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LYMPHOID NEOPLASIA
Epigenetic reprogramming reverses the relapse-specific gene expression signature
and restores chemosensitivity in childhood B-lymphoblastic leukemia
Teena Bhatla,1 Jinhua Wang,1 Debra J. Morrison,1 Elizabeth A. Raetz,1 Michael J. Burke,2 Patrick Brown,3 and
William L. Carroll1
1New
York University Cancer Institute, New York University Langone Medical Center, New York, NY; 2University of Minnesota Amplatz Children’s Hospital,
Minneapolis, MN; and 3Johns Hopkins University, Baltimore, MD
Whereas the improvement in outcome for
children with acute lymphoblastic leukemia
has been gratifying, the poor outcome of
patients who relapse warrants novel treatment approaches. Previously, we identified
a characteristic relapse-specific gene expression and methylation signature associated with chemoresistance using a large
cohort of matched-diagnosis relapse
samples. We hypothesized that “reversing”
such a signature might restore chemosensitivity. In the present study, we demonstrate
that the histone deacetylase inhibitor vorinostat not only reprograms the aberrant
gene expression profile of relapsed blasts
by epigenetic mechanisms, but is also synergistic when applied before chemotherapy
in primary patient samples and leukemia
cell lines. Furthermore, incorporation of the
DNA methyltransferase inhibitor decitabine
led to reexpression of genes shown to be
preferentially methylated and silenced at
relapse. Combination pretreatment with vorinostat and decitabine resulted in even
greater cytotoxicity compared with each
agent individually with chemotherapy. Our
results indicate that acquisition of chemoresistance at relapse may be driven in part
by epigenetic mechanisms. Incorporation of
these targeted epigenetic agents to the standard chemotherapy backbone is a promising approach to the treatment of relapsed
pediatric acute lymphoblastic leukemia.
(Blood. 2012;119(22):5201-5210)
Introduction
Relapsed acute lymphoblastic leukemia (ALL) is one of the leading
causes of death among children with cancer. A hallmark of relapsed
blasts is their intrinsic chemoresistance compared with what is
observed at initial diagnosis.1,2 Given the frequent failure of
conventional salvage chemotherapy, including intensified drug
schedules and stem cell transplantation, in the treatment of relapsed
ALL,3,4 innovative strategies are urgently needed.
In recent years, it has become clear that cancer can be driven by
patterns of altered gene expression mediated not by mechanisms
that affect the primary DNA sequence, but through the “epigenetic”
processes of DNA-promoter methylation and histone modification.5,6 DNA methylation is catalyzed by DNA methyltransferases
(DNMTs) and has been shown to be an important contributor to
carcinogenesis, acting by silencing tumor suppressor genes in
many tumor types, including hematologic malignancies.6,7 Chromatin structure is also regulated by the “histone code,” which refers to
posttranslational modifications (ie, methylation, acetylation, phosphorylation, and ubiquitination) of key lysine residues on core
histone proteins.8 These 2 epigenetic processes of DNA-promoter
methylation and histone modifications are clearly interdependent
and coordinated.9-12
DNMT inhibitors such as 5-azacitidine and decitabine have the
potential to reverse promoter hypermethylation in tumor cells,
leading to reexpression of aberrantly silenced genes and inducing
tumor cell death.13 DNMT inhibitors have been demonstrated to be
effective therapy for myelodysplastic syndrome, which is characterized by global promoter hypermethylation.14 Likewise, inhibition
of histone deacetylases (HDACs) with HDAC inhibitors such as
vorinostat can alter the balance in favor of histone acetyltransferases, resulting in an increased acetylation of histone H3K9 and
H3K14 and gene transcription.15
Recently, we performed an integrated analysis to identify
biological pathways underlying relapsed ALL using genome-wide
gene expression arrays, single nucleotide polymorphism arrays,
and DNA methylation arrays.16 We have identified a relapsespecific gene expression signature characterized by up-regulation
of genes involved in regulation of the cell cycle and apoptosis
(BIRC5, FOXM1, and GTSE1), DNA replication and repair
(FANCD2), and nucleotide biosynthesis (TYMS, CAD, PAICS,
ATIC, and DHFR), and by down-regulation of genes involved in
sensitivity to thiopurines and alkylators (MSH6) and glucocorticoids (BTG1 and NR3C1). In addition, our genome-wide methylation analysis revealed a distinctly higher cytosine-phosphateguanine methylation level in the relapsed cohort compared with
diagnosis,16 and for a subset of differentially methylated genes,
concordant down-regulation of mRNA expression was observed,
implicating epigenetic dysregulation in the acquisition of chemoresistance at relapse.
