours in complete media (n=5) with 3T3-L1 fibroblasts
(hatched bars) or adipocytes (solid bars), compared to culture alone (gray bars). Dashed line
indicates initial number of cells plated. (B) 8093 leukemia cells cultured for 72 hours in media
containing 1 IU/mL ASNase in direct (left, n=5) or TransWell separated (right, n=6) co-culture with
feeder layers. (B) 8093 cells plated as above with bone marrow-derived OP9 fibroblasts or
adipocytes as feeder layer (n=4). (C) BrdU incorporation was measured in 8093 cells by flow
cytometry after 48 hours of co-culture in TransWells over various feeder layers, with or without
ASNase treatment (n=4). *, P < 0.05 compared to No Drug, paired t test

Glutamine secretion by adipocytes protects from L-
Asparaginase
Since adipocytes produce both ASN and GLN, we next tested whether either of these
amino acids were responsible for adipocyte protection of ALL cells from ASNase. Twice
daily addition of ASN had no effect on ASNase cytotoxicity (Figure 6A), whereas GLN
supplementation partially blocked ASNase cytotoxicity (Figure 6B). Treating adipocytes

19
with dexamethasone to increase glutamine production tended to increase protection of
leukemia cells (Figure 6C).

No Drug 0 5 10 40 400
0
2.0×10
4
5.0×10
5
1.0×10
6
1.5×10
6
2.0×10
6
2.5×10
6
No Drug 0 50 100 500
0
4.0×10
5
8.0×10
5
2.0×10
6
3.0×10
6
*
A C B
Viable Cells
Asparagine (nmol) Glutamine (nmol)
N A Dex-A
0
2.0×10
5
4.0×10
5
6.0×10
5
8.0×10
5
1.0×10
6
*
*
*

Figure 6. Adipocytes protection depends on glutamine production.
(A+B) 8093 cells were cultured for 72 hours with 1 IU/mL ASNase, with ASN (A, n=3) or GLN (B, n=4)
added every 12 hours. * P < 0.05 relative to no addition. (C) 8093 cells plated over no feeder (N), 3T3-
L1 adipocytes (A), or Dexamethasone treated adipocytes (A-Dex) in the presence of 1 IU/mL ASNase.

Conversely, inhibition of adipocyte GS using MSO rendered adipocytes unable to protect
ALL cells from ASNase (Figure 7A). Similarly, use of Erwinase, a form of asparaginase
with five-fold greater glutaminase activity than E. coli ASNase (33), was able to inhibit
the protective effect of adipocytes (Figure 7B).

20
N A A+MSO
0
5.0×10
4
1.0×10
5
1.5×10
5
2.0×10
5
2.5×10
5
** *
A B
Viable Cells
ASNase ERWinase
0
5.0×10
4
1.0×10
5
1.5×10
5
2.0×10
5
2.5×10
5
3.0×10
5
*
*
*

Figure 7. Impaired GLN secretion by adipocytes reverses protection from ASNase
(A) 8093 cells plated over no feeder (N), 3T3-L1 adipocytes (A), or MSO treated adipocytes (A-MSO)
in the presence of 1 IU/mL ASNase. (B) 8093 leukemia cells cultured for 72 hours in 1 IU/mL ASNase
or Erwinase over no feeder layer (gray bars), 3T3-L1 fibroblasts (hatched bars), or adipocytes (solid
bars). n=3; *, P < 0.05 compared to No Drug, paired t test

We next evaluated the sensitivity of leukemia cells to asparagine and glutamine
starvation by culturing ten leukemia cell lines in media lacking ASN, GLN, or both
(Figure 8A). Only 3 of 10 human leukemia cell lines were sensitive to removal of ASN
from the media, while 8 of 10 were sensitive to GLN removal. All lines tested were most
sensitive to removal of both amino acids. In similar tests with four human leukemia cells
(BLQ1, Txl2, US7, US7R) (53), one line was sensitive to ASN removal, three were
sensitive to GLN removal, and all four were sensitive to removal of both amino acids
(Figure 8B). Sensitivity to either amino acid could not be explained by ASNS or GS
expression (Figure 8C) (35).

