Novel transcriptional targets of ETV6, a transcription
factor frequently altered in childhood pre-B acute
Camille Malouf1, 2, Karine Lagace1, 2, Claire Drullion1, Sylvie Langlois1, Josiane Larose1, Ekat A. Kritikou1, Christian
Sirard1, Simon Drouin1 and Daniel Sinnett*1-3
Received: 23 September, 2016; Accepted: 10 October, 2016; Published: 20 October, 2016
1Division of Hematology-Oncology, Sainte-Justine University Health Center Research Center, Montreal, Canada H3T 1C5
2Department of Biochemistry
3Department of Pediatrics, Faculty of Medicine, University of Montreal, Montreal, Canada, H3T 1C5
Daniel Sinnett, Sainte-Justine University Health Center Research Center, 3175 chemin de la Côte-Ste-Catherine, Montreal
(Quebec) Canada, H3T 1C5, Tel:+1-514-345-4931 ext. 2990; E-mail:
Malouf C, Lagace K, Drullion C, Langlois S, Sinnett D, et al. (2016) Novel transcriptional targets of ETV6, a transcription
factor frequently altered in childhood pre-B acute lymphoblastic leukemia. Cancer Sci Res Open Access 3(1): 1-11. DOI: http://dx.doi.org/10.15226/csroa.2016.00124
Pre-B Acute Lymphoblastic Leukemia (ALL), the most common
hematological malignancy in children, represents ~25% of all
pediatric cancer cases. The most frequent genetic alteration
associated with pre-B ALL is the t(12;21) translocation, which results
in the expression of the ETV6-RUNX1 chimera. The frequent deletion
of the residual ETV6 allele leads to the complete loss of wildtype
ETV6, a ubiquitously expressed Ets family transcriptional repressor
with few known target genes. We had previously identified candidate
ETV6 transcriptional targets using microarray gene expression
profiling. Here we show, using chromatin immunoprecipitation
experiments and reporter gene assays, that the sphingosine kinase 1
(SPHK1) and prostaglandin E2 receptor EP4 subtype (PTGER4) genes
are direct ETV6 transcriptional targets. Furthermore, ETV6-mediated
transcriptional repression of both genes requiresboth ETV6's pointed
(PNT) and Erythroblast Transformation Specific (ETS) functional
domains, and depends on Ets-Binding Sites (EBS) in the proximal
promoter region of the target genes. Functional studies in leukemic
cells implicated SPHK1 and PTGER4 in cell survival, proliferation,
clonogenic capacity and migration. This study is one of the first to
elucidate the functional role of ETV6 transcriptional targets and to
suggest their role in childhood leukemogenesis.
Acute Lymphoblastic Leukemia (ALL) is the most frequent
pediatric cancer in children and accounts for ~25% of all pediatric
cancers . Precursor B cell ALL (pre-B ALL), the predominant
form of childhood ALL, has been associated with many genetic
abnormalities including chromosomal translocations . The
t(12;21) translocation is the most common genetic aberration
in childhood pre-B ALL, occurring in 25% of pre-B ALL cases
. This translocation leads to the formation of the ETV6-
RUNX1 chimera, an in-frame fusion of an ETV6 (erythroblast
transformation-specific variant 6) allele with an allele of RUNX1
(runt-related transcription factor 1) [3, 4]. The expression of the
ETV6-RUNX1chimera is under the control of the ETV6 promoter,
which leads to the expression of a chimeric transcription factor composed of the N-terminal part of ETV6 fused to the near
complete RUNX1 protein.
ETV6 is a ubiquitously expressed transcription factor of the
Ets family . Unlike most Ets transcription factors that function
as transcriptional activators, ETV6 has been shown to act as a
transcriptional repressor . ETV6 is essential for embryonic
development as ETV6 knockout mice succumb to severe defects
in the vascular network of the yolk sac and to increased apoptosis
of mesenchymal and neural tissues . Using inducible gene
disruption and ETV6-/- chimeric mice, ETV6 was shown to be
involved in the survival/homing of the hematopoietic stem cells
and in the differentiation of the megakaryocytic precursors
within the bone marrow microenvironment [8, 9].
ETV6 has a C-terminal ETS DNA-binding domain and a
pointed (PNT) helix-loop-helix domain required for proteinprotein
interactions . The ETS domain has been shown to
recognize a consensus Ets-binding site (EBS), which consists of
a core GGAA/T sequence with adjacent purine-rich sequences
. The PNT domain is involved in the interactions of Ets
transcription factors with other proteins . Unlike other Ets
proteins, ETV6 has been shown to homodimerize in vivo. This
interaction requiresits PNT domain, which is also required for
its transcriptional repression activity . The central domain
of ETV6 is also implicated in protein-protein interactions with
members of the SMRT/N-CoR/mSin3A/HDAC corepressor
Several lines of evidence indicate that the inactivation of ETV6
is one of the early events in leukemogenesis of ALL . Studies
in transgenic mice [15, 16], zebrafish , and humanized NOD/
SCIDγ mice transplanted with cord blood  have clearly shown
that the ETV6-RUNX1 translocation is not sufficient for pre-B
ALL development, indicating that additional steps are required
for leukemic transformation . The non-translocated ETV6
allele is inactivated through deletion (loss of heterozygosity
(LOH)) in up to 90% of pre-B ALL cases carrying t(12;21) [19,
20]. Interestingly, ETV6 expression is also absent in patients that do not have LOH in the residual allele, suggesting that
other mechanisms contribute to the ETV6 inactivation [21, 22].
