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ODC1 is a critical determinant of MYCN oncogenesis and a
therapeutic target in neuroblastoma
Michael D. Hogarty1,2, Murray D. Norris3, Kim Davis1, Xueyuan Liu1, Nicholas F.
Evageliou1, Candace S. Hayes4, Bruce Pawel5, Rong Guo6, Huaqing Zhao6, Eric Sekyere3,
Joanna Keating3, Wayne Thomas3, Ngan Ching Cheng3, Jayne Murray3, Janice Smith3,
Rosemary Sutton3, Nicola Venn3, Wendy B. London7, Allan Buxton7, Susan K. Gilmour4,
Glenn M Marshall3,8, and Michelle Haber3
1Division of Oncology, The Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia,
PA 19104-4318, USA
2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
3Children’s Cancer Institute Australia for Medical Research, PO Box 81 (High St) Randwick NSW Australia
2031
4Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA
5Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
6Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia,
PA 19104, USA
7Department of Statistics, University of Florida and Children’s Oncology Group Statistics and Data Center
Department, Gainesville, FL 32611, USA
8Centre for Children’s Cancer and Blood Disorders, Sydney Children’s Hospital, High Street, Randwick
NSW Australia 2031
Abstract
Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly
as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream
of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that
MYCN-amplified neuroblastomas have co-ordinately deregulated myriad polyamine enzymes
(including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-
risk tumors without MYCN amplification also overexpress ODC1, the rate-limiting enzyme in
polyamine biosynthesis, when compared with lower risk tumors, suggesting this pathway may be
pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced
survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival
and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence
on this pathway in neuroblastoma. The Odc inhibitor α-difluoromethylornithine (DFMO) inhibited
neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of
neuroblastoma-prone genetically-engineered mice (TH-MYCN GEM) extended tumor latency and
survival in homozygous mice, and prevented oncogenesis in hemizygous mice. In the latter, transient
Odc ablation permanently prevented tumor onset consistent with a time-limited window for
Corresponding Author: Michael D. Hogarty, Division of Oncology, The Children’s Hospital of Philadelphia, 9 North ARC; Suite 902C,
3615 Civic Center Boulevard, Philadelphia, PA 19104-4318, USA, Phone: 215-590-3931 FAX: 215-590-3770, Email:
hogartym@email.chop.edu.
The authors have no conflicts of interest to report.
NIH Public Access
Author Manuscript
Cancer Res. Author manuscript; available in PMC 2009 December 1.
Published in final edited form as:
Cancer Res. 2008 December 1; 68(23): 9735–9745. doi:10.1158/0008-5472.CAN-07-6866.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
embryonal tumor initiation. Importantly, we show that DFMO augments anti-tumor efficacy of
conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of
MYCN oncogenesis and demonstrates initial efficacy for polyamine depletion strategies in
neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors.
Keywords
Embryonal tumors; metabolomics; polyamines; oncogene; experimental therapeutics
INTRODUCTION
Neuroblastoma is a common childhood tumor arising within the peripheral nervous system.
The genetic feature most consistently associated with treatment failure is amplification of the
MYCN proto-oncogene that strongly correlates with advanced disease (1,2). Even in otherwise
favorable localized disease MYCN amplification portends poor outcome underscoring its
biological importance (3). In high-risk neuroblastomas that lack MYCN amplification, MYC
itself may be deregulated (4,5). Myc genes (including MYCN, MYC and MYCL1) represent a
family of basic helix-loop-helix leucine zipper transcription factors that are among the most
frequently deregulated genes in cancer. Myc proteins form heterodimers with Max and are
recruited to CACGTG (E-box) recognition sequences to transactivate target genes, or enter
additional complexes to form repressors [reviewed in (6,7)]. It has been estimated that nearly
1/10th of all genes may be directly or indirectly regulated by the Myc:Max axis (8) yet the
determination of those necessary or sufficient to confer oncogenic properties remains empiric.
One compelling target that may be decisive in mediating MYC effects is ODC1 (9), a bona fide
oncogene that encodes the rate-limiting enzyme in polyamine synthesis (10). Polyamines are
organic cations that enhance transcription, translation and replication (11) and support many
cellular processes governed by MYC genes. Their maintenance is essential for cell survival as
depletion activates growth arrest or apoptotic checkpoints (12). Thus, intracellular polyamines
are kept under tight control through post-transcriptional as well as transcriptional regulation,
with the rate-limiting enzymes ODC1 and AMD1 having among the shortest half-lives of any
mammalian enzyme as a result (13). Odc activity is frequently elevated in cancer through
deregulation of MYC resulting in higher polyamine content to support rapid tumor cell
proliferation (11). Considerable evidence links elevated polyamines to colon, breast, prostate
and skin carcinoma progression (14) but not embryonal tumors to date. Recently the
contribution of Odc activity to MYC-induced lymphomagenesis was investigated using the
Eμ-MYC murine model in which transient biochemical ablation inhibited lymphomagenesis,
while restoration of Odc activity allowed for delayed tumor onset (15).
We therefore sought to define whether Odc-mediated polyamine biosynthesis was a requisite
metabolic biopathway supporting embryonal tumor initiation or progression. Most children
with high-risk neuroblastoma have tumors that manifest a lethal course despite intensive
multimodal therapy (16,17). Thus, elucidating novel therapeutic pathways is paramount. We
demonstrate that polyamine expansion through broad deregulation of regulatory enzymes,
including ODC1, is a hallmark of neuroblastomas with MYCN amplification. High ODC1
correlates with poor clinical outcome in a large cohort of patients, including those lacking
MYCN amplification. Further, we demonstrate that biochemically disabling Odc inhibits
neuroblastoma proliferation in vitro and has marked anti-tumor efficacy in a neuroblastoma-
prone transgenic mouse model. Together these data support that the adequate provision of
polyamines is critical for MYC-driven proliferation and that targeted disruption of this pathway
has therapeutic utility.