21

Figure 8. Leukemia cells show greater dependence on glutamine than asparagine.
(A) Proliferation of one murine and nine human leukemia cell lines when cultured in asparagine-free
media (white bars), glutamine-free media (horizontally lined bars), or media lacking both amino acids
(hatched black bars) compared to complete media (vertically lined bars, n=4). (B) Proliferation of four
human leukemia cells in direct coculture with OP9 feeder layers in media as in (A) (n=4). (C)
Representative western blot of ASNS and GS protein expression prior to and after an EC 50 dose of
ASNase in four human cell lines. *, P < 0.05

Since most human leukemia cell lines were more sensitive to GLN depletion than ASN
depletion, we tested whether Erwinase would prove more cytotoxic to these cells than
ASNase. Four of 6 human cell lines tested showed greater cell death when Erwinase
was used than ASNase (Figure 9A). Adipocytes also protected human leukemia cell
lines from death due to lacking ASN and GLN (AGF media, Figure 10B), and we
hypothesized that Erwinase treatment would be more effective than ASNase at reversing
the protection. Indeed, in 3 of 4 human leukemia cell lines we found that Erwinase
significantly reduced adipocyte protection of leukemia cells from ASN/GLN starvation

22
compared to ASNase treatment (Figure 9B).
SD-1
0
50,000
100,000
150,000
200,000
250,000
*
*
*
p=0.06
Cell Count
RS4;11
0
200,000
400,000
600,000
800,000
*
Cell Count
HL60
0
100,000
200,000
300,000
400,000
500,000
*
*
*
Cell Count
Molt-4
0
250,000
500,000
750,000
1,000,000
1,250,000
*
*
*
Cell Count
AGF ASNase ERW inase
RS4;11 SD-1 SupB15 Molt4 HL60
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
*
p=0.07 p=0.07
Cell Count
RCH-ACV
0
500,000
1,000,000
1,500,000
2,000,000
*
n=5
AGF ASNase ERW inase
A
B

Figure 9. Erwinase is more effective than ASNase in killing leukemia cells.
(A) Viability of six human cell lines exposed to either ASNase (white bar) or Erwinase (black bar) at a
dose of 0.3 IU/mL for 72 hours. (B) Viability of human leukemia cell lines 72 hours after culture in
media lacking both ASN and GLN in Transwells over adipocytes (black columns), or no feeder (gray
columns; n=4), with no drug, 0.3IU/mL ASNase, or 0.3IU/mL Erwinase. Dashed line indicates initial
number of cells plated. *, P < 0.05

23
Discussion
Although obesity has been recognized as a major factor in leukemia progression and
relapse, the precise mechanism(s) by which obesity impairs treatment outcome remains
unclear. In order to elucidate the role of obesity in leukemia treatment, we have
investigated the use of the front-line chemotherapy L-Asparaginase which, despite its
use clinically for over 50 years, is still being studied to determine ideal treatment
strategies. Several studies have shown improved patient outcome with more intense or
longer treatment with ASNase (59,60), while insufficient drug exposure, as in the case of
silent hypersensitivity, is associated with higher risk of relapse (61). ASNase cytotoxicity
relies on its ability to deplete ASN and GLN from plasma. This effectively starves
lymphoid cells, which unlike most other cells are unable to sustain themselves through
de novo production (32). Effective use of ASNase has traditionally been measured by
the depletion of plasma ASN and GLN, or its surrogate, plasma asparaginase activity
(62).
In our murine leukemia model, ASNase treatment was less effective in obese mice than
nonobese mice. Notably, there was no significant difference in plasma amino acid levels
between obese and nonobese mice at any timepoint, despite the dramatic difference in
survival. Regardless of diet group, plasma ASN remained suppressed, while GLN began
to recover within 12 hours. Similarly, in patient samples, GLN did not appear
suppressed, though early timepoints were not sampled in this study. Although a rebound
effect was found in other studies (63), its mechanism is unknown and may be the result
of an increase in endogenous GLN synthesis or release of tissue GLN during cachexia.
We documented a dramatic increase in GS in bone marrow collected from ALL patients
at the end of induction, but not in adipose tissue or bone marrow following ASNase