Consistent with its tumor suppressor activity, overexpression
of ETV6 in Ras-transformed fibroblasts inhibits cell growth and
enhances apoptosis in vitro and suppresses tumor formation
in nude mice [23-25]. Thus, the complete inactivation of ETV6
appears to be required for leukemogenesis .
A complete understanding of the impact of the loss of
ETV6 in the initiation and progression of leukemia requires
the identification of its downstream target genes. Very few
transcriptional targets of ETV6 are currently known: MCSFR
(macrophage colony-stimulating factor receptor) , GpIbα
and GpIX (platelet glycoprotein Ib alpha chain and IX) ,
stromelysin-1/MMP-3 (matrix metalloprotein-3)  and the
anti-apoptotic protein BCL-XL . Using a microarray-based
approach, we had previously identified several putative ETV6-
regulated genes, including SPHK1 (sphingosine kinase 1) and
PTGER4 (prostaglandin E2 receptor EP4 subtype) .
In the present study, we investigated ETV6-mediated
transcriptional regulation on the proximal promoters of SPHK1
and PTGER4 using Chromatin Immunoprecipitation (ChIP)
and gene reporter assays. We show that SSPHK1 and PTGER4
are direct ETV6 transcriptional targets with ETV6-mediated
transcriptional regulation requiring consensus EBS in the
proximal promoter region. Furthermore, functional studies
revealed that deregulated expression of these novel ETV6
transcriptional targets in Reh, an ETV6-deficient t(12;21) pre-B
leukemia cell line, play a role in the pathophysiology of childhood
Materials and Methods
HeLa cells (immortalized cervical cells from an adenocarcinoma
patient, ATCC #CCL-2TM) were cultured in DMEM 10% FBS (Wisent) at 37°C, 5% CO2. Jurkat cells (immortalized leukemic
T lymphocytes from a 14 years old patient, kindly provided by
Dr. José Menezes), IM-9 cells (Epstein-Barr virus-transformed B
lymphoblast from a multiple myeloma patient, ATCC #CCL-159)
and Reh cells (immortalized t(12;21)+ leukemic pre-B lymphocytes,
ATCC #CRL-8286TM) were cultured in RPMI-1640 10% FBS
(Wisent) at 37°C, 5% CO2. Unless otherwise specified, cell culture
conditions were the same for all experiments.
SPHK1 and PTGER4 promoters were amplified by PCR
(Platinum Taq, Invitrogen) and subcloned in the pGL3-basic
vector (Promega) upstream of the Firefly luciferase gene.
The proximal promoter region tested were as follows: SPHK1
promoter -858/+115 and PTGER4 promoter -855/+601
[Figure 2A]. Full-length ETV6 was subcloned downstream of
the pcDNA3.1 vector's (Invitrogen) CMV promoter. Mutation of
consensus EBS (GGAA→CCAA) was done using the Quick Change
Multi-Site Directed Mutagenesis kit (Stratagene). The PNT and
ETS domains of ETV6 were removed by restriction endonuclease
after inserting, respectively, a PacI or KpnI restriction site at each
end of the domain of ETV6 subcloned in pcDNA3.1. The pLKO.1
constructs harboring the SPHK1 shRNA and PTGER4 shRNA
were purchased from Open Biosystems (RHS4533, RHS4533 and
EHS1001-43247). The integrity of all constructs was verified by
direct sequencing of both strands with the ABI 3730 automatic
sequencer. Primers, shRNA and scrambled shRNA sequences are
presented in Table 1.
Gene reporter assays
HeLa and Jurkat cells were transfected using lipofectamineTM
2000 (Invitrogen) with a combination of constructs: promoter
constructs in pGL3-basic vector (Promega), pRL-CMV vector
Table 1: Primers used for molecular cloning, site-directed mutagenesis and chromatin immunoprecipitation
Sequence (5' to 3')
Cloning : promoters
Cloning : Site-directed mutagenesis
Cloning : shRNA and scrambled shRNA
SPHK1 shRNA (shSPHK1)
(Clone ID: TRCN0000036964)
scrambled SPHK1 shRNA
PTGER4 shRNA (shPTGER4)
(Clone ID: TRCN0000000204)
scrambled PTGER4 shRNA
14834 ChIP.F (+)
14834 ChIP.R (+)
Negative ChIP.F (-)
Negative ChIP.R (-)
(Promega) to normalize the transfection efficiency and ETV6
constructs in pcDNA3.1 vector. 1 μg/mL leucoagglutinin and
50 ng/mL phorbol 12-myristate 13-acetate were added to each
reaction 4-hour after transfection (Sigma Aldrich) in Jurkat cells.
Luciferase activity was measured 48-hour later using the Dual-
Luciferase® Reporter Assay System (Promega) and LLMAX384
LUMinometer (Molecular Devices).
Chromatin immunoprecipitation assays
HeLa cells with inducible ETV6-HA  were incubated for
48-hour with 2 μg/ml doxycycline and IM9 cells expressing
a lentivirus harboring ETV6-HA (pLenti-ETV6-HA) were
cross-linked with 1% formaldehyde for 10 minutes at room
temperature, glycine stopped, lysed, and sonicated to obtain 200-
1500bp DNA fragments. After immunoprecipitation with an anti-
HAantibody (Santa Cruz Biotechnology sc-7392), the precipitates
were reverse cross-linked and treated with proteinase K. The
purified endogenous DNA was analysed by quantitative real-time
PCR. Experimental controls were selected according to a previous
ChIP-chip experiment conducted by our group (unpublished
data): Negative maps to an exon-intron junction region of the
chromosome-associated protein D2 (NCAPD2), 14834 maps to
the proximal promoter of the activator of basal transcription 1
(ABT1) gene and 10505 maps to the proximal promoter of the
cAMP responsive element binding protein 1 (CREB1).
1 x 106 Reh cells were infected at a multiplicity of infection
of 1 in RPMI-1640 10% FBS 8 μg/mL polybrene (Sigma-Aldrich).
Forty-eight hours later 1 μg/μL puromycin (Sigma-Aldrich) was
added to enrich for transduced cells.
Quantitative PCR and immunoblot
Quantitative real-time PCR of mRNA was done as previously
described . For Western blots, 15 x 106 Reh cells were
homogenized in RIPA buffer and quantified using a Lowry assay
(BioRad). Proteins were denatured in sample buffer with SDS
(2%) and 2-mercaptoethanol (2 mM), separated on a 12% SDSPAGE
and electroblotted to a nitrocellulose membrane (Roche).
Non-specific binding sites were blocked using skimmed milk
followed by hybridization with the primary antibodies (SPHK1:
Abcam ab-56414; PTGER4 and GAPDH: Santa Cruz Biotechnology
sc-13602 and sc-31915, respectively) and the secondary
antibodies (Santa Cruz Biotechnology sc-166574 or sc-48167).
Enhanced chemiluminescence (Pierce) allowed protein detection
and their subsequent quantification by densitometry (Adobe
Cell Proliferation Reagent WST-1 (Roche) and cell counting
was used to study Reh cell proliferation. Reh cells were seeded
at 0.4 x 105 cells/mL and grown for four days without renewaing
the culture medium. Formazan dye absorbance was measured at
450 nm with a reference at 750 nm.
DNA damage-induced apoptosis and cell cycle
Reh cells were seeded at a density of 4 x 105 cells/mL in 24-well plates for 12 hours and treated with 2 μM camptothecin
for 4 hours (Tocris Bioscience). Harvested cells were stained
using the FITC AnnexinV/Dead Cell apoptosis kit (Invitrogen).
Cell cycle analysis was done using the FITC BrdU Flow Kit (BD
Pharmigen) where 4 x 105 Reh cells were synchronized for 24-
hour by serum-starvation and cultured in RPMI 1640 10% FBS
medium for another 24-hour. Stained cells were captured and
analyzed using BD Biosciences FACS Aria and BD FACSDiva
Migration of Reh cells was performed for 2-hour in a
ChemoTx® 3.2 mm diameter 96 well-plate (NeuroProbe) with an
input of 2 x 105 cells and CXCL12 as a chemoattractant (ProSpec).
Cell quantification was done using the Cell Proliferation
Reagent WST-1 (Roche) or by cell count using a Coulter counter
After 2 weeks of selection with 1 μg/μL puromycin, 500 Reh
cells infected with lentivirus were plated in quadruplicate in 1
ml of HSC-CFU Basic Human medium (Miltenyi Biotec inc) (1.2%
methylcellulose, 30% FBS, 1% BSA, 0.1mM 2-mercaptoethanol,
and 2mM L-glutamine) onto 35mm tissue culture dishes and
incubated for 18 days in a humidified atmosphere at 37oC and
5% CO2 . Colonies consisting of > 40 cells were counted using
an inverted microscope.
Graphs and statistical analysis were done using the GraphPad
5.0 software. For all experiments, unless otherwise mentioned,
statistical significance was indicated as follows: *: p ≤ 0.05, **: p
≤ 0.01, ***: p ≤ 0.001.
ETV6 expression induces a tumor suppressor phenotype in
ETV6 has previously been shown to have tumor suppressor
capabilities in NIH-3T3 cells [23-25]. To determine whether
ETV6 has tumor suppressor activity in human lymphoblastic
cells, we overexpressed ETV6 via lentivirus infection in REH cells,
with high levels of ETV6 protein in infected cells confirmed by
Western blot [Figure 1A, inset]. ETV6 overexpression reduced
cell proliferation and clonogenic potential of Reh cells compared
to mock infected cells [Figure 1A, B], suggesting that loss of ETV6
can increase the proliferative capacity of t(12;21)-positive pre-B
Etv6 mediates transcriptional repression of SPHK1
and ptger4 via its PNT and ETS functional domains
We have previously identified several genes that were
deregulated early after ETV6-induced expression using
microarray expression profiling . Two of these genes, SPHK1
and PTGER4, were retained for further analysis. To examine
whether ETV6 can bind to these promoters endogenously, we
first conducted ChIP assays in HeLa cells overexpressing HA tagged ETV6 (ETV6-HA). Quantitative PCR (qPCR) analysis
indicated that there was significant enrichment of chromatinbound
ETV6 to genomic fragments encompassing the proximal
promoter regions of SPHK1 and PTGER4 [Figure 2A] in HeLa cells
when using an antibody against HA compared to a non-specific
control antibody [Figure 2B]. Similar ChIP results were obtained
using the IM-9 B lymphoblastoid cell line for the promoters of
SPHK1 and PTGER4 [Figure 2C]. These results indicate that ETV6
can form a chromatin complex on the promoters of its target
To examine the ETV6-dependent modulation of these
genes, we used gene reporter assays in HeLa and Jurkat, two
highly transfectable cells lines, that were co-transfected with a
pcDNA3.1 expression vector overexpressing wildtype ETV6 or
versions lacking either the PNT domain, involved in proteinprotein
interaction, or the ETS domain, required for DNA
binding at the consensus EBS [Figure 3A]. An artificial promoter
containing three consensus EBS located upstream of the
thymidine kinase minimal promoter (EBS3tk) was used as control
for ETV6-mediated transcriptional repression. Following ETV6
overexpression, the activity of EBS3tk was dramatically reduced
in both HeLa and Jurkat cells [Figure 3B,C]. Previous studies on
ETV6 transcriptional targets have shown that ETV6 requires the
PNT and ETS functional domains to repress transcription [23,
25, 27, 28], which we confirmed in our system in both cell lines
[Figure 3B, C].
To assess the ETV6-mediated transcriptional repression of
the target genes, we subcloned a ~2 kb region of the SPHK1 and
PTGER4 proximal promoters along with a region downstream
of the transcription start site that contains putative EBS into
the pGL3 reporter vector [Figure 3A]. Overexpression of
ETV6 in both cell lines resulted in significant transcriptional
repression of all reporter constructs [Figure 3B, C], indicating
that ETV6 can mediate transcriptional regulation of SPHK1 and
PTGER4. Deletion of either PNT or ETS domain resulted in the
loss of ETV6-mediated repression of these target genes in both
cell lines [Figure 3B, C]. These findings indicate that both ETS
and PNT domains, through direct binding to the promoter and
protein-protein interactions, respectively, are required for ETV6-
mediated repressive function.
ETV6 recognizes consensus EBS for transcriptional
Similar to other Ets family members such as ETS1 and
FLI1, ETV6 mediates transcriptional regulation by binding
to a consensus EBS [30-32]. Close examination of the SPHK1
and PTGER4 proximal promoter regions using the Transfac
bioinformatics tool  predicted many consensus EBS. To
delineate the minimal region of the proximal promoter required
for ETV6-mediated repression, truncated versions of the SPHK1
and PTGER4 promoters [Figure 2A] were subcloned in the
pGL3 reporter vector. Co-transfection of the various reporter
constructs with ETV6 in both HeLa and Jurkat cell lines allowed
us to identify these regions:- 168/+115 for the SPHK1 promoter
and -337/+178 for the PTGER4 promoter (data not shown).
Figure 1: Tumor suppressor activity of ETV6 in Reh lymphoblastic cell
line. (A) Overexpression of ETV6 in the Reh ETV6-deficient cell line
inhibits cell growth. Cell proliferation assay of Reh cells (WT) stably
infected with an empty lentivirus (Empty) or expressing ETV6-HA
(ETV6) (inset). Cell counts were performed on triplicate samples and
a representative growth curve from three independent experiments is
shown. Statistical differences between the linear regression slopes (**)
were assessed (days 0 to 4) using pLenti-empty as a reference. (B) Expression
of ETV6 decreases the clonogenic potential of Reh cells. The
clonogenic surviving fraction corresponds to the plating efficiency of
the lentivirus-infected cells normalized to the plating efficiency of the
uninfected (WT) Reh cells (expressed in percentage). Colony counts
were performed in triplicate and shown is a representative clonogenic
assay from three independent experiments. Statistical significance was
assessed using one-way ANOVA with Bonferroni's multiple comparison
Figure 2: ETV6 binds SPHK1 and PTGER4 proximal promoters. (A)
Schematic diagrams depicting the promoter regions of SPHK1 and PTGER4
used for chromatin immunoprecipitation (ChIP) and reporter
gene assay experiments. The transcription start site (TSS) corresponds
to position +1. ETS binding site (EBS) predictions by Transfac are indicated
by the thich black vertical lines. ChIP of the indicated promoter
region in the HeLa (B) or the IM-9 lymphoblastoid (C) cell lines overexpressing
HA-tagged ETV6. ETV6 binding enrichment is depicted as
the fold enrichment of the HA antibody relative to the pre-immune
serum immuno-precipitate, normalized to the negative control. Results
were calculated using the ΔCT log values (mean ± SE). Statistical significance
was assessed using one-way ANOVA with a Dunnett's multiple
comparison post-test (n=2, in triplicates). Statistical significance: p <
0.05 *, p < 0.01 **, p < 0.001 ***.
Changing EBS consensus sequences from GGAA/T to
CCAA/T is sufficient to abolish their function . Using sitedirected
mutagenesis, we tested the ability of the various EBS to
mediate ETV6-induced transcriptional repression in the minimal
promoters of each gene. The SPHK1 minimal promoter has four
putative consensus EBS: -158/-104/-84/+60. Mutation of any of
these resulted in loss of ETV6-mediated repression in both HeLa
and Jurkat cells [Figure 4A, B]. The minimal PTGER4 promoter
has six predicted consensus EBS: -305/-97/-92/-55/-49/+35.
Because of the proximity of EBSs -97/-92 and of -55/-49, these
pairs were mutated simultaneously. All EBS mutant constructs,
with the exception of -305, abolished ETV6-dependant
transcriptional repression [Figure 4C, D]. These results indicate
that ETV6 mediates transcriptional inhibition by binding to EBS
in the proximal promoter region of both these genes.
SPHK1 is a positive regulator of leukemic cell survival,
proliferation, and migration
To assess the biological impact of ETV6 transcriptional
targets, we knocked down their expression in the ETV6-
deficient / t(12;21)-positive Reh cell line  and monitored
cell proliferation, DNA damage-induced apoptosis, clonogenic
potential, and cell migration. SPHK1 mRNA was reduced by
~40% by infecting Reh cells with lentiviruses expressing a
shRNA targeting its sequence (shSPHK1, Figure 5A). Western
blot analysis confirmed this knockdown (data not shown). A
scrambled sequence was used as a negative control (scSPHK1).
The proliferative properties of Reh cells were measured
using the WST-1 colorimetric assay. SPHK1 knockdown resulted
in a significantly slower proliferation of Reh cells, compared
to control over a four day period (linear regression y intercept
p=0.032) [Figure 5B]. However, downregulation of SPHK1 had no
detectable impact on the cell-cycle distribution of a synchronized
cell population 24 hours post-release (data not shown). These
results are consistent with the well characterized role of the
downstream effector of SPHK1, Sphingosine-1-Phosphate (S1P),
on cell proliferation . Indeed, higher levels of SPHK1 allow
for more sphingosine to be converted into S1P via its lipid
kinase activity, which results in enhanced cellular proliferation
. Sphingosine can also be transformed into pro-apoptotic
ceramide via ceramide synthase . Thus, SPHK1 levels are
believed to act as a survival rheostat that balances the cellular
level of the pro-apoptotic ceramide with the anti-apoptotic,
pro-proliferation S1P signal. The role of SPHK1 on apoptotic
pathways was therefore examined in Reh cells treated with
camptothecin, a DNA damaging agent inhibiting the DNA repair
ability of the DNA topoisomerase I. Downregulation of SPHK1
increased the sensitivity of Reh cells to camptothecin-induced
apoptosis [Figure 5C].
The clonogenic potential of leukemic cell lines kept in
suspension culture is reflected in the proportion of cells capable
of maintaining in vitro cell growth. Indeed, the higher the
clonogenic potential of a cell line, the higher the number of cells
capable of autonomous cell growth. To examine the role of SPHK1
on the clonogenic potential of Reh cells colony formation in methylcellulose was assessed on cells expressing SPHK1 shRNA.
Downregulation of SPHK1 considerably decreased the clonogenic
potential of Reh cells compared to cells expressing scrambled
control shRNA [Figure 5D].
Figure 3: ETV6-dependent transcriptional repression requires both the
ETS and PNT functional domains. (A) A schematic representation of the
coding sequence of wild-type ETV6 and its deleted versions (ETV6ΔPNT
and ETV6ΔETS). Amino acid positions are indicated. Reporter gene
assays conducted in HeLa (B) and Jurkat (C) cell lines. "Empty vector"
is the promoter-less pGL3-basic luciferase reporter vector and "EBS3tk"
contains 3 EBS and a thymidine kinase minimal promoter (ETV6 transcriptional
repression positive control). The luciferase value is depicted
as the firefly luciferase value of the pGL3-basic reporter gene vector
relative to the Renilla luciferase value of the pRL-CMV control vector
normalized to the cells transfected with empty pcDNA3.1 vector. Statistical
significance was assessed using two-way ANOVA with Bonferroni's
multiple comparison post-test (n=3, in triplicates).
Figure 4: EBS consensus sites are involved in the ETV6-mediated transcriptional
repression. Site-directed mutagenesis of predicted consensus
EBS located in the basal promoters of SPHK1 (A, B) and PTGER4
(C, D) were performed on the corresponding luciferase reporter constructs.
Gene reporter assays were conducted in HeLa and Jurkat cell
lines. The luciferase value is depicted as the firefly luciferase value of
the pGL3-basic reporter gene vector relative to the Renilla luciferase
value of the pRL-CMV control vector normalized to the cells transfected
with empty pcDNA3.1 vector. Statistical significance was assessed using
two-way ANOVA with Bonferroni's multiple comparison post-test (n=3,
Finally, we assessed the role of SPHK1 on the migration
potential of leukemic cellsusing the CXCL12 chemoattractant in a
transmigration assay. CXCL12 is the ligand for CXCR4, a G-protein
coupled receptor expressed in pre-B leukemic cell lines .