We hypothesized that there may be an existing drug that has an
effect on gene expression that might mimic reversal of the relapse
signature, which may therefore functionally restore chemosensitivity in leukemic blasts. We searched the Connectivity Map (cmap)
database17,18 to “query” our relapse signature and identified the
HDAC inhibitor vorinostat as the top candidate agent that could
potentially endow a chemosensitive gene expression profile. We
validated this finding by demonstrating reversal of the relapse
Submitted December 30, 2011; accepted March 17, 2012. Prepublished online as
Blood First Edition paper, April 11, 2012; DOI 10.1182/blood-2012-01-401687.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2012 by The American Society of Hematology
BLOOD, 31 MAY 2012 䡠 VOLUME 119, NUMBER 22
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BHATLA et al
signature in primary B-lymphoblastic leukemia patient samples
and cell lines treated with vorinostat that correlated with modulation of key epigenetic histone modifications. We further questioned
whether a DNMT inhibitor could reverse relapse-specific promoter
hypermethylation and reexpress aberrantly silenced genes, and
confirmed this in ALL cell lines treated with the DNMT inhibitor
decitabine. Finally, we tested whether “epigenetic reprogramming”
with each agent alone or with the combination of HDAC inhibitor
and DNMT inhibitor may functionally restore chemosensitivity. By
treating primary patient samples and cell lines with vorinostat and
decitabine, we demonstrated in the present study enhanced chemosensitivity that was correlated with modulation of an aberrant
relapse-specific signature. Our data suggest that incorporation of
epigenetic agents into conventional treatment regimens may improve the outcome of relapsed childhood ALL.
Methods
Cells
Primary patient samples were collected from patients treated at the New
York University Medical Center and from the Children’s Oncology Group
cell bank. Samples were collected under the respective center’s institutional
review board–approved cell-procurement protocols for children with ALL.
Informed consent was obtained in accordance with the Declaration of
Helsinki protocol. Of the total 7 samples, 4 were from initial diagnosis and
3 were collected at the time of relapse. Samples were enriched from
diagnostic BM collections by Ficoll-Hypaque centrifugation and resuspended in fresh culture medium (RPMI 1640) containing 20% FBS; 0.01%
insulin, transferrin, and sodium selenide (Sigma-Aldrich) solution; and 1%
penicillin-streptomycin. The diagnosis of ALL was based on morphology
and flow cytometric analysis of the immunophenotype.
The B-lineage leukemia cell lines Reh, RS4:11, and UOCB1; the
B-lymphoma cell line Raji; and the B-myelomonocytic leukemia cell line
MV4:11 were grown in RPMI1640 medium supplemented with 10% FBS,
10mM HEPES buffer, 1% penicillin/streptomycin under 5% CO2 at 37°C.
All cell lines were purchased from the ATCC and were authenticated
according to their protocols (http://www.atcc.org). Stock solutions of
vorinostat (Cayman Chemical) and etoposide were prepared in DMSO,
whereas doxorubicin, cytarabine, and decitabine (Sigma-Aldrich) were
prepared in double-distilled water. Prednisolone (Pharmacia) was suspended in 0.9% NaCl. Drugs were serially diluted in RPMI and added to the
culture medium at the indicated concentrations. Cells were incubated with
chemotherapy for 24-48 hours.
Connectivity map
The cmap is a collection of genome-wide transcriptional expression data
from cultured human cells treated with various bioactive small molecules.
Using a nonparametric, rank-based, pattern-matching strategy based on the
Kolmogorov-Smirnov statistic, this analysis provides a ranked order of
individual treatment instances based on their similarity to a given gene
expression profile. The current version of the cmap dataset (build02;
www.broadinstitute.org/cmap) was used to “query” our relapse-specific
gene expression signature. The relapse gene expression profile was derived
from 49 diagnosis/relapse patient pairs analyzed on Affymetrix U133plus2.0
microarrays.16 Three hundred top-ranking genes (150 down-regulated and
150 up-regulated genes at relapse compared with diagnosis) were chosen
based on a false discovery rate ⬍ 10% and a P value ⬍ .002. Because cmap
contains gene expression signatures derived from the Affymetrix U133A
platform, close to half of our probe-sets could not be analyzed. Therefore,
the final list included 154 probe sets consisting of 56 up-regulated and 99
down-regulated genes (supplemental Table 1, available on the Blood Web
site; see the Supplemental Materials link at the top of the online article).
Gene expression analysis
RNA was isolated from 3 primary patient samples and 3 leukemia cell lines
with or without treatment with 1␮M vorinostat for 24 hours using RNeasy
Mini Kits (QIAGEN), and quality was verified with an Agilent 2100 Bioanalyzer.