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METHODS
Patient samples, expression profiling, array-CGH, and real-time Q-PCR
Transcriptome profiles from 101 primary neuroblastomas from COG and the Children’s
Hospital of Philadelphia were obtained by our groups previously. Clinical and genetic features
have been reported (18). Briefly, risk class was defined using COG criteria: 28 were localized
biologically favorable neuroblastoma (low-risk), 21 were intermediate-risk and 52 were high-
risk tumors (of which 20 had MYCN amplification). cRNA was hybridized to Affymetrix
U95Av2 oligonucleotide arrays containing 12,625 probe sets (Affymetrix, Santa Clara, CA)
and statistical modeling of probe set behavior was conducted using Probe Profiler (Corimbia,
Berkeley, CA). A quantitative expression score (e-score) was calculated for each probe set.
The data from this experiment are available at http://www.ncbi.nlm.nih.gov/geo/ [accession
number GSE3960]. Differential gene expression was measured with the Patterns from Gene
Expression (PaGE; (19)) algorithm using binary comparisons of e-scores. All runs were done
with 2001 permutations on unlogged data. A confidence level of 0.95 (1-FDR) was used to
define differential expression for these analyses.
A subset of tumors (N=80) had DNA available for detection of copy number alterations (CNA)
and have been applied to a BAC-array-CGH platform. The platform is described in detail in
(20) and its application and methodology for neuroblastoma CNA detection in (21). Briefly,
normalized log2 intensity ratios were averaged within slide for each BAC clone using the
DNAcopy package within Bioconductor (www.bioconductor.org). A mean log2 ratio >= 1.0
was considered a high-level amplification. Amplification of both the MYCN-containing BAC
and the CTD-2603D17 clone that contains ODC1 and was used to define co-amplification.
A second independent cohort of 265 neuroblastomas from COG with available RNA was
studied. Outcome data was available for 209 (79%). Informed consent was obtained from all
subjects. Clinical characteristics and RNA/cDNA isolation procedures were previously
described (22). ODC1 and MYCN expression was determined by real-time quantitative PCR
(Q-PCR). The β2-microglobulin gene served as an internal control and the primers and probe
sequences for β2-microglobulin and MYCN have been reported (22). ODC1 primer and probe
sequences were: ODCF 5′-GATGACTTTTGATAGTGAAGTTGAGTTGA-3′; ODCR 5′-
GGCACCGAATTTCACACTGA-3′; ODC Probe 5′-
CGGATTGCCACTGATGATTCCAAAGC-3′. Q-PCR data was collected using a Prism 7700
Sequence Detection System (Applied Biosystems; Foster City, CA) and the level of target gene
expression was determined using the ΔΔCt method (22). For all tumors in these studies,
MYCN status was defined as >4-fold copies of MYCN compared to a 2p reference probe using
fluorescence in situ hybridisation (FISH).
Cell lines and tissue culture
Neuroblastoma cell lines were obtained from the Children’s Hospital of Philadelphia Cell Line
Bank courtesy of Garrett M. Brodeur and have been previously reported (23). Cells were grown
in RPMI Media 1640 (Life Technologies) supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 U/ml of penicillin and 100 mcg/ml gentamicin. Tissue culture was at 37° C
in a humidified atmosphere of 5% CO2 as previously described (24). Cell line transcriptome
profiles were obtained using the Affymetrix U133+2 oligonucleotide array and analyzed as
above.
Cell line ODC1 expression
Real-time Q-PCR (TaqMan) was used to quantify ODC1 mRNA. Total RNA (2 μg) was
reverse-transcribed in a 20 μL reaction using the SuperScript III First Strand Synthesis System
(Invitrogen, Carlsbad, CA). The probe and primer set used for ODC1 detection was Assay-on-
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Demand Hs00159739_m1 (Applied Biosystems). The genes SDHA and HPRT served as
housekeeping genes for normalization. Triplicate reactions were performed and the mean
expression values were used for calculating relative expression.
Cytotoxicity assays
Cells were seeded into multi-well sensor microplates at 3 × 104 cells per well and allowed to
adhere overnight. Cell index was obtained for each test condition in duplicate over 96 hours
using the real-time cell electronic sensor system (RT-CES, ACEA Biosciences, San Diego,
CA). This label-free dynamic monitoring system uses electrical impedance to quantify viable
adherent cell number in real time (25). 2-difluoromethyl-DL-ornithine (DFMO; Eflornithine)
was added to culture media at a final concentration of 2.5, 5 or 10 μM. DFMO for all studies
was generously provided by Patrick Woster (Wayne State University) courtesy of ILEX
pharmaceuticals (San Antonio, TX). All experiments were replicated three times.
TH-MYCN mouse trials
129X1/SvJ mice transgenic for the TH-MYCN construct (26-28) were graciously provided by
Bill Weiss (Department of Neurology, UCSF) for establishment of TH-MYCN breeding
colonies in both Philadelphia and Sydney. All studies were approved by the Institutional
Animal Care and Utilization Committee at The Children’s Hospital of Philadelphia
(Philadelphia) and the Animal Care and Ethics Committee of the University of New South
Wales (Sydney).