24
treatment in mice. While it is possible that this difference results from species-specific
response to ASN and/or GLN depletion in the plasma, it is more likely that it results from
the use of combination chemotherapy in human ALL. In particular, glucocorticoids have
been shown to induce GS in some tissues (58), and we found that dexamethasone
treatment induced an increase in GS protein levels in 3T3-L1 adipocytes.
GLN is the most abundant amino acid in plasma, and necessary for nucleotide and
amino acid synthesis. Although a nonessential amino acid, a variety of human cancer
cell lines, including pancreatic cancer, colon cancer, small cell lung cancer, and
leukemia have been shown to be highly dependent on GLN for proliferation and survival
(39). GLN depletion can induce apoptosis in cancer cells, with a higher selectivity than
glucose depletion (64). In addition, leukemia cells adapt to ASNase treatment by
increasing synthesis and transport of GLN, and inhibition of GS has been shown to re-
sensitize resistant leukemia lines to ASNase (38). These studies highlight the possibility
of targeting GLN metabolism to combat ASNase resistance. However, studies aimed at
systemic inhibition of GLN metabolism have been limited due to toxicity (65,66).
Using our in vitro system we found that adipocytes protect leukemia cells both from L-
asparaginase and from ASN/GLN starvation, primarily through secretion of GLN. As
adipose tissue secretes significant amounts of GLN into interstitial fluid (44), and, as we
have previously shown, leukemia cells infiltrate adipose tissue (19,26), it is possible that
adipose tissue is a sanctuary where ALL cells are protected from ASNase activity. This
may also happen in the bone marrow after initiation of chemotherapy, when the number
of adipocytes and their expression of GS both increase dramatically. As obese patients
have large amounts of body fat, it might increase the probability that the adipocyte-
mediated protection from ASNase may become clinically relevant, and be one of the
factors leading to increased relapse rate in obese ALL patients.

25
Our results complement the recent finding that bone marrow-derived mesenchymal cells
(MSCs) protect leukemia from ASNase treatment through ASN secretion (41,50,51).
Laranjeira et al. showed that leukemia-cell secretion of IGFBP-7 increased ASN
synthesis by stromal cells (51). Interestingly, this effect was further increased by the
addition of insulin and IGF-1, both of which are elevated in obesity (67).
Two recent publications have questioned the role of bone marrow MSCs as a source of
ASN during ASNase treatment in patients (68,69). These papers found that upon
treatment, the extent of depletion of ASN and GLN were similar in the bone marrow and
the peripheral blood. Interestingly both studies showed higher aspartic acid
concentrations in the marrow than in the peripheral blood, possibly indicating either high
rates of de novo aspartic acid production or a greater turnover of ASN in the
microenvironment. Further investigation into the extent to which known sanctuary sites
may counteract the depletion of ASN and GLN from blood should be performed. In
particular, it is possible that tissues with poor capillarization, such as adipose tissue in
obese patients (70), may provide an environment more removed from ASNase
treatment.
Several groups have been developing alternative ASNase preparations with lower
glutaminase activity (71–73). The goal is minimizing the side effects associated with
GLN starvation such as immunosuppression and pancreatitis. One study looked into the
possibility of supplementing the diet of rats treated with ASNase with alanyl-glutamine to
counteract the immunosuppressive effects of GLN depletion (63). In our study 8 out of 9
commercial cell lines, and 4 out of 4 human leukemia cells, were more sensitive to GLN
than ASN starvation and, in nearly all cases, depletion of both amino acids had a
stronger effect than either amino acid individually. This dependence on glutamine was
reflected in the greater sensitivity several human leukemia cells had to Erwinase, a form

26
of l-asparaginase with higher glutaminase activity commonly used in cases of allergy to
E. coli l-asparaginase. These results are in line with a study performed by Offman (73),
who showed that, in their recombinant ASNase, some cell lines responded better to wild
type ASNase than an asparaginase with decreased glutaminase activity. Additionally, we
have shown here that Erwinase was able to impair the ability of adipocytes to protect
leukemia cells in vitro. These findings suggest that strategies to develop alternative
ASNase preparations with lower glutaminase activity may in fact be detrimental to the
cytotoxicity of ASNase and should be done with caution.
Conclusions and Future Studies
The findings here present several avenues for investigation to improve current leukemia
treatment. Our findings highlight that new treatment regimens utilizing ASNase
preparations should not only focus on the suppression of plasma ASN and GLN levels,
but also on the effectiveness of the drug on the cancer microenvironment. Adipose
tissue may have a key role to maintain a leukemia cell population during ASNase
treatment. Given the rising prevalence of obesity worldwide, pharmacological strategies
aimed to modulate adipocyte effects on malignant cells might become important in
cancer treatment.
In addition, our finding that leukemia cells showed a great deal of variability in their
dependence to asparagine and glutamine opens the door for research to improve
personalized treatment regimens. In the United States, the high-glutaminase enzyme
Erwinase is only used in cases of allergy to L-asparaginase. Our results suggest that
patients with leukemia cells that are highly dependent on exogenous glutamine may be
treated more effectively through the use of a high-glutaminase preparation such as
Erwinase. Conversely, given the side effects associated with glutamine depletion a low-
glutaminase preparation may prove a safer option for patients whose leukemia cells are