In vitro transcription was completed with biotinylated UTP and CTP for labeling
using the ENZO BioArray HighYield RNA Transcript Labeling kit (Enzo
Diagnostics). Thirteen micrograms of labeled cRNA was fragmented and
hybridized to Affymetrix U133Plus2 microarrays according to the Affymetrix
protocol. Raw Affymetrix CEL files were processed with the standard Affymetrix
probe modeling algorithm RMA. Background correction and quantile normalization were applied during the process. The processed data were stored in a matrix
containing one intensity value per probe set in the gene cluster text (GCT) format
file. Analyses were based on the GCT format files with a class label file created in
the categorical class file (CLS) format. Different versions of the CLS files were
created comparing different subsets of samples using applications of gene pattern.
Two independent statistical methods, the ␹2 test and random sampling, were used
to test the significance of changes observed in the relapse-specific signature
(154 probes) compared with the whole gene set (54 676 probes). Expression data
discussed herein were deposited in the National Center for Biotechnology
Information Gene Expression Omnibus (http://www.ncbi.nlm.gov/geo) as accession number GSE34880.
Quantitative RT-PCR analysis
To determine the relative expression of each gene of interest, total RNA was
extracted using the RNeasy Mini Kit (QIAGEN), and RT-PCR was
performed using the I-Script II complementary DNA Synthesis kit (BioRad) and the PerfeCTA SYBR Green FastMix (Quanta Biosciences).
Synthesis of PCR products was monitored by the DNA Engine Opticon
System (MJ Research) and normalized to ␤2 microglobulin levels. Data
were plotted relative to mRNA levels in control samples using the ⌬Ct or
⌬⌬Ct method. PCR primers are listed in the supplemental data (supplemental Table 2). The Wilcoxon paired signed-rank test was used to test the
significance of change.
Western blotting
Reh cells were treated with or without vorinostat (1␮M) and cell lysates
were prepared using the histone extraction protocol (acid extraction)
following the manufacturer’s instructions (Abcam). Lysates were probed
with purified rabbit polyclonal anti-acetyl histone H3 at a 1:5000 dilution
(06-599; Millipore) and the levels of acetyl-H3 were determined relative to
a housekeeping gene, actin, at a 1:2000 dilution (Abcam). Signals were
visualized using the Odyssey infrared imaging system (LI-COR).
ChIP
ChIP was carried out following the protocol provided by EZ-ChIP
(Millipore) with a few modifications. Briefly, Reh cells (1 ⫻ 106) were
grown in RPMI medium with 10% FBS with or without vorinostat (1␮M)
for 24 hours. Proteins were cross-linked to DNA with 1% formaldehyde
added directly to the culture medium for 10 minutes at 37°C, followed by
cell lysis with SDS. The cell lysates were sonicated to shear DNA to the
length of 200-1000 bp. Chromatin solutions were precipitated overnight at
4°C using 2 ␮g of anti-H3K9ac (334481; QIAGEN), anti-H3K27me2
(ab24684; Abcam), anti H3K4me2/me3 (ab6000; Abcam), and antiH3K9me3 (ab8898; Abcam). For the negative control, species-specific IgG
was used. For the positive control, anti-RNA polymerase II (05-623;
Millipore) was used. DNA from protein-associated complexes and corresponding input samples were recovered using the QIAquick PCR purification kit (QIAGEN), and assayed by real-time PCR under standard
conditions. Primers were targeted to the transcription start site (TSS) and
the upstream promoter regions of the genes of interest. Full details,
including the primer sequences, are provided in supplemental Table 2.
Methylation-specific PCR
Genomic DNA was extracted from B-lineage leukemia cell lines (Reh and
UOCB1) and B-lymphoma cell lines (Raji), and sodium bisulfite modification was performed using the Epitect bisulfite kit (QIAGEN) according to
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BLOOD, 31 MAY 2012 䡠 VOLUME 119, NUMBER 22
EPIGENETIC REPROGRAMMING IN RELAPSED CHILDHOOD ALL
the manufacturer’s protocol. Then, methylation-specific PCR (MSP) was
performed as described previously.19 Completely methylated and unmethylated control DNA (Epitect PCR control DNA set; QIAGEN) was tested for
each primer pair. PCR products were analyzed after electrophoresis on 2%
agarose gels containing ethidium bromide. MSP primers are provided in
supplemental Table 2.
relapse-specific genes were reversed after vorinostat treatment,
compared with only 12.8% (mean; 95% confidence interval across
6 samples, 8.8%-16.7%) genes were affected in the whole-genome
analysis (P ⫽ .025 by ␹2 test). This result was also validated using
a random sampling method in which 10 000 random combinations
showed that in only 185 instances could such a finding be
attributable to chance alone (P ⫽ .019). These 2 independent
methods showed a specific effect of vorinostat on the genes
included in the relapse-specific signature. Microarray data were
then validated by RT-PCR in a selected set of candidate chemoresistance genes from our previous study,16 which showed significant
down-regulation of the expression of BIRC5, FOXM1, TYMS, and
FANCD2 (genes up-regulated at relapse; Figure 2A), and an
increase in the expression of NR3C1, HRK, and SMEK2 (genes
down-regulated at relapse; Figure 2B).