Pre-emptive therapy trial (Philadelphia)—TH-MYCN hemizygous mice were bred and
litters randomized to water or water with 1% DFMO from birth onward. DFMO passes in breast
milk so treated pups received DFMO from birth. After weaning at ∼day 28 mice continued ad
libitum water or water with 1% DFMO as initially randomized through day 70. Mice were
genotyped from 1 cm tail-snip isolated DNA. Primers to detect the human MYCN transgene
were: 5′-CACAAGGCCCTCAGTACCTC-3′ (forward) and 5′-
AGGCATCGTTTGAGGATCAG-3′ (reverse). Hemizygous or homozygous transgene status
was determined using real-time Q-PCR (TaqMan) with the following: TM-MYCNF1 primer
5′-CGACCACAAGGCCCTCAGT-3′ (within exon 2) and TM-MYCNF2 primer 5′-
AGGAGGAACGCCGCTTCT-3′ (exon 3); MYCN probe FAM-
ATCGCTCAGGGTGTCCTCTCCGG-TAMRA; murine B-actinF1 primer 5′-
TGCGTCTGGACTTGGCTG-3′ and murine B-actinR1 5′-TAGCCACGCTCGGTCAGG-3′;
and murine B-actin probe VIC-CGGGACCTGACTGACTACCTCATGA-TAMRA. For each
analysis, 10 ng template DNA was used. All unknowns, FISH confirmed homozygous and
hemizygous TH-MCYN controls, and non-template controls were loaded in triplicate. The
average transgene dose was calculated using ΔΔCT and calls made based on comparison to
control values within each run.
Palpation for intra-abdominal tumors was performed thrice weekly. Animals with palpable
tumors underwent serial abdominal ultrasonography under isoflurane sedation to determine in
situ tumor volume using a Vevo660 imaging system with 3D Acquisition and Visualization
software (VisualSonics; Toronto, Canada). Mice were screened by experienced animal
personnel and sacrificed for pathological signs of tumor burden (predominantly hunching and
poor mobility).
Delayed treatment trial (Sydney)—The specific characteristics of the Sydney breeding
colony of TH-MYCN transgenic mice, including their maintenance, genotyping, and tumor
incidence and latency, have been described previously (26). Cohorts of mice received plain
water or 1% DFMO in their water ad libitum from day 25 (post-weaning) onward. Animals
were abdominally-palpated twice weekly by experienced staff. Time to onset of tumor
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development and time to sacrifice according to well defined humane endpoints was determined
for all animals (26).
Combination chemotherapy treatment in TH-MYCN mice—Homozygous TH-
MYCN mice with palpable tumors of 5-7 mm diameter (N≥10 per group) were treated with
cisplatin (Pharmacia) intraperitoneally daily × 5 days at 2 mg/kg, or cyclophosphamide IP daily
× 5 days at 20 mg/kg. The control groups were treated with chemotherapy alone, while DFMO-
treated groups received either continuous 1% DFMO in drinking water after completion of the
cisplatin course, or 1% DFMO in drinking water simultaneously with the cisplatin or
cyclophosphamide and continuously thereafter. Animals were sacrificed when tumors
recurred, according to well-defined humane endpoints (26), or otherwise at 140 days of age.
Tumor histolopathology and polyamine content
Animal tumors and tissues were harvested at sacrifice and fixed in 10% neutral buffered
formalin and paraffin embedded for histologic studies, as well as flash frozen in liquid nitrogen
for metabolic assays. Tissue sections were stained with hematoxylin and eosin and assessed
histologically by a pathologist (B.P.) for confirmation of tumor type, and percentage of necrosis
(average of five 40X microscopic fields) and mitotic/karyorrhectic cells (average of five 600X
microscopic fields).
For immunohistochemistry, 5 μm sections were stained with antibodies to caspase-3 at a 1:1000
dilution (R&D Systems AF835, Minneapolis MN) and Ki67 at a 1:1000 dilution (Santa Cruz
SC-7846, Santa Cruz, CA) on an Autostainer Plus staining system (DAKOCytomation,
Carpinteria, CA) using standard methods, including microwave antigen retrieval for 5 minutes
in 0.01 M Citrate buffer at pH 7.6. Both Caspase-3 and Ki-67 staining were scored as the
percentage of stained tumor nuclei using the average of five 600X microscopic fields.
Tumor tissues were frozen in liquid nitrogen, ground to a fine powder, and stored at -80°C.
For polyamine analyses, ground tissues were homogenized in 0.2 N perchloric acid and
incubated at 4°C overnight. Dansylated polyamines were separated on a reversed-phase C18
HPLC column (29). Polyamine values were normalized to the amount of DNA in the tissue
extracts.
Murine paravertebral ganglia studies
To study the in vivo effects of perinatal Odc1 inhibition, litters from TH-MYCN mice were
randomized and treated with 1% DFMO in maternal drinking water as above. Pups were
euthanized at postnatal day 0 (n= 26), 7 (n=40) and 14 (n=26). Additionally, a cohort of
pregnant females received DFMO prenatally from embryonic day 14 to 21 and ganglia from
pups were obtained at day 0. Tissues were formalin-fixed and paraffin-embedded. A histologic
audit was performed with each section scored for the presence of ≥30-cell neuroblast
hyperplasia. Animals were TH-MYCN genotyped as previously described (30).
Paravertebral sympathetic ganglia were dissected from normal and TH-MYCN mice at postnatal
day 14 and cultured for 3-5 days as described previously (27). DFMO (1 mM) was added to
half the wells for 7 days. On day 5, cells were washed twice and 10 μg/ml anti-NGF antibody
(Chemicon) or isotype control (Cedarlane Laboratories) was added to alternate wells for 48
hours. The number of neurons surviving withdrawal of NGF was quantitated using
immunofluorescent staining for the neuron-specific marker ßIII-tubulin, expressed as a
percentage of neurons positive for ßIII-tubulin before and after NGF withdrawal.