27
sensitive exclusively to asparagine starvation. Future in vivo studies should compare the
efficacy of ASNase compared to Erwinase in mice with leukemia cells of varying degrees
of glutamine dependence.
Our observation that dexamethasone increases both glutamine synthesis by adipocytes
and protection from L-asparaginase presents an intriguing possibility for altering
chemotherapy. Common childhood leukemia treatment protocols include 3-5 days of
glucocorticoid treatment prior to the start of L-asparaginase. These results suggest that
such a protocol may in fact be detrimental as glucocorticoids increase GS within the
non-malignant cells that make up the leukemia microenvironment. The potential for
glucocorticoid induction of GS to impair L-asparaginase efficacy, particularly in the
context of the tumor microenvironment, may be the target of future studies.

28
References

1. Menifield CE, Doty N, Fletcher A. Obesity in America. ABNF J. 2008;19:83–8.
2. Ogden CL, Carroll MD, Kit BK, Flegal KM. Prevalence of Obesity and Trends in
Body Mass Index Among US Children and Adolescents, 1999-2010. JAMA: The
Journal of the American Medical Association [Internet]. 2012 [cited 2012 Jan 30];
Available from: http://jama.ama-
assn.org/content/early/2012/01/11/jama.2012.40.abstract
3. Finkelstein EA, Khavjou OA, Thompson H, Trogdon JG, Pan L, Sherry B, et al.
Obesity and Severe Obesity Forecasts Through 2030. American Journal of
Preventive Medicine. 2012;42:563–70.
4. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic
burden of the projected obesity trends in the USA and the UK. The Lancet.
378:815–25.
5. Mokdad AH, Marks JS, Stroup DF, Gerberding JL. Actual causes of death in the
United States, 2000. JAMA. 2004;291:1238–45.
6. Polednak AP. Estimating the number of U.S. incident cancers attributable to obesity
and the impact on temporal trends in incidence rates for obesity-related cancers.
Cancer Detect. Prev. 2008;32:190–9.
7. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and
mortality from cancer in a prospectively studied cohort of U.S. adults. N. Engl. J.
Med. 2003;348:1625–38.
8. Butturini AM, Dorey FJ, Lange BJ, Henry DW, Gaynon PS, Fu C, et al. Obesity and
Outcome in Pediatric Acute Lymphoblastic Leukemia. JCO. 2007;25:2063–9.
9. Gaynon PS, Qu RP, Chappell RJ, Willoughby ML, Tubergen DG, Steinherz PG, et
al. Survival after relapse in childhood acute lymphoblastic leukemia: impact of site
and time to first relapse--the Children’s Cancer Group Experience. Cancer.
1998;82:1387–95.
10. Hursting SD, Margolin BH, Switzer BR. Diet and Human Leukemia: An Analysis of
International Data. Preventive Medicine. 1993;22:409–22.
11. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: A Cancer Journal for
Clinicians. 2013;63:11–30.
12. Mathers C, Boschi-Pinto C, Lopez A, Murray C. Cancer incidence, mortality and
survival by site for 14 regions of the world. [Internet]. World Health Organization;
2001 [cited 2013 Jun 6]. Available from: http://www.who.int/healthinfo/paper13.pdf