Cell-viability assays
CellTiter-Glo Luminescent Cell Viability assays (Promega) were performed
on primary patient samples and cell lines grown in the appropriate survival
conditions. Briefly, cells were seeded in 96-well plates and treated with
vorinostat and/or decitabine (0, 0.1, 0.5, 1, 2.5, and 5␮M). After 24 or
48 hours of incubation, prednisolone, etoposide, doxorubicin, and cytarabine were added to the medium for an additional 24 hours of incubation.
Finally, CellTiter-Glo reagent was added and luminescence was recorded
using a Synergy HT multidetection microplate reader (BioTek Instruments).
Drug-combination effects were analyzed with CalcuSyn software (Biosoft)
using the combination index (CI) equation of Chou-Talalay.20 This application describes the drug interaction as: CI ⬎ 1.1, antagonism; CI ⫽ 0.9-1.1,
additive; CI ⫽ 0.85-0.9, slight synergism; CI ⫽ 0.7-0.85, moderate synergism; CI ⫽ 0.3-0.7, synergism; CI ⫽ 0.1-0.3, strong synergism; and
CI ⬍ 0.1, very strong synergism.
Results
Connectivity map analysis identifies vorinostat as the top
candidate agent that could reverse the relapse-specific gene
expression signature
After the identification of the relapse-specific drug-resistance
signature, we searched the current version of the cmap database to
identify agents that could potentially reverse this signature and
restore chemosensitivity. All treatment instances from the database
were ranked according to their negative connectivity scores with
P ⬍ .05. Remarkably, compounds that ranked among the top were
the HDAC inhibitors vorinostat and trichostatin A. The average
connectivity score of the 12 treatment instances of vorinostat in
various cancer cell lines in the cmap database was ⫺0.659 (P ⫽ 0),
whereas the 182 instances of trichostatin A had a connectivity score
of ⫺0.452 (P ⫽ 0; supplemental Figure 1). This analysis indicated
that the genetic makeup of relapsed ALL might be epigenetically
driven and, therefore, its reversal by HDAC inhibitors could
potentially restore chemosensitivity.
Gene expression microarrays and RT-PCR validates the cmap
results
To validate and confirm the cmap findings, primary patient samples
(n ⫽ 3) and leukemia cell lines (Reh, RS4:11, and MV4:11) were
treated with 1␮M vorinostat and incubated for 24 hours. RNA was
extracted and gene expression profiling was performed using the
Affymetrix U133Plus2 array. The expression of 38.7% (mean; 95%
confidence interval across the 6 samples, 30.51%-46.18%) of genes
differentially expressed at relapse was reversed after vorinostat
treatment using the fold change of 0.8 and 1.2 for down- and
up-regulated genes, respectively, and a P value ⬍ .05 (Figure 1).
Details of individual genes and corresponding reversal on vorinostat treatment are provided in supplemental Table 1. To determine
whether the relapse signature is enriched for genes that have an
expression that is epigenetically regulated compared with the
whole genome, we examined expression changes in genes using a
more stringent fold change of 2. The expression of 17.8% (mean;
95% confidence interval across the 6 samples, 12.7%-22.8%) of the
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Association of histone marks is correlated with vorinostat
exposure as HDAC inhibitor
The biologic impact of vorinostat was validated by showing an
increase in global histone acetylation at the protein level as
measured by Western blot in Reh cells (Figure 3A). ChIP experiments were performed to examine the effect of vorinostat on
specific activating (H3K4me2/3 and H3K9ac) and repressive
(H3K9me3 and H3K27me3) histone marks that modulate gene
expression. Chromatin from the vorinostat-treated and untreated
Reh cells were immunoprecipitated with these Abs, and DNA was
subjected to PCR amplification using primer sets designed to
amplify the transcriptional start site and/or 5⬘ region of the genes of
interest. We observed significant enrichment of the acetylation
histone mark H3K9ac on the promoters of all of the genes tested
(NR3C1, HRK, BIRC5, and FOXM1), indicating a direct effect of
vorinostat in the accumulation of acetylated lysine 9 of histone H3
(Figure 3B). This observation was correlated with the transcriptional activation of NR3C1 and HRK in patient samples and cell
lines after vorinostat treatment. In addition, we observed modest
enrichment of H3K4me2/3 (activation mark) at the NR3C1 and
HRK promoters, supporting the activation of these genes after
vorinostat exposure. Of the repressive marks, we observed a
decrease in the H3K9me3 mark on the promoters of all genes
except FOXM1. The interplay of posttranslational methylation and
acetylation at the same lysine residue (K9) further supports the
effect of vorinostat in regulating the expression of NR3C1 and
HRK, although this effect is not consistent with the observed
down-regulation of BIRC5 and FOXM1. The H3K27me3 mark did
not show significant modulation with vorinostat treatment in any of
the genes tested (Figure 3B).