For Odc1 immunohistochemistry, cells from murine ganglia were cultured as described (27),
then treated with 1 mM DFMO for 7 days. Media was replaced every 2-3 days with complete
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media containing anti-NGF and DFMO. Cells were fixed with 4% formaldehyde (20 minutes
at RT), followed by methanol permeabilization (20 minutes at RT). Immunofluorescent
staining was performed using a mouse Odc antibody (clone MP 16-2, Sapphire Biosciences,
Sydney) at 1:25 dilution, or isotype control in conjunction with the Vector Labs M.O.M kit
(Australian Laboratory Services, Sydney). DFMO competitively inhibits binding of this
antibody to Odc1 (31). Cells were imaged using an Axioplan 2 microscope (Zeiss, Oberkochen,
Germany) using a Sensicam Charged Coupled Device (CCD) camera (PCO Imaging, Kelheim,
Bavaria, Germany).
Statistical analyses
Pearson’s correlation coefficients were calculated to assess gene expression correlations. Two-
tailed students T-test was used to test significance unless otherwise stated. Survival analyses
were performed according to the method of Kaplan and Meier (32) with standard errors
according to Peto (33). Comparisons of outcome between subgroups were performed by a 2-
sided log-rank test. Event-free survival time was calculated as described previously (22) and
death was the only event considered for the calculation of overall survival time. A continuous
range of ODC1 Q-PCR values was used in outcome analyses. To categorize expression as
either high or low, the following cut-points were tested: the mean, median, upper quartile and
upper decile values (34). The cut-point that maximized the difference in EFS between the two
groups was selected, and that cut-point was applied to analyses of the overall cohort as well as
the subgroups.
RESULTS
ODC1 expression correlates with survival in neuroblastoma
Odc1 is rate-limiting for polyamine biosynthesis, a bona fide oncogene, and direct MYCN
target. We therefore compared ODC1 mRNA expression with MYCN gene status, MYCN
expression, and outcome in a large cohort of neuroblastoma patients. ODC1 expression was
significantly higher in MYCN amplified tumors (Figure 1A) and strongly correlated with
MYCN expression (r=0.80; p<0.0001). Event-free survival (EFS) for patients with high
ODC1 expression (defined as the upper decile and determined by an optimal cut-point analysis)
was significantly poorer than that of patients with low ODC1, with 5-year rates of 38%±11%
and 76%±3% (Figure 1B). Similarly, worse overall survival (OS) was associated with high
ODC1 (p<0.001). High ODC1 was associated with worse EFS and OS when the groups were
dichotomised around the mean, median or upper quartile as well (p<0.05 for each). In patients
with stage 4 metastatic disease high ODC1 expression was again associated with reduced EFS
or OS (Figure 1C).
Since MYCN amplification has a negative influence on survival through regulation of numerous
target genes (in addition to ODC1) we assessed tumors without MYCN amplification. Again,
high ODC1 was associated with a worse EFS and OS, with 5-year EFS rates of 43%±19%
compared to 80%±3% (Figure 1D) suggesting a role for ODC1 in promoting an aggressive
phenotype (for dichotomization at the mean, median or upper quartile the p value was again
≤0.05). In non-amplified neuroblastomas there is no correlation between MYCN expression
and outcome so it is unlikely this is a surrogate for MYCN activity (30). To assess the
independent prognostic significance of ODC1 while controlling for known powerful
prognostic factors such as tumor stage (stages 1,2,4S versus 3,4), age at diagnosis (<1 year
versus ≥1 year) and MYCN status these factors were tested in a Cox proportional hazards model
with dichotomised ODC1 expression. The addition of ODC1 expression by itself did not add
independent significance to this highly prognostic model.
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Neuroblastomas with MYCN amplification demonstrate co-ordinate pathway alterations that
enhance polyamine biosynthesis
To further explore polyamine metabolism (see pathway, Figure 2A) we mined transcriptome
profiles from neuroblastomas of diverse risk classes (17). As predicted, MYCN and ODC1
mRNA were significantly higher in HR-A tumors in comparison with all other groups (Figure
2B). Odc1 is additionally regulated by antizymes (OAZs) that direct its degradation, while
OAZIN inhibits this activity. OAZ2 was significantly reduced in HR-A neuroblastomas further
promoting Odc activity. Notable were alterations in numerous polyamine regulators in HR-A
tumors, all in a direction promoting biosynthesis. Each pro-synthetic enzyme was upregulated
(confidence level >0.95) while there was a reduction in SMOX that catabolizes polyamines
(note, no PAOX probe sets were on the array). Together these data demonstrate systematic
alterations in polyamine metabolism correlated with MYCN amplification. High-risk tumors
without MYCN amplification (HR-NA) also had higher ODC1 and reduced OAZ2 compared
with low and intermediate risk tumors, suggesting pathway enhancement in these tumors as
well.
ODC1, SRM, and AMD1 have been posited as Myc targets (Myc Cancer Gene Database;
www.myccancergene.org) and our data support this. ODC1 was strongly correlated with
MYCN across the entire cohort (r=0.53; p<0.0001; Figure 2C). SRM and AMD1 yielded similar
correlations (r=0.30; p=0.001 and r=0.59, p=0.001, respectively). SMS, despite no prior
evidence as a MYC target, had the strongest correlation (r=0.69, p<0.0001), while OAZ2 was
inversely correlated (r= -0.42, p<0.0001; see Supplementary Figure S1) though not a previously
identified repressed target. Thus, MYCN-amplified neuroblastomas directly or indirectly
promote polyamine pool expansion through co-ordinate alteration of multiple polyamine
regulators through mechanisms that may include de novo transcriptional initiation or mRNA
stability.