29
13. Paget S. THE DISTRIBUTION OF SECONDARY GROWTHS IN CANCER OF THE
BREAST. The Lancet. 1889;133:571–3.
14. Witz IP, Levy-Nissenbaum O. The tumor microenvironment in the post-PAGET era.
Cancer Letters. 2006;242:1–10.
15. Manabe A, Coustan-Smith E, Behm FG, Raimondi SC, Campana D. Bone marrow-
derived stromal cells prevent apoptotic cell death in B-lineage acute lymphoblastic
leukemia. Blood. 1992;79:2370–7.
16. Vianello F, Villanova F, Tisato V, Lymperi S, Ho K-K, Gomes AR, et al. Bone marrow
mesenchymal stromal cells non-selectively protect chronic myeloid leukemia cells
from imatinib-induced apoptosis via the CXCR4/CXCL12 axis. Haematologica.
2010;95:1081–9.
17. Jin L, Tabe Y, Konoplev S, Xu Y, Leysath CE, Lu H, et al. CXCR4 up-regulation by
imatinib induces chronic myelogenous leukemia (CML) cell migration to bone
marrow stroma and promotes survival of quiescent CML cells. Molecular Cancer
Therapeutics. 2008;7:48–58.
18. Fei F, Stoddart S, Muschen M, Kim Y, Groffen J, Heisterkamp N. Development of
resistance to dasatinib in Bcr/Abl-positive acute lymphoblastic leukemia. Leukemia
[Internet]. 2010 [cited 2010 Mar 4]; Available from:
http://dx.doi.org/10.1038/leu.2009.302
19. Pramanik R, Sheng X, Ichihara B, Heisterkamp N, Mittelman SD. Adipose tissue
attracts and protects acute lymphoblastic leukemia cells from chemotherapy.
Leukemia Research. 2013;37:503–9.
20. Somasundar P, Yu AK, Vona-Davis L, McFadden DW. Differential effects of leptin on
cancer in vitro. J. Surg. Res. 2003;113:50–5.
21. Barb D, Neuwirth A, Mantzoros CS, Balk SP. Adiponectin signals in prostate cancer
cells through Akt to activate the mammalian target of rapamycin pathway. Endocr.
Relat. Cancer. 2007;14:995–1005.
22. Pamuk GE, Demir M, Harmandar F, Yesil Y, Turgut B, Vural O. Leptin and resistin
levels in serum of patients with hematologic malignancies: correlation with clinical
characteristics. Exp. Oncol. 2006;28:241–4.
23. Yang G, Lu G, Jin F, Dai Q, Best R, Shu XO, et al. Population-based, case-control
study of blood C-peptide level and breast cancer risk. Cancer Epidemiol.
Biomarkers Prev. 2001;10:1207–11.
24. Shimon I, Shpilberg O. The insulin-like growth factor system in regulation of normal
and malignant hematopoiesis. Leuk. Res. 1995;19:233–40.
25. Halberg N, Wernstedt-Asterholm I, Scherer PE. The adipocyte as an endocrine cell.
Endocrinol. Metab. Clin. North Am. 2008;37:753–768, x–xi.

30
26. Behan JW, Yun JP, Proektor MP, Ehsanipour EA, Arutyunyan A, Moses AS, et al.
Adipocytes Impair Leukemia Treatment in Mice. Cancer Research. 2009;69:7867 –
7874.
27. Avramis VI, Tiwari PN. Asparaginase (native ASNase or pegylated ASNase) in the
treatment of acute lymphoblastic leukemia. Int J Nanomedicine. 2006;1:241–54.
28. Patil S, Coutsouvelis J, Spencer A. Asparaginase in the management of adult acute
lymphoblastic leukaemia: is it used appropriately? Cancer Treat. Rev.
2011;37:202–7.
29. Kidd JG. REGRESSION OF TRANSPLANTED LYMPHOMAS INDUCED IN VIVO
BY MEANS OF NORMAL GUINEA PIG SERUM. J Exp Med. 1953;98:565–82.
30. Broome JD. EVIDENCE THAT THE L-ASPARAGINASE OF GUINEA PIG SERUM
IS RESPONSIBLE FOR ITS ANTILYMPHOMA EFFECTS. J Exp Med.
1963;118:99–120.
31. Kaspers GJL, Veerman AJP, Pieters R, Van Zantwijk CH, Smets LA, Van Wering
ER, et al. In Vitro Cellular Drug Resistance and Prognosis in Newly Diagnosed
Childhood Acute Lymphoblastic Leukemia. Blood. 1997;90:2723–9.
32. Oettgen HF, Old LJ, Boyse EA, Campbell HA, Philips FS, Clarkson BD, et al.
Inhibition of leukemias in man by L-asparaginase. Cancer Res. 1967;27:2619–31.
33. Müller HJ, Boos J. Use of L-asparaginase in childhood ALL. Crit. Rev. Oncol.
Hematol. 1998;28:97–113.
34. Kitoh T, Kubota M, Takimoto T, Hashimoto H, Shimizu T, Sano H, et al. Metabolic
basis for differential glutamine requirements of human leukemia cell lines. Journal
of Cellular Physiology. 1990;143:150–3.
35. Stams WAG, Boer D, L M, Beverloo HB, Meijerink JPP, Stigter RL, et al. Sensitivity
to L-Asparaginase Is Not Associated with Expression Levels of Asparagine
Synthetase in T(12;21)+pediatric ALL. Blood. 2003;101:2743–7.
36. Appel IM, den Boer ML, Meijerink JPP, Veerman AJP, Reniers NCM, Pieters R. Up-
regulation of asparagine synthetase expression is not linked to the clinical response
L-asparaginase in pediatric acute lymphoblastic leukemia. Blood. 2006;107:4244–
9.
37. Stams WAG, den Boer ML, Holleman A, Appel IM, Beverloo HB, van Wering ER, et
al. Asparagine synthetase expression is linked with L-asparaginase resistance in
TEL-AML1-negative but not TEL-AML1-positive pediatric acute lymphoblastic
leukemia. Blood. 2005;105:4223–5.
38. Aslanian AM, Kilberg MS. Multiple adaptive mechanisms affect asparagine
synthetase substrate availability in asparaginase-resistant MOLT-4 human
leukaemia cells. Biochem J. 2001;358:59–67.