SPHK1 downregulation reduced the migration potential of Reh
cells at low CXCL12 concentration [Figure 5E], indicating that
higher expression of SPHK1 can be favourable to the mobilisation
of leukemic progenitors.
These results indicate SPHK1 is an important regulator of
cell survival, proliferation, and migration in leukemic cells and
suggest that ETV6, by repressing its expression, acts as a tumor
PTGER4 expression promotes leukemogenesis
PTGER4 is a G-protein coupled receptor responding to its
ligand prostaglandin E2 (PGE2). To assess the functional role
of PTGER4 during leukemogenesis its expression was knocked
down by ~65-70% using shRNA infection, as detailed above
[Figure 6A]. Western blot analysis confirmed this knockdown
(data not shown). PTGER4 downregulation, with both shRNAs, led
to a significant decrease in clonogenic capacity and proliferation
rate of Reh cells [Figure 6B], D but did not affect the cell cycle
distribution of synchronized Reh cells over a 24-hour period
(data not shown). Furthermore, PTGER4 repression inhibited
CXCL12-induced migration Figure 6E. Interestingly, it had no
effect on camptophecin-induced apoptosis [Figure 6C].
These data were confirmed by specifically inhibiting PTGER4
with the L-161,982 antagonist, which prevents PTGER4-mediated
signaling . Consistent dose-dependent growth inhibition
was observed in the presence of PTGER4 inhibitor [Figure 7A],
confirming that PTGER4 signaling plays a positive role in Reh cell
proliferation. Cell migration was also inhibited in a dose-dependent
manner with the addition of increasing concentrations of
L-161,982 [Figure 7B]. Camptothecin-induced apoptosis was unaffected
by PTGER4 chemical inhibition [Figure 7C], confirming
the results obtained using shRNA knockdown. These results indicate
that PTGER4 transduces a positive leukemic signal enhancing
cell proliferation and migration in REH cells.
Several childhood pre-B ALL harbor the t(12;21)
translocation, yielding an ETV6-RUNX1 fusion protein thought
to confer ETV6-dependant repressive activity to RUNX1
transcriptional targets. Experimental evidence indicates that
the ETV6-RUNX1 fusion is insufficient to initiate leukemia, which
instead requires the allelic loss of the remaining wildtype ETV6
allele, suggesting that the deregulation of ETV6 transcriptional
targets play an important role in leukemogenesis. However,
functional targets of ETV6 involved in leukemogenesis remain
elusive. Using a combination of ChIP, gene reporter assays, and
loss-of-function analyses we demonstrated that SPHK1 and
PTGER4 are direct transcriptional targets of ETV6 and that they
have a functional role in promoting or maintaining a leukemic
phenotype. Although increased expression of both these genes
has been associated with many types of cancer, this is the first demonstration that their expression could be modulated by the
ETV6 tumor suppressor.
ETV6 binds directly to the promoter of SPHK1 and
ETV6 can both interact directly with the histone deacetylase
co-repressor complex SMRT/N-CoR/mSin3A/HDAC and bind
DNA in a sequence-specific manner [10-13]. Using ChIP analysis,
we have shown that ETV6 can bind the promoter region of SPHK1
and PTGER4 loci. The molecular mechanisms used by ETV6 to
Figure 5: The ETV6-transcriptional target SPHK1 is a positive regulator
of leukemia cell survival, clonogenic potential, and migration. (A) qPCR
analysis of SPHK1 expression in Reh cells stably expressing a SPHK1-
specific shRNA (shSPHK1) or a scrambled shRNA (scSPHK1). Expression
levels were normalized to GAPDH and expressed relative to sc-
SPHK1-expressing cells. Western blot analysis of SPHK1 protein levels
confirms the knockdown of the endogenous protein (data not shown).
(B) Knockdown of SPHK1 expression decreases Reh cell proliferation.
Formazan dye absorbance is proportional to cell density (mean ± SE).
Statistical significance of differences between the linear regression
slopes (*)(days 0 to 4) were assessed, using scSPHK1 as reference (n =
4). (C) SPHK1 is a negative regulator of DNA damage-induced apoptosis
in Reh cells. The percent of apoptotic cells was determined by adding
the fractions that were Annexin V+/ PI+ and Annexin V+/PI- gated outside
the cellular debris (mean ± SE). Statistical significance was assessed
using an unpaired t-test (n = 3). (D) Knockdown of SPHK1 expression
decreases the clonogenic potential of Reh cells. The clonogenic surviving
fraction corresponds to the plating efficiency of shSPHK1-infected
cell normalized to the plating efficiency of the scSPHK1-infected Reh
cells (expressed in percentage). (E) Knockdown of SPHK1 expression
decreases transmigration potential of Reh cells. Relative cell migration
is measured as the ratio of migrated cells/input (mean ± SE). Statistical
significance was assessed using a non-parametric Mann-Whitney t-test
(n = 4).
regulate transcription require both its PNT protein-protein
interaction domain and its ETS DNA-binding domain. In this study,
we have shown that the overexpression of ETV6 in two different
cell lines can actively repress the proximal promoterregion of
both SPHK1 and PTGER4. This repression was dependent on both
ETV6 functional domains, indicating that these genes behave like
typical ETV6 transcriptional targets. Mutational analysis of the
Figure 6: The ETV6 transcriptional target, PTGER4, is a positive regulator
of leukemia cell survival, clonegenic potential, and migration. (A)
qPCR analysis of PTGER4 expression in Reh cells stably expressing PTGER4-
specific shRNA (shPTGER4) or a scrambled shRNA (scPTGER4).
Expression levels were normalized to GAPDH and expressed relative
to scPTGER4-expressing cells. Western blot analysis of PTGER4 protein
levels confirms the knockdown of the endogenous protein (data
not shown). (B) Knockdown of PTGER4 expression decreases Reh cell
proliferation. Formazan dye absorbance is proportional to cell density
(mean ± SE). Statistical differences between the linear regression slopes
(***) were assessed (days 0 to 4) using scPTGER4 as reference (n = 4).
(C) Knockdown of PTGER4 expression has no DNA damage-induced
apoptosis cytoprotective effect. The percent of apoptotic cells was
determined by adding the fractions that were Annexin V+/ PI+ and Annexin
V+/PI- gated outside the cellular debris (mean ± SE). Statistical
significance was assessed using an unpaired Student's t-test (n = 3). (D)
Knockdown of PTGER4 expression decreases the clonogenic potential
of Reh cells. The clonogenic surviving fraction corresponds to the plating
efficiency of shPTGER4-infected cells normalized to the plating efficiency
of scPTGER4-infected Reh cells (expressed in percentage). Statistical
significance was assessed using an unpaired Student's t-test (n
= 3). (E) Knockdown of PTGER4 expression decreases transmigration
potential of Reh cells. Relative cell migration is measured as the ratio of
migrated cells/input (mean ± SE). Statistical significance was assessed
using a non-parametric Mann-Whitney t-test (n = 4).
Figure 7: Inhibition of PTGER4 using a specific inhibitor reveals a
functional role in the leukemic transformation of Reh cells. (A) The
PTGER4 inhibitor L-161,982 prevents proliferation of Reh cells in a
dose-dependent manner. The inhibitor was added at the indicated concentrations
and cell counts were performed over a 5-day period. No
significant difference was observed on cell proliferation exposed to the
different DMSO concentration corresponding to that used with the inhibitors.
Data for the DMSO control, equivalent to that of 40μM inhibitor
is shown. Statistical significance was assessed using two-way ANOVA
with Bonferroni's multiple comparison post-test. (B) The PTGER4 inhibitor
L-161,982 inhibits the migration capability of the Reh cells
through transwells containing 10 ng/mL of CXCL12 chemoattractant.
The controls (C) contain DMSO concentrations equivalent to the ones
used for the various inhibitor concentrations. The negative control (-)
has no CXCL12 attractant, while the positive control (+) has CXCL12.
(C) The PTGER4 inhibitor L-161,982 induces no cytoprotective effect
to DNA damage-induced apoptosis. The percent of apoptotic cells was
determined by adding the fractions that were Annexin V+ and PI+ gated
outside the cellular debris (mean ± SE). Statistical significance for the
migration and apoptosis assays was assessed using one-way ANOVA
with Bonferroni's multiple comparison post-test. All experiments were
repeated three time and representative results shown.
EBS on the minimal promoter region still responsive to ETV6
revealed that they were required for ETV6-mediated repression.
Taken together these data strongly suggest that ETV6 binds
directly to the promoter of these target genes to downregulate
A functional role of SPHK1 in the progression of ETV6-
deficient pre-B ALL
Since the reporter assays revealed that the promoters of both
target genes could be repressed by ETV6, our functional studies
were conducted in the ETV6-deficient pre-B ALL Reh cell line.
We found that the downregulation of SPHK1 expression reduced
cellular proliferation and migration andenhancedapoptosis
in response to DNA damage. SPHK1 is a lipid kinase that
catalyzes the phosphorylation of sphingosine to S1P, which in
turn stimulates cell proliferation, survival and migration by
binding to various S1P receptors (S1PR) . S1PRs activate G
proteins which interact with numerous intracellular pathways
including MAPK/ERKs and PI3K, involved in cell proliferation and survival, as well as with PLC and Rac, both involved in cell
migration. Sphingosine can also be transformed into ceramide,
a pro-apoptotic signal, by ceramide synthase. Increased
expression of SPHK1 would therefore shift sphingosine towards
S1P rather than ceramide and would consequently favor cellular
proliferation, survival, and migration. Our results are consistent
with the known biological function of SPHK1 and attribute a
functional role to SPHK1 in the progression of ETV6-deficient
pre-B ALL. We have also observed that downregulation of SPHK1
results in decreased clonogenic potential of Reh cells, suggesting
SPHK1 levels regulate autonomous cell growth, which would also
contribute to leukemogenesis.
Leukemia can originate from a blockage of differentiation
process in early progenitors and a premature mobilization of
these defective progenitors in the circulation (egress). Gradients
of S1P are directly involved in hematopoietic stem cell (HSC)/
progenitor cell (HPC) egress. High concentration of S1P in the
peripheral blood and low levels of S1P in the bone marrow and
other tissues promote the mobilization of HPC into the circulation
[41, 42]. Recently, crosstalk has been uncovered between CXCR4/
CXCL12 pathway, responsible for the homing of the HPC to the
bone marrow, and the S1P signaling pathway . Increased
levels of S1P activate there lease of the CXCL12 ligand from the
bone marrow stroma tothe circulation and work in concert with
S1P gradients to promote HPC egress from the bone marrow.
Therefore, it is conceivable that increase of SPHK1 expression
in ETV6-deficient ALL progenitors would favor their release
from the bone marrow which, combined with the enhanced selfrenewal
potential and the increase in proliferation, migration,
and survival signals, would contribute to leukemogenesis.
Our findings are consistent with previous observations
documenting the up-regulation of SPHK1 in many cancers,
including acute leukemia . Moreover, although the expression
of SPHK1 has been shown to be regulated by a range of growth
factors and protein kinases, this study provides the first evidence
of SPHK1 regulation by a transcriptional repressor involved in
A functional role of PTGER4 in the progression of
ETV6-deficient pre-B ALL
PTGER4 expression is elevated in numerous cancers including
breast, colon, and endometrial adenocarcinoma and is known to
enhance cell survival and cell proliferation by activating PKB(Akt),
ERK1/2, and early growth response factor-1 (EGR-1) [44-47].
PTGER4 is one of four PGE2 receptors, which in turn is synthesized
by COX enzymes . Overexpression of COX2 has a main role in
the tumorigenesis of breast and colorectal cancer [48, 49], and
has been the target in several therapeutic approaches using nonsteroidal
anti-inflammatory drugs (NSAIDs) similar to aspirin
. Because of side effects related to the use of NSAIDS, mostly
affecting the heart, PTGER4 inhibitors have gained increasing
attention. In this study, we have demonstrated that ETV6 directly
binds to the PTGER4 promoter and represses its expression.
Down-regulation of PTGER4 activity by shRNA or receptor
antagonists in an ETV6-deficient cell line resulted in decreased proliferation, migration, and clonogenic potential, indicating
that the increased expression of PTGER4 could contribute to
leukemogenesis. These results indicate that the PGE2/PTGER4
pathway is involved in leukemogenic transformation and that
increased PTGER4 receptor levels are sufficient to sensitize cells
to transforming processes.
A recent study has uncovered a pivotal role for PGE2/PTGER4
signaling in HSC egress from the bone marrow. While the inhibition
of PGE2/PTGER4 signaling in the stromal microenvironment
causes HSC egress, inhibition of the PGE2/PTGER4 signaling
within the hematopoietic cells favors myeloid hematopoietic
progenitor cell, but not HSC, egress from the bone marrow .
It is difficult to consolidate these results with enhanced PGE2/
PTGER4 signaling in leukemic cells since it would favor homing
of progenitor cells to the marrow. However, the role of PGE2/
PTGER4 in pre-B lymphoid cells was not assessed in that study
and repression of this pathway in the bone marrow stroma in
pre-B ALL has never been investigated. It is noteworthy that
murine ETV6 hematopoietic cells are defective in homing to the
bone marrow of transplanted mice , suggesting that ETV6-
deficient tumor precursor cells could egress rather than enter the
bone marrow. The role of PTGER4 in the egress of these ETV6-
deficient tumor precursor cells remains to be examined.
Overall, this study provides evidence that deregulated
expression of ETV6 transcriptional targets following the loss of
ETV6 might be involved in the development of childhood pre-B
ALL. Further evidence assessing the impact of PTGER4 and SPHK1
dysregulation in patients that lack functional ETV6 could provide
further insights into the mechanisms underlying ETV6-mediated
leukemogenesis. Furthermore, inhibition of the PGE2/PTGER4
pathway or S1P production could provide interesting avenues for
the development of alternative therapeutic approaches.
This work was supported by the Canadian Institutes of Health
Research (#MOP-93518 to D.S.); and the Cancer Research Society
(#14162 to D.S.).
The authors have no potential conflicts of interest and no
competing financial interests. The authors wish to thank the
Canadian Institutes of Health Research and the Cancer Research
Society for their financial support. At the time of the study, C.M.
held a Ph.D. scholarship from the Fonds de Recherche en Santé du
Québec (FRSQ). D.S. holds the François-Karl Viau Chair in Pediatric
Oncogenomics and is a scholar of the FRSQ. C.M. designed and
performed the reporter gene assays and the functional studies,
analyzed the results and wrote the paper. K.L. and C.D. designed
and performed functional studies. S.L. performed the chromatin
immunoprecipitation experiments. J.L. and E.A.K. participated to
the design of the experiments and revised the manuscript. C.S. and
S.D. participated in the design of the experiments with inhibitors
and writing the manuscript. D.S. is the principal investigator of
the research and participated in the design and interpretation of
the results as well as in the writing of the paper.
Conflict of interest statement
The authors report no conflict of interest.
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