The correlation between MYCN and ODC1 expression was less evident in tumors with
MYCN amplification (compared with other bona fide MYCN targets) due to the presence of
outliers with exceptionally high ODC1 (Figure 2C). ODC1 maps ∼5.5 Mb telomeric to
MYCN (2p24) using UCSC Genome Browser coordinates. We sought whether ODC1 was co-
amplified with MYCN as has been reported (35). Eighty of the 101 tumors had DNA available
for determination of MYCN and ODC1 genomic copy number using a BAC-array-CGH
platform (21). No tumors without MYCN amplification (N=64) had ODC1 amplification.
Sixteen MYCN-amplified tumors (by FISH) were confirmed to have high-level MYCN
amplification using array-CGH. Three of these (19%) had high-level ODC1 co-amplification
and each was an outlier with extremely high ODC1 expression (Figure 2C, arrowheads; the
fourth outlier did not have DNA for CNA determination). Thus, a subset of neuroblastomas
co-amplify both the transcriptional regulator (MYCN) and target gene (ODC1) to augment
effects on polyamine biosynthesis, a putative “amplification loop” that has not been previously
postulated.
Disabling ODC1 in neuroblastoma cell lines inhibits proliferation
Across 26 neuroblastoma cell lines there was a trend for higher ODC1 with MYCN-
amplification (p=0.10; p=0.06 if NBL-S with prolonged MycN half-life (36) is included in the
“amplified” cohort; Supplemental Figure S2). We assessed Odc1 inhibition in vitro using
DFMO, an irreversible Odc inhibitor. DFMO-mediated growth inhibition correlated with
ODC1 mRNA expression and proliferative rates (Figure 3A), were apparent by early time-
points (48 hrs), and was seen in cells both with and without MYCN amplification. Indeed,
Affymetrix expression data for neuroblastoma cell lines and fetal brain cDNA shows
upregulation of polyamine synthetic enzymes and downregulation of catabolic enzymes in non-
amplified cells (Figure 3B). This growth inhibition is not surprising as most cell types
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demonstrate cytostasis when Odc is inhibited in vitro, including neuroblasts {Wallick, 2005
#1863}. Tissue culture conditions do not provide the same rescue opportunities present to cells
depleted of polyamines in vivo, where many markedly increase polyamine uptake, an option
not present in polyamine-poor culture media. We therefore focused on polyamine depletion
effects in vivo.
Disabling Odc1 prevents MYCN-mediated oncogenesis
We determined the impact of disabling Odc (to impede polyamine synthesis) on both tumor
initiation and progression using a MYCN transgenic mouse model. Mice homozygous for a
neural-crest targeted MYCN transgene (TH-MYCN) develop tumors with complete penetrance,
while hemizygous TH-MYCN mice have reduced (∼30%) tumor penetrance (27,28). Tumors
develop within hyperplastic rests that are transiently present even in wild-type animals (27).
Their number and persistence correlate with tumor penetrance and MYCN dosage. Tumors
share biochemical features and orthologous genomic alterations with human neuroblastomas,
suggesting preferred secondary pathways are recapitulated (28,37,38). Thus, the model
provides a platform for evaluating biopathway targeted therapies (39).
We evaluated whether Odc activity was required for MYCN-initiated oncogenesis by treating
mice with DFMO from birth. All mice homozygous for the transgene (highest MYCN)
developed tumors, however, tumor latency (mean 31±2 versus 43±7 days; p<0.001) and overall
survival (mean 43±4 versus 59±9 days; p<0.001) were markedly extended by DFMO (Figure
4A). Moreover, hemizygous mice (high MYCN) were protected from tumor initiation. Seven
of 16 untreated hemizygous mice (44%) developed tumors, consistent with the penetrance
observed historically, whereas only 6 of 38 DFMO-treated mice (16%) developed tumors
(p=0.035). Importantly, DFMO was removed at day 70 yet no tumors arose beyond this time-
point. This is consistent with a finite vulnerable period for embryonal oncogenesis and suggests
that transiently inhibiting Odc1 provides long-lasted tumor protection.
Delaying Odc inhibition until after tumor onset also had a measurable effect. In a second trial,
DFMO therapy was initiated after weaning when small tumors are invariably present in
homozygous animals and in the majority of hemizygous animals (27). DFMO treatment of
homozygous mice again inhibited progression (time to palpable tumor burden: mean 47.5±1.3
days versus 38.6±1.5 days) and time to death (mean 49.2±1.3 days versus 42.6±1.2 days;
p=0.001; Figure 4B). Delayed DFMO treatment in hemizygous mice did not reduce penetrance
(as the majority of tumors were present prior to therapy) yet there was a modest trend toward
tumor inhibition based on a reduction in penetrance and extended tumor free and overall
survival.
DFMO enhances the therapeutic effect of chemotherapy
DFMO has been shown to induce cell cycle arrest in neuroblasts (40) and therefore may
interfere with chemotherapy effects. We assessed Odc inhibition in combination with cisplatin,
vincristine or cyclophosphamide, first-line agents with high single-agent activity in
neuroblastoma. TH-MYCN homozygous mice with palpable intra-abdominal tumors (75-150
mm3) were treated with the chemotherapeutic alone or in combination with DFMO. Cisplatin
induced transient tumor regression with a mean latency to recurrence of 32 days. DFMO started
concurrently or following cisplatin and continued thereafter did not interfere with cisplatin-
induced regression and led to an extended relapse-free survival (p<0.01; Figure 4C). Similar
findings were obtained using vincristine (data not shown), and a more marked augmentation
of chemotherapy efficacy was seen with cyclophosphamide, where cyclophosphamide alone
resulted in long term cure of ∼20% of neuroblastoma-bearing mice. Concurrent administration
of DFMO with cyclophosphamide increased overall survival to 80% (p=0.03; Figure 4D).