31
39. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in cancer.
Trends in Biochemical Sciences. 2010;35:427–33.
40. Gaurav K, Goel RK, Shukla M, Pandey M. Glutamine: A novel approach to
chemotherapy-induced toxicity. Indian J Med Paediatr Oncol. 2012;33:13–20.
41. Iwamoto S, Mihara K, Downing JR, Pui C-H, Campana D. Mesenchymal cells
regulate the response of acute lymphoblastic leukemia cells to asparaginase.
Journal of Clinical Investigation. 2007;117:1049–57.
42. Miller RE, Hackenberg R, Gershman H. Regulation of glutamine synthetase in
cultured 3T3-L1 cells by insulin, hydrocortisone, and dibutyryl cyclic AMP. Proc Natl
Acad Sci U S A. 1978;75:1418–22.
43. Frayn KN, Karpe F, Fielding BA, Macdonald IA, Coppack SW. Integrative physiology
of human adipose tissue. Int J Obes Relat Metab Disord. 27:875–88.
44. Patterson BW, Horowitz JF, Wu G, Watford M, Coppack SW, Klein S. Regional
muscle and adipose tissue amino acid metabolism in lean and obese women. Am J
Physiol Endocrinol Metab. 2002;282:E931–936.
45. Maggs DG, Jacob R, Rife F, Lange R, Leone P, During MJ, et al. Interstitial fluid
concentrations of glycerol, glucose, and amino acids in human quadricep muscle
and adipose tissue. Evidence for significant lipolysis in skeletal muscle. J Clin
Invest. 1995;96:370–7.
46. Mittelman SD, Klier K, Braun S, Azen C, Geffner ME, Buchanan TA. Obese
Adolescents Show Impaired Meal Responses of the Appetite-Regulating Hormones
Ghrelin and PYY. Obesity (Silver Spring). 2010;18:918–25.
47. Frayn KN, Khan K, Coppack SW, Elia M. Amino acid metabolism in human
subcutaneous adipose tissue in vivo. Clin. Sci. 1991;80:471–4.
48. Cooney DA, Capizzi RL, Handschumacher RE. Evaluation of L-asparagine
metabolism in animals and man. Cancer Res. 1970;30:929–35.
49. Ding Y, Li Z, Broome JD. Epigenetic changes in the repression and induction of
asparagine synthetase in human leukemic cell lines. Leukemia. 2005;19:420–6.
50. Fung K-L, Liang RH-S, Chan GC-F. Vincristine but not imatinib could suppress
mesenchymal niche’s support to lymphoid leukemic cells. Leuk. Lymphoma.
2010;51:515–22.
51. Laranjeira ABA, de Vasconcellos JF, Sodek L, Spago MC, Fornazim MC, Tone LG,
et al. IGFBP7 participates in the reciprocal interaction between acute lymphoblastic
leukemia and BM stromal cells and in leukemia resistance to asparaginase.
Leukemia. 2012;26:1001–11.
52. Heisterkamp N, Jenster G, ten Hoeve J, Zovich D, Pattengale PK, Groffen J. Acute
leukaemia in bcr/abl transgenic mice. Nature. 1990;344:251–3.