Treatment with the demethylating agent decitabine leads to
reexpression of relapse-specific hypermethylated genes
Having identified the “methylome signature” of relapsed ALL
previously,16 we sought to determine the impact of the demethylating agent decitabine in reexpression of relapse-specific silenced
hypermethylated genes. Reh and UOCB1 cell lines were treated
with 1␮M decitabine and incubated for 48 (Reh) or 72 (UOCB1)
hours. RNA was extracted and RT-PCR was performed on relapsespecific target genes. We focused on the genes that were identified
from the integrated results of the cross-platform analysis (CDKN2A,
COL6A2, PTPRO, and CSMD116) that were hypermethylated and
down-regulated at relapse and focally deleted in a subset of
samples. In addition, we examined other gene promoters that
showed concordant hypermethylation and down-regulation in our
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BLOOD, 31 MAY 2012 䡠 VOLUME 119, NUMBER 22
Figure 1. Gene expression microarrays in primary B-lymphoblastic leukemia patient samples and cell lines reveal reversal of relapse-specific signature. Heat map
showing the effect of vorinostat in reversing the relapse-specific gene expression profile. Rows represent individual genes (154 probes); relative overexpression is shown in red
and underexpression in green. The first 3 columns are primary patient samples (1, 2, and 3), followed by the Reh, RS4:11, and MV4:11 cell lines, respectively, before treatment
with vorinostat; the next 6 columns correspond to each sample after treatment.
relapse cohort, such as WT1, APC, GATA4, and HOXA9. We
observed a 2- to 200-fold induction of expression of PTPRO,
COL6A2, WT1, GATA4, and HOXA9 (Figure 4A). Reexpression of
CDKN2A was examined in Raji (B-lymphoma cell lines) cells
because both alleles are deleted in Reh cell lines,21 whereas its
expression is lost in UOCB1 cell lines,22 and again observed a 2- to
4-fold induction in its expression. A 6-fold increase in expression
of CSMD1 was observed in UOCB1 cells, whereas no change was
observed in the Reh cell line. No change in the expression of APC
was observed in either cell line tested. As a negative control, we
tested the NR3C1 gene, which is down-regulated at relapse and was
not found to be hypermethylated in our relapse cohort, although it
has been shown to be hypermethylated in other cancer types.23 No
change in the expression of NR3C1 was observed after decitabine
treatment in either cell line.
some unmethylation was also observed at baseline and this was
significantly enriched after treatment with decitabine. The APC
promoter was preferentially unmethylated in both Reh and UOCB1
cell lines, but had a faint methylation band in Reh cells at baseline.
After decitabine treatment, the intensity of the methylation band
was decreased and the unmethylated product was modestly increased in Reh cells, whereas it was unchanged in the UOCB1
cells. Preferential unmethylation of this promoter at baseline in
these cell lines could be the reason for unaltered expression of this
gene as determined by RT-PCR. The NR3C1 promoter was
completely unmethylated before and after exposure to decitabine in
both cell lines, which is the likely explanation for its insensitivity to
decitabine treatment (Figure 4B). MSP for CDKN2A promoter
performed in Raji cell lines showed enrichment of unmethylated
product after decitabine exposure, which is consistent with the
increase in expression shown by RT-PCR.
MSP demonstrates the effect of decitabine on promoter
hypermethylation and validates RT-PCR results
To demonstrate the direct effect of decitabine on promoter hypermethylation, MSP was performed with cell lines before and after
treatment with decitabine. The PTPRO and HOXA9 genes were
completely methylated in Reh and UOCB1 cell lines before
treatment, and an enrichment of the unmethylated amplicon was
observed after treatment with decitabine (1␮M). The GATA4
promoter was preferentially methylated in both cell lines, although
Pretreatment with vorinostat and/or decitabine induces
chemosensitivity in primary patient samples and leukemia
cell lines
Finally, to examine whether epigenetic priming translates to
increased chemotherapy-induced cytotoxicity of leukemic blasts,
we assessed cytotoxicity with a panel of chemotherapeutic agents
commonly used in ALL therapy. Primary patient samples (n ⫽ 7)
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Figure 2. Relative mRNA expression of genes differentially expressed at relapse after exposure to vorinostat. Quantitative RT-PCR validation of selected targets on 3 primary patient samples and Reh, RS4:11, and
MV4:11 cell lines is shown. The x-axis represents the
vorinostat concentration. The y-axis represents normalized ⌬CT values (CT of gene of interest ⫺ CT of the
housekeeping gene). CT indicates threshold cycles for
amplification. Each experiment was performed in triplicate. Mean expression is indicated by the horizontal bars.
P values correspond to Wilcoxon signed-rank test comparing the expression levels before and after treatment.
and Reh and RS4:11 cell lines were treated sequentially with
increasing concentrations of vorinostat and/or decitabine at 0 hours,
followed by application of conventional chemotherapeutic agents
(prednisolone, doxorubicin, cytarabine, and etoposide) at various
concentrations at 24 or 48 hours. Cytotoxicity assays were
performed at 48 or 72 hours. Vorinostat alone induced 40%-60% of
the cytotoxic effect at the maximal concentration of 2.5␮M. An
additive or synergistic effect was observed when cells were
pretreated with vorinostat followed by chemotherapy in Reh and
RS4:11 cell lines (CI ⫽ 0.5-1.05; supplemental Figure 2A-B).
Similarly, decitabine alone induced 25%-40% of the cytotoxic
effect at its maximal concentration used, and the addition of
chemotherapy after decitabine pretreatment significantly increased the amount of cytotoxicity (CI ⫽ 0.34-0.9; supplemental Figure 2C-D). The 2 epigenetic drugs by themselves showed
a mostly additive effect to moderate synergism (CI ⫽ 0.57-1.1),
whereas pretreatment with both vorinostat and decitabine followed by prednisolone had the most robust cytotoxicity compared with any other combination, showing strong synergism
(CI ⫽ 0.17-0.5; Figure 5A-D). As predicted from the cmap
results, we did observe increased cytotoxicity in the relapse
samples (n ⫽ 3) when vorinostat alone was followed by prednisolone (CI ⫽ 0.36-0.6) compared with the diagnosis samples
(n ⫽ 4; CI ⫽ 0.43-1.0; Figure 5C-D), although the comparison
was limited because of the small sample size. These data suggest
that although both the diagnosis and relapse blasts can be
sensitized to chemotherapy, the effect of vorinostat was more
pronounced in the relapsed blast population. We did not see this
effect with decitabine, with which the impact was equivalent at
both initial diagnosis and relapse.
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Figure 3. Histone modifications at the promoters of genes of interest. (A) Western blot analysis of acetyl-H3 (and actin loading control) in Reh cells with and without
vorinostat exposure. (B) ChIP was carried out with Abs specific for key H3 modifications associated with transcriptional activation (K9ac and K4me2/3) and transcriptional
repression (K9me3 and K27me3) and purified DNA was subjected to RT-PCR using primer sets designed to amplify the transcriptional start site and/or the 5⬘ region of NR3C1,
HRK, BIRC5, and FOXM1. Each graph shows the modification of these marks after vorinostat exposure normalized to log input (1%) and the corresponding mRNA expression
of the gene by quantitative RT-PCR. TSS indicates transcriptional start site.
Discussion
ALL is the most common childhood cancer and the majority of
children can be cured with current therapies; however, up to 20% of
children relapse and their outcome remains dismal. Historically,
there have been several obstacles to the successful treatment of
relapsed ALL, and numerous efforts to improve outcome after
relapse have been unsuccessful. Contemporary reinduction regimens have relied on significantly more aggressive approaches with
higher doses and/or compacted drug schedules. These have not
only failed to improve remission rates, but have also reached
tolerability limits, with toxic death rates generally ranging from
3%-8%, but with reports of rates up to 19%.24 Therefore, further
dose intensification is not a viable option for improving outcome.
One of the major challenges faced at relapse is intrinsic chemoresistance.1,2 The results of the present study indicate that the chemoresistance that drives relapse in ALL may be reversible by
epigenetic reprogramming with DNMT and HDAC inhibitors. Our
results indicate that vorinostat reverses significantly the expression
of genes that are differentially regulated at relapse, some of which
are linked to tumor formation and progression, and, most importantly, may have a role in chemoresistance. Of particular interest
are BIRC5 and FOXM1, which are associated with a wide range of
cancers25-28 and were up-regulated in our relapse signature. We
demonstrated that expression of both of these genes could be
decreased after vorinostat exposure. Improving chemosensitivity
by performing knock-down experiments of BIRC5 has been shown
by us26 and others29 and has further supported its role in chemoresistance. Similarly, down-regulation of genes such as NR3C1 and
BTG1 have been linked previously to glucocorticoid resistance,30
and we observed herein increased expression of these genes after
vorinostat treatment. In addition, up-regulation of genes involved
in nucleotide biosynthesis and folate metabolism identified in the
late-relapse (relapse ⬎ 36 months from diagnosis) cohort, such as
TYMS, CAD, and DHFR, was reversed by treatment with vorinostat. The effect of HDAC inhibition on BIRC5, TYMS, and DHFR
expression in B-ALL patient samples and cell lines is consistent
with the results of previous studies in a variety of other tumor
types.31-33 Therefore, we have demonstrated that many genes
differentially expressed at relapse compared with diagnosis are
epigenetically regulated and can be reprogrammed with HDAC
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EPIGENETIC REPROGRAMMING IN RELAPSED CHILDHOOD ALL
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Figure 4. Decitabine treatment of ALL cell lines
shows reexpression of hypermethylated genes at
relapse. (A) Real-time PCR was performed on Reh,
UOCB1 (B-lineage), and Raji (lymphoma) cells treated
with decitabine at a 1␮M concentration for 48 hours (Reh
and Raji) or 72 hours (UOCB1). Results are expressed
relative to the levels observed at baseline without decitabine exposure. Each experiment was performed in
triplicate. Error bars represent SD. (B) Determination of
concordant changes in promoter methylation levels by
MSP in Reh and UOCB1 cell lines. *CDKN2A mRNA
expression levels and MSP were performed in Raji cells
only.
inhibitors. This reversal of gene expression results functionally in
enhanced chemosensitivity of leukemic blasts.
Posttranslational modifications of histone tails, especially acetylation and methylation on lysine residues, play a pivotal role in
regulating gene expression by controlling the access of key
regulatory factors and complexes to chromatin.34,35 Although
HDAC inhibitors are known to activate gene expression by
antagonizing HDACs and opening up the chromatin structure, their
effect on global gene expression patterns remains elusive and needs
further investigation. Whereas our ChIP experiments offer an
explanation for the vorinostat-mediated activation of repressed
genes such as NR3C1 and HRK, we did not observe enrichment of
repressive histone marks as a possible mechanism of vorinostatinduced down-regulation of BIRC5 and FOXM1. Conversely, we
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5208
BHATLA et al
BLOOD, 31 MAY 2012 䡠 VOLUME 119, NUMBER 22
Figure 5. In vitro cell-viability assays in primary patient samples and cell lines (Reh and RS4:11). Summary graph representing the effect of all agents at 48-hour time
point either individually or in combination in Reh cell lines (A), RS4:11 cell lines (B), relapse patient samples (C), and diagnosis patient samples (D) are shown. The x-axis
represents the vorinostat and decitabine concentrations at 0, 0.1, 0.5, 1, 2.5, and 5␮M and prednisolone at 0, 100, 200, 300, 400, and 500 ␮g/mL, respectively. The y-axis
shows the percent survival.
observed reciprocal interaction of acetylation and methylation
marks at lysine 9 on histone H3, which is in agreement with
previous studies showing their mutual exclusivity.35 In light of
several reports suggesting the effect of these agents on various
nonhistone proteins and transcription factors, we speculate that
down-regulation of these genes may be mediated by an intermediate transcriptional regulator restored by HDAC inhibitor exposure.
For example, p21 expression increases with vorinostat treatment in
a dose-dependent manner,36 which in turn might repress FOXM1.37
The resultant decrease in the levels of FOXM1 may affect the levels
of BIRC5, CCNB1, and FANCD2, as described previously.38 The
fact that similar alterations of gene expression patterns were seen
repeatedly and consistently in different primary patient samples
and cell lines strongly favors its unique and specific action.
Recently, Stumpel et al has also demonstrated HDAC inhibitor–
mediated down-regulation of a subset of hypomethylated protooncogenes and resultant chemosensitivity in MLL-rearranged
ALL, which supports our findings (although clear underlying
mechanisms remain undetermined).39 Future studies involving
ChIP sequencing might provide further insight to understand this
complex network.
Genome-wide methylation assays have identified many cancerrelated genes as substrates of DNMTs leading to epigenetic
silencing. In the present study, we were able to show reexpression
of many genes that were hypermethylated at relapse by both
RT-PCR and concordant promoter hypomethylation by MSP. Of
these, CDKN2A, PTPRO, and CSMD1 have been identified previously as tumor suppressors in many cancers,40,41 and reactivation
with demethylating agents is a logical approach. Furthermore, WT1
and APC are inhibitors of the ␤-catenin/TCF/LEF complex and
were differentially down-regulated and hypermethylated in the
relapse cohort. In the present study, we have demonstrated a
significant up-regulation of WT1 after decitabine exposure. However, there was no significant baseline hypermethylation of APC in
the Reh and UOCB1 cell lines, and therefore, our results failed to
demonstrate a decitabine effect. Finally and most importantly, not only
was aberrant gene expression reprogrammed by these 2 agents, but
combination treatment with vorinostat and decitabine synergized with
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BLOOD, 31 MAY 2012 䡠 VOLUME 119, NUMBER 22
EPIGENETIC REPROGRAMMING IN RELAPSED CHILDHOOD ALL
standard chemotherapy agents to result in enhanced chemosensitivity. A
larger sample size would be needed to further compare the effect of
these agents on the diagnosis versus relapsed blasts.
The combination of vorinostat and decitabine has been tested
previously in human leukemia and showed evidence of cell growth
inhibition and gene silencing.42 Recently, Kalac et al also reported a
highly synergistic combination of HDAC inhibitor (panobinostat)
and DNMT inhibitor (decitabine) and association with unique gene
expression and epigenetic profiles in large B-cell lymphoma.43
Although newer HDAC and DNMT inhibitors are under active
investigation, in the present study, we focused on vorinostat and
decitabine not only because they are Food and Drug Administration–
approved, but also because phase 1 trials using these agents have
shown their safety in the pediatric population.44,45 Reversal of the
gene expression and methylation signature in our study was seen
with 1␮M concentrations of vorinostat and decitabine, respectively, a plasma concentration shown to be clinically achievable
and tolerable in these phase 1 clinical trials. Ravandi et al reported
this combination to be well tolerated in a phase 1 study in
hematologic malignancies in adults.46 Additional support for the
feasibility of this combination has been described in adults with
solid tumors and non-Hodgkin lymphoma.47 Moreover, it has been
shown in previous studies that apoptosis induced by these epigenetic agents is specific to the malignant cell population and has
comparatively little or no activity against their normal counterparts
(nonmalignant cells),48-50 suggesting that incorporating these agents
episodically in the standard reinduction chemotherapy for the
treatment of relapsed ALL is feasible.
Overall, the results of the present study reveal an attractive
approach to reversing the drug resistance gene signature to restore
chemosensitivity in relapsed ALL and we validated this approach
in preclinical assays. The data described herein have led to a
multi-institutional phase 1 clinical trial evaluating the feasibility
and safety of using the combination of these epigenetic agents that
is set to accrue patients through the Therapeutic Advances in
Childhood Leukemia and Lymphoma Consortium (http://www.clinicaltrials.gov as NCT01483690). Gene expression and methylation
arrays and subsequent validation studies will be undertaken as part
of correlative biology assays for this clinical protocol. Our data
provide a strong rationale for undertaking such a trial to improve
the cure rates in relapsed childhood ALL, which otherwise has a
dismal prognosis.
5209
Acknowledgments
The authors thank Matthias Karajannis, Julia Meyer, and Laura
Hogan for reviewing the manuscript and Rana Lamisa for technical
assistance.
This research was supported by the National Institutes of Health
(5 R01CA 140729-02 to W.L.C.), the New York University Cancer
Center (support grant 5 P30 CA16087-30 and R21 CA161688 to
M.J.B. and P.B. and K12 NCI CA96028 to M.J.B.), the Pediatric
Cancer Foundation, an Ira Sohn Foundation fellowship grant, and
the Leukemia & Lymphoma Society.
Authorship
Contribution: T.B. and D.J.M. designed and performed the research, collected, analyzed, and interpreted the data, performed the
statistical analysis, and wrote the manuscript; J.W. analyzed and
interpreted the data and performed the statistical analysis; E.A.R.,
M.J.B., and P.B. designed the research and wrote the manuscript;
and W.L.C. designed and directed the research, analyzed and
interpreted the data, and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: William L. Carroll, MD, NYU Cancer Institute, Smilow 1201, 522 First Ave, New York, NY 10016; e-mail:
[email protected].
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2012 119: 5201-5210
doi:10.1182/blood-2012-01-401687 originally published
online April 11, 2012
Epigenetic reprogramming reverses the relapse-specific gene
expression signature and restores chemosensitivity in childhood
B-lymphoblastic leukemia
Teena Bhatla, Jinhua Wang, Debra J. Morrison, Elizabeth A. Raetz, Michael J. Burke, Patrick Brown
and William L. Carroll
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