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We noted no overt toxicity attributable to DFMO therapy in these trials. Wild-type mice
receiving DFMO from birth weighed less than untreated littermates following weaning (∼day
28), yet they gained weight at the same rate or better thereafter, despite ongoing DFMO
exposure (average weight gain of 0.45 gm/wk in DFMO-treated mice versus 0.28 gm/wk for
control mice between weeks 14 and 20; p=0.12; Supplemental Figure 3). Mice receiving
DFMO delayed until after weaning showed modest growth inhibition through 5 months of life
compared with untreated animals (weight gain of 0.92 ± 0.04 versus 1.05 ± 0.04 gm/wk;
p=0.034).
Odc activity is not required for MYCN-mediated death resistance
Tumors in TH-MYCN mice arise within hyperplastic rests in sympathetic ganglia. These are
transiently present in wild-type mice but are increased in number and persist longer in a
MYCN dose-dependent manner (27). We assessed the effect of DFMO on this process by
performing a histologic audit. Prenatal DFMO treatment of pregnant mothers from embryonic
day 14-21 did not affect the incidence of hyperplasia noted at day 0 (data not shown). Post-
natal DFMO treatment of newborn pups did not have a demonstrable effect on neuroblast
hyperplasia by day 7, however, by day 14 homozygote mice (highest MYCN) treated with
DFMO demonstrated a significant reduction (Figure 5A). Together with in vivo data, this
suggests that Odc activity is not required for basal neuroblast hyperplasia, however, MYCN
supported maintenance and progression to tumor is at least partially Odc-dependent.
MYCN not only drives cell cycle entry but also protects against deprivation-induced apoptosis
in TH-MYCN neural cells. Cultured perinatal ganglia from TH-MYCN mice demonstrate
resistance to NGF withdrawal (27) analogous to that seen in postmitotic sympathetic neurons
(41). Paravertebral ganglia from untreated normal and TH-MYCN homozygote mice at day 14
were cultured in the presence of NGF with or without DFMO for 7 days, after which NGF was
withdrawn. While DFMO-mediated Odc inhibition was supported using a conformation-
specific antibody (Figure 5B) there was no effect on the death resistance of ganglia cells,
demonstrating that Odc-mediated polyamine synthesis was not a critical component of
MYCN-mediated apoptosis resistance (Figure 5C).
Neuroblastomas arising under DFMO may circumvent the polyamine depletion barrier
We assessed whether tumors arising under Odc inhibition overcame this blockade or took an
alternative route to transformation less dependent on polyamines. DFMO-treated and untreated
mice developed cellular tumors that were undifferentiated (with the exception of one lymph
node that had fibrillary neuropil) similar to poorly differentiated human neuroblastoma.
DFMO-treated tumors had larger cells with reduced hemorrhage and necrosis, but no
differences in mitosis/karyorrhexis index (Figure 6A). Tumors were notable for the large
percentage of cycling cells (Ki67+) and caspase activation although neither differed between
groups. Serial ultrasonography in homozygous TH-MYCN mice confirmed similar tumor
volume at the time of ascertainment (mean 227 ± 61 mm3 versus 232 ± 64 mm3; p=0.83) though
tumors arose later in DFMO-treated mice. Tumors grew at similar rates (Δvolume/week of 166
± 68 mm3 versus 156 ± 79 mm3 with DFMO; p=0.75) and lethality. Thus, aside from being
delayed in onset, DFMO-treated tumors manifested a similar aggressive phenotype in
homozygous mice.
Polyamine assays from tumors harvested at culling demonstrated reduced putrescine in DFMO-
treated tumors, a trend toward reduced spermidine and no effect on spermine (Figure 6B).
These effects are consistent with experimental models of DFMO-induced Odc inactivation
(42) including effects on NB cell lines (40) and support that polyamine depletion is at least
partially maintained. However, maintenance of spermidine and spermine through enhanced
polyamine uptake, compensatory Amd1 induction, or altered metabolism cannot be formally
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excluded as a mechanism for circumventing the polyamine depletion barrier. Further studies
are ongoing to determine whether neuroblastomas circumvent Odc inactivation to support
polyamine homeostasis or take an alternate route to oncogenesis.
DISCUSSION
Most children with high-risk neuroblastoma die from tumor progression and innovative
treatment approaches are needed (16,43). We show that neuroblastomas, particularly those
with MYCN amplification, have altered polyamine metabolism and may be vulnerable to
therapeutic polyamine depletion. Although MYCN amplification has long been associated with
poor outcome (1,2) the transcriptional targets governing this remain elusive as MYCN regulates
thousands of genes (7). We show that MYCN amplification is correlated with deregulation of
numerous enzymes that drive polyamine expansion, and such concerted alterations may be a
hallmark of MYC oncogenesis (15). Odc1 is rate-limiting in this pathway and, importantly, we
show that its biochemical inhibition alone has measurable consequences on tumor progression
in a transgenic model.
While MYCN amplified tumors deregulate diverse polyamine enzymes, high-risk tumors
without MYCN amplification also have elevated ODC1 and reduced Odc antizyme (OAZ2).
Deregulation of this rate-limiting step in polyamine biosynthesis may provide a therapeutic
vulnerability here as well. This is supported by demonstration that polyamine regulator
expression in MYCN non-amplified cells parallel those for amplified cells, and by in vitro data
showing DFMO-mediated growth inhibition in non-amplified cells. Of interest, we defined
MYCN and ODC1 high-level co-amplification in a subset of MYCN-amplified tumors
associated with markedly elevated ODC1 expression. As lesser copy number gain of the
ODC1 locus (2p25) is reported in half of high-risk neuroblastomas without MYCN
amplification (21), we speculate that ODC1 gene dosage gain is a potential mechanism for
pathway upregulation in this subset. Alternative MYC deregulation in high-risk neuroblastomas
may also transcriptionally drive ODC1 expression (4,5). Importantly, we show that ODC1
expression is inversely correlated with survival in a large validation cohort, even in tumors
lacking MYCN amplification. Together these data suggest polyamine depletion strategies may
be more broadly effective against high-risk tumors rather than selectively synthetic-lethal for
MYCN-amplified tumors.
Myc genes induce Cks1, a component of the SCFSkp2 E3 ubiquitin ligase complex that degrades
p27Kip1 (44). Since Odc is required both for Myc-induction of Cks1 (44) and for polyamine
biosynthesis, its activity licenses cell cycle entry at multiple steps. In vitro, Odc inhibition
causes polyamine depletion, increased p27Kip1 and Rb hypophosphorylation with G1-arrest in
neuroblasts (40). In vivo we show that Odc activity is not required for basal hyperplastic rest
formation (pre-neoplastic) nor neuroblast resistance to deprivation-induced apoptosis.
However, Odc supports hyperplastic rests and promotes their oncogenic conversion. It is likely
that DFMO-enforced growth arrest alters the stoichiometry of cycling cells within the
peripheral sympathetic compartment to impede tumor initiation.
This selective pressure can prevent tumor onset in TH-MYCN hemizygous mice, in which
neuroblastoma initiation requires MYCN transgene amplification indicating a higher threshold
of MYCN permissive for oncogenesis (27). Of greater interest is that transient Odc inhibition
through day 70 is capable of providing durable tumor protection. This contrasts with Eμ-
MYC mice where DFMO withdrawal leads to lymphomagenesis at the expected interval from
treatment cessation (15). This is consistent with a finite window for embryonal tumorigenesis
beyond which the specific tissue milieu may be incapable of supporting transformation and
suggests chronic Odc inhibition may not be required for sustained therapeutic benefit.
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In homozygous TH-MYCN mice, disabling Odc from birth delays but does not prevent tumors.
The polyamine depletion barrier imposed by Odc inactivation may be bypassed as only
putrescine remains depressed and the rate of end-stage tumor progression is reminiscent of
untreated animals. DFMO-mediated Odc inhibition is often accompanied by compensatory
Amd1 upregulation that may partly compensate. Alternatively, Odc inhibition may force pre-
malignant neuroblasts to adopt an alternate pathway to transformation, as has been shown for
lymphomas arising in Eμ-MYC:Odc+/- mice (15). Still, Odc inhibition after tumor onset delays
progression and augments sensitivity to cytotoxic stressors, providing clinical relevance to
these studies.
The utility of therapeutic polyamine depletion has been limited to date (14). However, DFMO
doses sufficient to inhibit Odc are well tolerated chronically and polyamine depletion as an
anti-cancer strategy is in the midst of a re-evaluation. Newer targeted agents are under
development including those that inhibit polyamine uptake form extra-cellular sources or target
additional regulatory enzymes (45-47). That DFMO augments chemotherapy efficacy in our
model allays concerns that enforced growth arrest via polyamine depletion will subvert
traditional chemotherapeutics. This is reassuring as the entre of polyamine depletion agents
into the clinic would likely be in concert with conventional cytotoxics. Interestingly,
cisplatinum has been shown to alter AMD1, ODC1, SRM and SAT in directions that antagonize
polyamine synthesis (48). Yet the dramatic responses with vincristine or cyclophosphamide
and DFMO, including improved overall survival rates, suggest this effect does not result from
a unique synergistic opportunity provided by platinators alone. Taken together, our data
strongly support that polyamine depletion may provide an important addition to the
neuroblastoma armamentarium, and perhaps other embryonal malignancies governed by
MYC. Potentiation of these effects with complimentary polyamine-targeted agents may further
improve efficacy and deserve further evaluation.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the Children’s Oncology Group (COG) for providing tumor specimens; Pat
Woster (Wayne State University) and ILEX Pharmaceuticals (San Antonio, TX) for DFMO; William Weiss (UCSF)
for TH-MYCN mice; Qun Wang for Affymetrix dataset assistance; Edward Attiyeh, Sharon Diskin and Yael Mosse
for array-CGH analyses; and Rosalind Barr, Eric Rappaport and the Nucleic Acids and Protein Core facility at the
Children’s Hospital of Philadelphia for technical assistance.
Funding to support this work came from the National Institutes of Health (CA97323 to M.D.H, CA070739 to S.K.G.);
the Richard and Sheila Sanford Chair in Pediatric Oncology (to M.D.H.); the National Health and Medical Research
Council Australia (to M.D.N., M.H., G.M.M.); the Cancer Institute New South Wales (to M.D.N., M.H., G.M.M.);
and the Cancer Council New South Wales (to M.D.N., M.H., G.M.M.).
Abbreviations
DFMO, α-difluoromethylornithine; INSS, International Neuroblastoma Staging System;
COG, Children’s Oncology Group.
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Figure 1. ODC1 expression is correlated with MYCN and inversely correlated with survival
(A) MYCN and ODC1 expression by Real-time Q-PCR in a set of 265 primary NBs stratified
by MYCN amplification status. Data represent the mean ± SE of triplicate assays and two-tailed
p-values are indicated. Event Free Survival curves for (B) all 209 neuroblastoma patients with
outcome data available, (C) the subset of metastatic stage 4 patients, and (D) the subset with
tumors without MYCN amplification (N=183), dichotomised around the upper decile for
ODC1 expression (with p-values using the method of Kaplan-Meier). EFS and OS were also
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significantly reduced in the high ODC1 cohort when dichotomized at the mean, median or
upper quartile.
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Figure 2. Polyamine regulation in neuroblastoma
(A) Polyamine metabolism: polyamines (putrescine, spermidine and spermine) are synthesized
from ornithine through decarboxylation and condensative processes. Synthetic (green) and
catabolic (red) enzymes are shown. Underlined enzymes are highly regulated with the shortest
half-lives of any mammalian enzymes. ODC1, ornithine decarboxylase; AMD1, S-
adenosylmethionine decarboxylase; SRM, spermidine synthetase; SMS, spermine synthetase;
SAT, spermine/spermidine-N-acetyltransferase; SMOX, spermine oxidase; OAZ1, ODC
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antizyme 1; OAZ2, ODC antizyme 2; OAZIN, ODC antizyme inhibitor. (B) Polyamine
regulator expression in primary neuroblastomas: LR, 28 low-risk; IR, 21 intermediate-risk;
HR-NA, 32 high-risk tumors without MYCN amplification; HR-A, 20 high-risk tumors with
MYCN amplification; *Differential expression between HR-A and HR-NA; **differential
expression between HR-A and all others; ***differential expression between HR-NA and LR
and IR (all with a confidence level >0.95 using PaGE analyses); filled circle on y-axis, fetal
brain (control) expression. (C) Correlation between ODC1 and PTMA with MYCN expression
using representative probe-sets. All 101 tumors are shown in left panels; right panels segregate
HR-A tumors from the other groups. Pearson correlation coefficient and p-values are given.
Arrowheads identify the three tumors that have both high-level MYCN and ODC1 co-
amplification.
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Figure 3. Polyamine dependence in neuroblastoma cell lines
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(A) DFMO-mediated growth inhibition in vitro. Cell Index, proportional to the viable cell
number, was obtained with the RT-CES platform. Viable cell number following short-term
exposure to DFMO is shown normalized to no treatment at 72 hours. White bars, control; light
grey, 2.5 mM DFMO; dark grey, 5 mM DFMO; black, 10 mM DFMO. Triplicate wells are
assessed for each experiment and multiple experiments are done for each condition; error bars
represent the standard deviation. (B) Affymetrix expression (e-score) for polyamine regulatory
enzymes in select neuroblastoma cell lines, segregated by MYCN status, and compared with
fetal brain cDNA as a normal reference. Both amplified and non-amplified cells have
expression alterations in a direction promoting polyamine expansion (more pronounced in the
amplified cells).
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Figure 4. Extended Tumor Free Survival in neuroblastoma-prone mice treated with DFMO
(A) Tumor Free Survival curves for homozygous (TH-MYCN +/+) or hemizygous (TH-
MYCN +/-) mice stratified by DFMO therapy. DFMO-treated mice (blue lines) received DFMO
from birth onward (pre-emptive treatment trial). DFMO therapy was stopped at day 70 in
tumor-free mice. (B) Delayed treatment trial: TH-MYCN homozygous and hemizygous mice
were randomized to DFMO (blue dashed lines) or control (red lines) following weaning at day
25. (C) Tumor Free Survival for TH-MYCN homozygous mice with advanced tumor from the
time of treatment with: cisplatin alone (black line), cisplatin followed by DFMO (red line), or
cisplatin administered simultaneously with DFMO (blue line), or (D) cyclophosphamide alone
or combined with DFMO. P-values using the method of Kaplan-Meier are shown.
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Figure 5. DFMO inhibits Odc activity and MYCN-driven hyperplasia but does not revert the
apoptosis resistance provided by MYCN
(A) Neuroblast hyperplasia following DFMO-mediated Odc1 inhibition in TH-MYCN
homozygous (+/+) or hemizygous (+/-) mice, and wild-type (-/-) littermates. The percentage
of ganglia with ≥30-cell neuroblast hyperplasia at each time-point is shown. DFMO-treated
mice differed significantly from untreated mice among homozygotes at P14 (p<0.001), but not
hemizygote or normal pups at either time-point. (B) Relative Odc1 activity in normal ganglia
following DFMO-mediated inhibition. Protein expression was detected using an Odc activity-
specific antibody (clone MP16-2). Green fluorescence represents uninhibited (active) Odc1;
blue fluorescence is DAPI nuclear stain; (i) untreated ganglia cells, (ii) DFMO-treated cells,
(iii) isotype control. (C) Relative ganglia cell survival in vitro, both before or after NGF
withdrawal in normal (white bars) or TH-MYCN homozygote (black bars) cells, in the presence
(hatched bars) or absence of 1 mM DFMO. Disabling Odc1 activity with 1 mM DFMO did
not diminish the survival advantage governed by MYCN in ganglia cells from homozygote
animals.
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Figure 6. Neuroblastomas arising under Odc blockade differ histologically but maintain partial
polyamine depletion
Analysis of tumors arising in TH-MYCN +/+ mice with or without polyamine depletion
imposed by Odc1 inhibition. (A) DFMO therapy led to tumors with reduced necrosis or
hemorrhage as well as larger cell size but no significant alterations in mitosis/karyorrhexis
index, proliferation (Ki67 staining), or apoptosis (activated Caspase 3 staining). (B) Evidence
for Odc inhibition was apparent by significantly reduced putrescine and a trend toward
spermidine reduction in DFMO-treated mice (N=6 per group). This pattern supports an on-
target effect of DFMO, although partial circumvention of polyamine synthesis inhibition
through uptake of spermidine or spermine cannot be excluded.
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