32
53. Fei F, Stoddart S, Groffen J, Heisterkamp N. Activity of the Aurora Kinase Inhibitor
VX-680 against Bcr/Abl-Positive Acute Lymphoblastic Leukemias. Molecular
Cancer Therapeutics. 2010;9:1318–27.
54. Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I, et al. Role of
Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-
Asparaginase. Journal of Biological Chemistry. 2006;281:31222–33.
55. Sherwood RA. Amino acid measurement in body fluids using PITC derivatives.
Methods Mol. Biol. 2000;159:169–75.
56. Gelelete CB, Pereira SH, Azevedo AMB, Thiago LS, Mundim M, Land MGP, et al.
Overweight as a Prognostic Factor in Children With Acute Lymphoblastic
Leukemia. Obesity. 2011;19:1908–11.
57. Georgiou KR, Scherer MA, Fan C, Cool JC, King TJ, Foster BK, et al. Methotrexate
chemotherapy reduces osteogenesis but increases adipogenic potential in the bone
marrow. Journal of Cellular Physiology. 2011;227:909–18.
58. Wang Y, Watford M. Glutamine, insulin and glucocorticoids regulate glutamine
synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1
adipocytes. Biochim Biophys Acta. 2007;1770:594–600.
59. Abshire TC, Pollock BH, Billett AL, Bradley P, Buchanan GR. Weekly polyethylene
glycol conjugated L-asparaginase compared with biweekly dosing produces
superior induction remission rates in childhood relapsed acute lymphoblastic
leukemia: a pediatric oncology group study. Blood. 2000;96:1709–15.
60. Silverman LB, Gelber RD, Dalton VK, Asselin BL, Barr RD, Clavell LA, et al.
Improved outcome for children with acute lymphoblastic leukemia: results of Dana-
Farber Consortium Protocol 91-01. Blood. 2001;97:1211–8.
61. Panosyan EH, Seibel NL, Martin-Aragon S, Gaynon PS, Avramis IA, Sather H, et al.
Asparaginase antibody and asparaginase activity in children with higher-risk acute
lymphoblastic leukemia: Children’s Cancer Group Study CCG-1961. J. Pediatr.
Hematol. Oncol. 2004;26:217–26.
62. Pinheiro JPV, Boos J. The best way to use asparaginase in childhood acute
lymphatic leukaemia - still to be defined? Br J Haematol. 2004;125:117–27.
63. Bunpo P, Murray B, Cundiff J, Brizius E, Aldrich CJ, Anthony TG. Alanyl-Glutamine
Consumption Modifies the Suppressive Effect of L-Asparaginase on Lymphocyte
Populations in Mice. J. Nutr. 2008;138:338–43.
64. Yuneva M, Zamboni N, Oefner P, Sachidanandam R, Lazebnik Y. Deficiency in
glutamine but not glucose induces MYC-dependent apoptosis in human cells. The
Journal of Cell Biology. 2007;178:93 –105.
65. Jayaram HN, Cooney DA, Milman HA, Homan ER, Rosenbluth RJ. DON, CONV and
DONV--I. Inhibition of l-Asparagine synthetase in vitro. Biochemical Pharmacology.
1976;25:1571–82.

33
66. Ahluwalia GS GJ, Ahluwalia GS, Grem JL, Hao Z, Cooney DA. Metabolism and
action of amino acid analog anti-cancer agents. Pharmacol. Ther. 1990;46:243–71.
67. Nam SY, Lee EJ, Kim KR, Cha BS, Song YD, Lim SK, et al. Effect of obesity on total
and free insulin-like growth factor (IGF)-1, and their relationship to IGF-binding
protein (BP)-1, IGFBP-2, IGFBP-3, insulin, and growth hormone. Int. J. Obes.
Relat. Metab. Disord. 1997;21:355–9.
68. Steiner M, Hochreiter D, Kasper DC, Kornmüller R, Pichler H, Haas OA, et al.
Asparagine and aspartic acid concentrations in bone marrow versus peripheral
blood during Berlin–Frankfurt–Münster-based induction therapy for childhood acute
lymphoblastic leukemia. Leukemia & Lymphoma. 2012;1–6.
69. Tong WH, Pieters R, Hop WCJ, Lanvers-Kaminsky C, Boos J, van der Sluis IM. No
evidence of increased asparagine levels in the bone marrow of patients with acute
lymphoblastic leukemia during asparaginase therapy. Pediatric Blood & Cancer.
2012;n/a–n/a.
70. Goossens GH, Bizzarri A, Venteclef N, Essers Y, Cleutjens JP, Konings E, et al.
Increased Adipose Tissue Oxygen Tension in Obese Compared With Lean Men Is
Accompanied by Insulin Resistance, Impaired Adipose Tissue Capillarization, and
InflammationClinical Perspective. Circulation. 2011;124:67–76.
71. Kumar S, Venkata Dasu V, Pakshirajan K. Purification and characterization of
glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428.
Bioresour. Technol. 2011;102:2077–82.
72. Durden DL, Distasio JA. Comparison of the Immunosuppressive Effects of
Asparaginases from Escherichia coli and Vibrio succinogenes. Cancer Research.
1980;40:1125 –1129.
73. Offman MN, Krol M, Patel N, Krishnan S, Liu J, Saha V, et al. Rational engineering
of L-asparaginase reveals importance of dual activity for cancer cell toxicity. Blood.
2011;117:1614–21.

Abstract (if available)

Abstract Obesity is a significant risk factor for cancer. A link between obesity and a childhood cancer has been identified: obese children diagnosed with high-risk acute lymphoblastic leukemia (ALL) had a 50% greater risk of relapse than their lean counterparts. L- asparaginase (ASNase) is a first-line therapy for ALL that breaks down asparagine and glutamine, exploiting the fact that ALL cells are more dependent on these amino acids than other cells. In the present study, we investigated whether adipocytes, which produce significant quantities of glutamine, may counteract the effects of ASNase. In children being treated for high-risk ALL, obesity was not associated with altered plasma levels of asparagine or glutamine. However, glutamine synthetase was markedly increased in bone marrow adipocytes after induction chemotherapy. Obesity substantially impaired ASNase efficacy in mice transplanted with syngeneic ALL cells, and, like in humans, without affecting plasma asparagine or glutamine levels. In co- culture, adipocytes inhibited leukemic cell cytotoxicity induced by ASNase, and this protection was dependent on glutamine secretion. These findings suggest that adipocytes work in conjunction with other cells of the leukemia microenvironment to protect leukemia cells during ASNase treatment.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The role of adipocytes in acute lymphoblastic leukemia cell migration and survival against daunorubicin

PDF

The role of adipocyte-derived free-fatty acids in acute lymphoblastic leukemia

PDF

Induction of hypersignaling as a therapeutic approach for treatment of BCR-ABL1 positive Acute Lymphoblastic Leukemia (ALL) cells

PDF

SGLT2 inhibitors reverse adipocyte differentiation and reduce daunorubicin metabolism in acute lymphoblastic leukemia treatment

PDF

Immune infiltrates in papillary thyroid carcinomas

PDF

Tumor necrosis therapy–interleukin-12 fusion proteins and cancer therapy

PDF

A transgenic mouse model with overexpression of human RAGE in endothelial cells presents enhanced amyloid-beta transport into the brain

PDF

Therapeutic resistance in acute lymphoblastic leukemia: cIAP2 inhibition sensitizes B cell acute lymphoblastic leukemia to anti-CD19 chimeric antigen receptor T cell

PDF

Clinical outcomes of allogeneic hematopoietic stem cell transplant in acute lymphoblastic leukemia patients: a quality improvement project and systematic review meta-analysis

PDF

Ectopic fat and adipose tissue inflammation in overweight and obese African Americans and Hispanics

PDF

Role of integrin α4 in drug resistant acute lymphoblastic leukemia

PDF

The role of survivin in drug resistant pediatric acute lymphoblastic leukemia

PDF

Increased abdominal adiposity in adolescents with classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency

PDF

T cell regulation of HLA-DR

PDF

The physiologic and pathophysiologic role of sympathetic nervous system induced pulsatile lipolysis in metabolism

PDF

The effect of cytomegalovirus on gene expression of pediatric acute lymphoblastic leukemia

PDF

Native American ancestry among Hispanic Whites is associated with higher risk of childhood obesity: a longitudinal analysis of Children’s Health Study data

PDF

Gene therapy for the prevention and treatment of leukemia

PDF

Exploring the interplay of birth order and birth weight on leukemia risk

PDF

A pantothenate (vitamin B5) metabolite promotes intestinal neuropeptide secretion as part of the gut-neural axis-mediated stress response

Asset Metadata

Creator Ehsanipour, Ehsan Ali (author)

Core Title Adipocyte production of glutamine protects leukemia cells from L-asparaginase

School Keck School of Medicine

Degree Master of Science

Degree Program Physiology and Biophysics

Publication Date 07/15/2013

Defense Date 06/20/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag adipocyte,All,chemotherapy,glutamine,L-asparaginase,leukemia,OAI-PMH Harvest,obesity

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mittelman, Steven D. (committee chair), Avramis, Vassilios I. (committee member), Kaslow, Harvey R. (committee member), Kim, Yong-Mi (committee member)

Creator Email eehsanipour@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-288746

Unique identifier UC11294907

Identifier etd-Ehsanipour-1769.pdf (filename),usctheses-c3-288746 (legacy record id)

Legacy Identifier etd-Ehsanipour-1769.pdf

Dmrecord 288746

Document Type Thesis

Format application/pdf (imt)

Rights Ehsanipour, Ehsan Ali

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA