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Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma

Authors:

Abstract

Background Diffuse intrinsic pontine glioma is an infiltrative, often high-grade glioma of the brainstem that is not amenable to surgical resection. The current treatment of DIPG by radiation therapy showed dramatically improvement of patient’s condition, however, the tumor recurs rapidly. More and more studies are focused on the genetic and epigenetic drivers of DIPGs, which may provide more and more novel therapy target for DIPG. EZH2 has been proved to be a potential therapeutic target for H3K27M-mutant pediatric gliomas recently. Meanwhile, BET family protein is a hot target in many different types of cancers, including DIPG. In this study, we performed the treatment of both EZH2 and BET inhibitor for DIPG cells. ResultsThe combination of these two inhibitors exhibited better inhibition of the tumor growth both in vitro and in vivo compared to use the inhibitor individually. This inhibition was performed by blocking the proliferation and promoting the cell apoptosis. Meanwhile, combination treatment of these two inhibitors would also affect the epigenetic markers which were abnormal in the tumors of the certain set of genes. Conclusion Thus we provided a novel therapy strategy for clinical treatment of DIPG.
Zhang et al. Cell Biosci (2017) 7:56
DOI 10.1186/s13578-017-0184-0
RESEARCH
Combination ofEZH2 inhibitor andBET
inhibitor fortreatment ofdiuse intrinsic
pontine glioma
Yaqin Zhang1, Weijie Dong2, Junying Zhu3, Lizhu Wang1, Xinjian Wu2* and Hong Shan4*
Abstract
Background: Diffuse intrinsic pontine glioma is an infiltrative, often high-grade glioma of the brainstem that is not
amenable to surgical resection. The current treatment of DIPG by radiation therapy showed dramatically improve-
ment of patient’s condition, however, the tumor recurs rapidly. More and more studies are focused on the genetic and
epigenetic drivers of DIPGs, which may provide more and more novel therapy target for DIPG. EZH2 has been proved
to be a potential therapeutic target for H3K27M-mutant pediatric gliomas recently. Meanwhile, BET family protein is a
hot target in many different types of cancers, including DIPG. In this study, we performed the treatment of both EZH2
and BET inhibitor for DIPG cells.
Results: The combination of these two inhibitors exhibited better inhibition of the tumor growth both in vitro and
in vivo compared to use the inhibitor individually. This inhibition was performed by blocking the proliferation and pro-
moting the cell apoptosis. Meanwhile, combination treatment of these two inhibitors would also affect the epigenetic
markers which were abnormal in the tumors of the certain set of genes.
Conclusion: Thus we provided a novel therapy strategy for clinical treatment of DIPG.
Keywords: DIPG, EZH2 inhibitor, BET inhibitor, Epigenetics, Tumor therapy
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Background
Diffuse intrinsic pontine glioma (DIPG), a tumor located
in the middle of the brain stem, is a fatal malignant pedi-
atric brain tumor, which is the leading cause of can-
cer-related mortality in children [1]. e 5-year survival
rate of DIPG is<1%. e median overall survival of chil-
dren diagnosed with DIPG is approximately 9 months
and the 1- and 2-year survival rates are approximately
30% and less than 10%, respectively [2]. So far, radiother-
apy, which offers a significant but transient improvement,
is the standard treatment of DIPG, while chemotherapy
has not shown any benefit [1, 3]. So more understanding
of the molecular mechanism of DIPG is required to find
the new target and develop more specifically therapeutic
approaches for DIPG.
Nearly 80% of DIPGs harbor histone H3 muta-
tions, wherein lysine 27 is substituted with methionine
(H3K27M) [48]. DIPGs expressing H3K27M mutant
will reduce the global levels of H3K27me3 [9], which is
mediated by PRC2 [10]. Polycomb repressive complexes
(PRCs), including PRC1 and PRC2, mediate gene silenc-
ing by posttranslational modification of histones [11, 12].
e PRC2 complex takes responsibility for trimethylation
of Lys 27 of histone H3 (H3K27me3), this modification
is catalyzed by its enzymatic subunits EZH1 and EZH2
[13]. EZH2 is actively involved in many cellular processes
such as cell cycle progression, cell proliferation, cell dif-
ferentiation and apoptosis [14]. EZH2 mutations have
been found to relate to multiple human cancers [15].
Recent study shows that EZH2 activity is required for the
growth of mouse DIPG tumor cells invitro [16].
Open Access
Cell & Bioscience
*Correspondence: wuxj514@sina.com; shanhong020@gmail.com
2 Neurosurgery Department, The 1st Affiliated Hospital of Sun Yat-
sen University, No. 58 Zhongshan No. 2 Road, Guangzhou 510030,
Guangdong Province, People’s Republic of China
4 Department of Interventional Medicine, The 5th Affiliated Hospital
of Sun Yat-sen University, No. 52 Meihua Dong Road, Zhuhai 519000,
Guangdong Province, People’s Republic of China
Full list of author information is available at the end of the article
Page 2 of 10
Zhang et al. Cell Biosci (2017) 7:56
e bromodomain and extraterminal (BET) family pro-
teins, which playing a key role as epigenetic regulators,
are responsible for the transcriptional activation by inter-
action with acetylated [17, 18] chromatin [19, 20]. BET
proteins regulate the expression of many important onco-
genes, which involved in apoptosis and cell cycle arrest-
ing [2123]. erefore, small molecule inhibitors of BET
proteins have been developed and proved to be active in
both solid and hematologic malignancies, including brain
tumors [24, 25]. JQ1, reported by Filippakopoulos etal.
is a small molecule that competitively binds to bromo-
domains with high potency and specificity. Taylor etal.
found that combination targeting MYCN and NOTCH
by JQ1 and MRK003 inhibited DIPG growth and induced
apoptosis, suggesting this may work as an effective thera-
peutic strategy in DIPG.
In this study, we performed the treatment of both
EZH2 and BET inhibitor for DIPG cells in order to exam-
ine whether combination treatment would be better than
the treatment of the inhibitor individually. is study
was aim to find the new strategy of chemotherapy for the
treatment of DIPG.
Methods andmaterials
Cell lines andculture
NSCs were isolated from the dorsal forebrain of mouse
embryos at E12.5. After the embryos were isolated, the
skin is removed, then the dorsal forebrains were dissected
out and incubated in 0.25% trypsin–EDTA (GIBCO,
Grand Island, NY,USA) at 37°C for 20min. e tissue
was dissociated by pipette thoroughly, and then cultured
in the poly--lysine (PDL, Sigma-Aldrich, St. Louis, MO,
USA)- and laminin (Sigma-Aldrich, USA)-coated plates
in neural stem cell medium. e neural stem cell medium
contained 50% DMEM-F12, 50% neurobasal medium,
N2 and B27 supplements, sodium pyruvate, glutamax,
HEPES, β-mercaptoethanol, non-essential amino acids,
bovine serum albumin, heparin, 100 U/ml penicillin,
100g/ml streptomycin, human recombinant epidermal
and basic fibroblast growth factors. After 3days culture,
the cells were treated with 0.25% trypsin–EDTA and snap
freezed by liquid nitrogen in NSC medium supplemented
with 10% DMSO.
Reagents
DMEM-F12 was bought from GIBCO (USA). e char-
coal-stripped fetal calf serum (FCS) was purchased from
HyClone Laboratories (Inc., Logan, UT, USA). Anti-
mouse Flag-tag, HA-tag, H3, H3K27me3 and GAPDH
were bought from Sigma-Aldrich (Inc, USA). Cell count-
ing kit-8 was bought from Dojindo Laboratories (Inc,
Japan). e EZH2 inhibitor EPZ6438 and BET inhibitor
JQ-1 were from Selleck Chemicals.
Flow cytometry andapoptosis
Cells with different treatments were washed twice in
FACS medium phosphate buffered PBS containing 1%
FCS and 0.1% NaN3. en the cells were washed by
AnnexinV binding buffer for 3 times. After centrifuga-
tion and discuss the supernatant, cells were incubated
for 30min at 4°C with FITC-AnnexinV according to the
standard procedure. PI was added before testing. Fluo-
rescence was measured by using a FACSCalibur (Bec-
ton–Dickinson, San Diego, CA) and data were analyzed
by using the Flowjo Software (Becton–Dickinson, San
Diego, CA).
Chromatin immunoprecipitation (ChIP)
Around 5×107 cells were incubated with 37% Formal-
dehyde diluted to a 1% final concentration for crosslink-
ing for 15min at room temperature. 1M Glycine diluted
to a final concentration of 125mM was added to stop
the crosslink at room temperature for 5min. Cells were
pelleted and resuspent in 5ml of Lysis Buffer (10g/ml
Leupeptin, 10 g/ml Aprotinin, and 1mM PMSF) and
aliquoted to the 1.5 mL eppendorf tubes 500 l each,
then the samples were incubated on ice for 10min. Sam-
ples were sonicated by Bioruptor UCD-200 to generate
500bp–1kb length DNAs. en 500l of each sample
was centrifuged for 10min at 12,000g. Supernatant was
collected and diluted by adding 1ml of Dilution Buffer
(containing the same amount of protease inhibitors as in
Lysine Buffer) and add 5g of the antibody or normal IgG
to the samples. Tubes were incubated at room tempera-
ture for 15min and then secondary antibody was added
into the tubes, after another incubation at room tempera-
ture for 15min, 4 times of washing were performed by
Wash Buffers pre-chilled to 2 to 8°C. After the final wash
and centrifugation, 120l of deionized or distilled water
was added to the system to resuspend the DNAs. 2–10l
of the DNA sample was used in the q-PCR reactions.
Soft‑agar colony formation assay
Cells with different treatments were harvested and pipet-
ted well to become single-cell suspension in complete
culture media in a concentration of 1× 106/ml. en
the cells were incubated at room temperature for using.
10% FBS DMEM was pre-warmed at 37°C and 4% agar
was melted by microwave and keep warm in 56°C water
bath. 0.9ml 4% agar and 4.1ml pre-warmed of 10% FBS
DMEM were mixed well and put into 60-mm culture
dish in the hood. After it became solid, 3 × 104 cells,
2.73ml pre-warmed 10% FBS DMEM and 270l of 4%
pre-warmed agar were mixed together to form the top
gel. After the gel became solid, the dish were incubated
at 37°C for 3weeks. en the colonies were stained with
0.04% crystal violet-2% ethanol in PBS.
Page 3 of 10
Zhang et al. Cell Biosci (2017) 7:56
Western blot analysis
Cells (1×107) were lysed in a buffer containing 20mM
Tris–HCl (pH 7.6), 250mM NaCl, 0.5% NP-40, 3 mM
EDTA and 1.5 mM EGTA with 10 g/ml Aprotinin,
10g/ml leupeptin, 1mM DTT, 1mM PNPP and 0.1mM
Na3VO4 as protease and phosphatase inhibitor. After
centrifugation, cell lysates (100g/lane) were subjected
to 10% SDS-PAGE and transferred onto polyvinylidene
difluoride membranes (Roche, Germany). e mem-
branes were blocked for 1h in TBST (25mM Tris–HCl,
pH 7.6, 125 mM NaCl, 0.1% Tween-20) containing 5%
nonfat dried milk, and then the membrane was incubated
with antibodies against Flag-tag, HA-tag, H3K27me3, H3
or GAPDH was diluted in TBST containing 5% nonfat
dried milk at 4°C overnight. HRP conjugated goat anti-
rabbit or anti-mouse antibodies were used as second
antibodies. All the antibodies were from Sigma. Protein
bands were detected by the Immobilon Western Chemi-
luminescent HRP Substrate (Millipore, Billerica, MA,
USA) and images were taken by FluorChem FC2 System
(Alpha Innotech Corporation, USA).
The cell proliferation assay
e cell proliferation was detected cell counting or by
a Cell Counting Kit-8 according to the manufacturer’s
instructions.
Animals andsurgical procedures
Six-week-old female BALB/c mice were provided by the
animal center in Sun Yat-sen University. All protocols,
described below, were approved by the Animal Care
and Use Committee of Sun Yat-sen University. 1×105
PDGFB/H3K27wt or PDGFB/H3K27M cells suspended
in 1l Hank’s balanced salt solution without Ca2+ and
Mg2+ were injected slowly (over 1min) into the pontine
tegmentum at 5-mm depth from the inner base of the
skull. Inhibitors were injected by intraperitoneal (i.p.)
injection with the amount as indicated in the figure leg-
end 5days after the tumor generation. Mice were moni-
tored daily and recorded the survival rate.
Statistical analysis
Results were expressed as mean ± SD. p values were
determined using two-tailed Student’s t test. p values
were indicated in each figure.
Results
H3K27M is sucient togenerate tumors
As reported, H3K27M-mutant expression in DIPGs is
associated with up-regulation of PDGF signaling [4,
5], so we expressed Flag-tagged K27M H3.3 mutant or
Flag-tagged H3K27wt in mouse neural stem cells (NSCs)
expressing HA-tagged-PDGFB. Figure 1a showed the
NSC cells expressed with both Flag-tagged H3K27M
and HA-tagged-PDGFB exhibited a global reduction of
H3K27me3. Consistent with other’s reports [4, 9, 16, 26],
these PDGFB/H3K27M NSCs had an improved ability of
colony-forming compared to the PDGFB/H3K27wt cells
(Fig.1b). Meanwhile, the PDGFB/H3K27M NSCs grew
faster than the PDGFB/H3K27wt cells, which made the
PDGFB/H3K27M NSCs could generate the larger tumor
when implanted to the pons of the mice (Fig.1c). en we
used mice model to evaluate the tumor formation ability
of the modified NSCs. e mice implanted the PDGFB/
H3K27M NSCs developed larger tumors than the wt cells
(Fig.2a), and PDGFB/H3K27M group had poor survival
rate compare to the PDGFB/H3K27wt (Fig. 2b). ese
results were consistent with the recent research and indi-
cated that H3K27M is sufficient to generate DIPG.
Combination ofEZH2 andBET inhibitors onthe tumor cells
proliferation andapoptosis
Although H3K27M mutant tumor cells exhibited the
global reduction in H3K27me3 levels (Fig. 1a), recent
studies showed that several genes, especially several
tumor-suppressor genes, retained or even showed
increased H3K27me3 levels [27, 28]. Due to this point,
EZH2 activity has been proved to be required for the
growth of mouse DIPG cells invitro and invivo [16]. In
the other hand, BRD2 and BRD4 proteins were found to
co-occupy with H3K27M-K27ac, then logically, the BET
inhibitor was also demonstrated could efficiently inhibit
tumor progression [29]. erefore, we thought these two
inhibitor may both be potential for clinical trial, so we
tested the effect of combination of these two inhibitors.
e cell counting (Fig.3a) and cell viability presented by
cck-8 kit (Fig.3b) indicated that both of EZH2 inhibitor
(EPZ6438) and BET inhibitor (JQ-1) could reduce the
proliferation of PDGFB/H3K27M NSCs. Interestingly,
combination of these two inhibitors exhibited better
reduction compare to only using one inhibitor (Fig.3a,
b). We got the similar results on the apoptosis assay by
FITC-AnnexinV and PI staining. e PDGFB/H3K27M
NSCs showed the very low basal apoptotic ratio (Fig.4a).
Treatment of EPZ6438 or JQ-1 remarkably promoted
the apoptosis and the combination of these two inhibi-
tors showed the further induction of apoptosis (Fig.4a,
b). us, we have demonstrated that EZH2 and BET pro-
teins activity were required for the growth of PDGFB/
H3K27M NSCs, inhibition of these two group of proteins
showed an impressive interfere in tumor progression.
Combination ofEZH2 andBET inhibitors epigenetically
regulated several tumor‑suppressors
Several tumor-suppressors, including p16INK4A and
CDKN2A, have been reported to have the reduced
Page 4 of 10
Zhang et al. Cell Biosci (2017) 7:56
expression due to the retain H3K27me3 activity in
H3K27M induced DIPG tumors [16]. So we were inter-
ested whether combination of EPZ6438 and JQ-1 would
also work on these epigenetic markers. We used ChIP-q-
PCR to test the H3K27me3 levels on the p16INK4A pro-
moter. We could see the increased H3K27me3 on the
p16INK4A promoter and EPZ6438 or JQ-1 would dra-
matically reduce the H3K27me3 level while both of them
showed totally abolish of the H3K27me3 activity (Fig.5a).
Igf2bp2 is a typical gene that will lose H3K27me3 with
the expression of H3K27M, ChIP-q-PCR result showed
that there is no effect of the inhibitors on this type of
genes (Fig. 5b). H3K27M is reported to correlate with
H3K27ac, however, this correlation is excluded by the
PRC2 targets [29]. So we took the HOXA10 as an exam-
ple to test the epigenetic changes in the PRC2 targets by
the treatment of EPZ6438 and/or JQ-1. To our surprise,
although the PRC2 targets also retained the H3K27me3
activity, neither of these two inhibitors would affect
the H3K27me3 levels in the PDGFB/H3K27M NSCs
(Fig.5c). ese results suggested that EPZ6438 and JQ-1
would change the epigenetic marks of the genes which
been abnormally repressed in PDGFB/H3K27M NSCs,
they would not affect the PRC2 targets to change their
original repression pattern. Meanwhile, we detected the
mRNA levels of these three different types of the genes,
Fig. 1 H3K27M make the NSCs gain the tumor activity. a Western blot showing the expression level of Flag-tagged-H3K27M or Flag-tagged-
H3K27wt, HA-tagged-PDGFB, H3K27me3, H3 and GAPDH. b Soft agar colony assay of PDFGB/H3WT or PDGFB/H3K27M NSCs. Data were repre-
sented as mean ± SD, n = 3 independent experiments. ***p < 0.001. c Cck-8 kit was used to evaluate the viability of PDFGB/H3WT or PDGFB/
H3K27M NSCs. Data were represented as mean ± SD, n = 3 independent experiments. ***p < 0.001
Page 5 of 10
Zhang et al. Cell Biosci (2017) 7:56
the expression levels were consistent with the ChIP
results for these three types of the genes (Fig.6).
Combination ofEZH2 andBET inhibitors improved survival
inmice model
Finally, we tested the combination effect of EPZ6438
and JQ-1 on the mice model. e mice were implanted
the PDGFB/H3K27M NSCs in the pons and treated with
EPZ6438 and/or JQ-1 by tail vein injection. Treatment
of the inhibitors individually showed the improvement
in survival and the combination of these two inhibitors
showed a prolonged survival which still had half of the
mice alive after 150days (Fig.7).
Discussion
DIPG is a highly aggressive pediatric brainstem tumor
characterized by rapid and uniform patient demise.
DIPGs usually grow quickly and affect important parts of
the brain. e standard treatment for DIPG is radiation
therapy, although it can dramatically improve patient’s
condition, it usually recur after 6–9months and progress
rapidly. To find the new target and develop more specifi-
cally therapeutic approaches for DIPG became important
to this tumor.
Previous studies uncovered that nearly 78% DIPG
contained histone H3 gene mutations on lysine 27.
Since human has dozens copies of Histones, H3K27M
Fig. 2 H3K27M is sufficient to generate DIPG tumors. a Tumor generated by PDFGB/H3WT or PDGFB/H3K27M NSCs was measured in diameters
and calculated to volume according to the time point. b Survival curve of the mice injected into the pons with PDFGB/H3WT or PDGFB/H3K27M
NSCs (1 × 105). Each group contains 20 mice
Fig. 3 Combination of EZH2 and BET inhibitors reduced the cell proliferation in DIPG cells. a Cell proliferation of different groups as indicated was
determined by cell counting. The concentration of EPZ6438 was 3 μM and JQ-1 was 300 nM, the following in vitro assay used the same amount of
the inhibitors. **p < 0.01 and ***p < 0.001. b Cck-8 kit was used to evaluate the viability of each group of the cells as indicated. Data were repre-
sented as mean ± SD; n = 3 independent experiments. **p < 0.01 and ***p < 0.001
Page 6 of 10
Zhang et al. Cell Biosci (2017) 7:56
therefore constitutes a minor part (3.6–17.6%) of
total Histone 3, this minor part of H3K27 mutation
would make the global reduction of H3K27me3 lev-
els [9]. However, recent work by ChIP-seq analysis
showed that, several sets of genes retain H3K27me3
in H3K27M-mutant DIPGs [16, 2729]. is is quite
interesting cause some of these genes are tumor sup-
pressors and the retaining of the H3K27me3 makes
them keep silencing when the tumor under progress,
which may be the potential target for the clinical treat-
ment. Due to this, EZH2, which is the core member in
PRC2 complex, was found to be required for the DIPG
growth and it inhibitor had the effect on the tumor
transplanted to the mice model. Meanwhile, when the
nucleosomes lose H3K27me3 because of H3K27M
occupation, they usually acquire H3K27ac, which
results in the formation of H3K27M-K27ac heterotypic
nucleosomes [9]. Piunti etal. found that bromodomain-
containing protein 2 (BRD2) and BRD4 showed highly
overlap with H3K27M-occupied sites [29]. is co-
occupancy between bromodomain-containing protein
and H3K27M-K27ac heterotypic nucleosomes indicates
a potential role of BRD proteins in DIPG pathogenesis.
Due to this, they used a well-known bromodomain and
extra-terminal domain (BET) inhibitor, JQ-1, to treat
the DIPG cells and animal model and demonstrated
JQ-1 inhibited the tumor growth both in vitro and
invivo. us, BET inhibitors may also be a promising
therapeutic strategy in DIPG.
Since DIPG is not a single gene disorder, develop more
targets may be benefit in the clinical treatment. In this
study, we test the combination effect of EZH2 inhibi-
tor and BET inhibitor, in order to evaluate whether this
combination is better for the treatment of DIPG. We gen-
erated H3K27M over-expressed mouse NSCs with the
expression of PDGFB, which is proved to be similar to
the human DIPG. en we treated the cells with EPZ6438
and/or JQ-1. Our results showed that each inhibitor
had the effect on the proliferation and apoptosis of the
PDGFB/H3K27M NSCs, interestingly, combination of
these two inhibitors exhibited better effect on suppressing
growth of the tumor cells. In order to provide the detail
mechanism, we examined the epigenetic markers of dif-
ferent kind of genes. p16INK4A is a tumor-suppressor pro-
tein which acts as a cell-cycle inhibitor. Expression level of
p16 will be strongly induced by stress and oncogene acti-
vation. ChIP-q-PCR showed p16 would has an increased
H3K27me3 activity when H3K27M was expressed, this
induction of H3K27me3 levels could be dramatically
inhibited by EPZ6438 or JQ-1. Combination of these two
inhibitors showed the better inhibition of H3K27me3
activity, which can activate the gene expression of p16
and thus to suppress the tumors. Igf2bp2 is another type
of genes, which would lost H3K27me3 activity in the
presence of H3K27M. is is the most cases because
H3K27me3 is globally reduced when H3K27M exists in
the cells. Under this condition, EPZ6438 or JQ-1 would
not affect the H3K27me3 levels, since it is already lost.
Fig. 4 Combination of EZH2 and BET inhibitors promoted the cell apoptosis in DIPG cells. a The apoptosis of the DIPG cells with different treatment
as indicated was determined by using the Annexin-V-FITC & PI Apoptosis Kit and assessed by flow cytometry. n = 3 independent experiments and
this panel presented one of these repeats. b Statistic of percentage of the apoptosis cells performed in a. Data showed the Annexin-V and PI double
positive cells. Data were represented as mean ± SD; n = 3 independent experiments. **p < 0.01 and ***p < 0.001
Page 7 of 10
Zhang et al. Cell Biosci (2017) 7:56
Fig. 5 Combination of EZH2 and BET inhibitors epigenetically regulated tumor-surpressor like p16Inka4. ChIP-q-PCR analysis showing the enrich-
ment of H3K27me3 (left panel) or H3 (right panel) as control over the p16Ink4a gene (a), Igf2bp2 (b) and HOXA10 (c) in the NSCs with different
treatment as indicated. The genes and the primer locations were presented on the top of the panel. Data were represented as mean ± SD; n = 3
independent experiments
Page 8 of 10
Zhang et al. Cell Biosci (2017) 7:56
e last type of genes is the PRC2 targets. HOXA10 is a
typical PRC2 target, which would retain its H3K27me3
levels even in the presence of H3K27M. is retaining of
the H3K27me3 activity would make it keep silencing in
the tumor cells. Interestingly, neither of the inhibitors of
EZH2 and BET works on this type of genes. e results
from SF8628 human DIPG cells revealed a genome-
wide distribution of H3.3K27M that is highly correlated
with active transcription, which were acetylated H3K27
(H3K27ac) and RNA polymerase II (RNA pol II) [29].
However, further study showed that H3K27M colocal-
izes with transcriptionally active chromatin regions were
largely excluded from regions that are occupied by PRC2
and H3K27me3. In this case, PRC2 is almost entirely con-
fined to chromatin regions marked with H3K27me3, and
this mark is mutually exclusive with H3K27ac. If we com-
pare the overlap between H3K27M and H3K27ac binding
regions, it looks like H3K27M occupies many chromatin
regions which EZH2 or PRC2 is unlikely [30]. at is the
differences between the different loci in our Fig. 5 and
maybe the reasons why cells transfected by H3K27M
would be sensitive to EPZ6438 and JQ-1 in the loci like
Fig.5a. us, we demonstrated combination of EPZ6438
and JQ-1 would affected on the tumor suppressors which
is abnormally repressed by H3K27M.
At last, we performed our results on the animal model,
the mice transplanted with PDGFB/H3K27M NSCs were
treated with EPZ6438 and/or JQ-1. Consistent with the
invitro data, the treatment of the inhibitors proved the
survival of the mice and the combination showed the
best effect. We realized it is better to generate DIPG
xenograft model by primary human DIPG cell lines such
as DIPG007, DIPG012, SF7761, SF8628, DIPG017 or
DIPG018, unfortunately, we don’t have the way to get
these cell lines. We also try to generate the cell lines for
the human DIPG, but the specimen is limited to us. e
xenograft model may provide the further information on
the combination effect of these two inhibitors.
Conclusions
In conclusion, in this study, we used combination of
two inhibitors, one response on the inhibition of EZH2,
another is for BET proteins. e combination of two
inhibitors showed the better effect on the treatment of
H3K27M DIPG cells by inhibiting the cell proliferation
and promoting the apoptosis through epigenetic regula-
tion of several tumor suppressor genes. ese may pro-
vide EZH2 and BET proteins as the combined clinical
therapy target for DIPG.
Fig. 6 Gene expression level under the condition of combination of EZH2 and BET inhibitors. Q-PCR analysis showing the mRNA level of p16 (a),
Igf2bp2 (b) and HOXA10 (c) gene with the different treatment as indicated. Data were represented as mean ± SD; n = 3 independent experiments.
*p < 0.05, **p < 0.01 and #p > 0.05
Fig. 7 Combination of EZH2 and BET inhibitors improved survival in
mice model. Survival curve of the mice injected into the pons with
PDFGB/H3WT or PDGFB/H3K27M NSCs (1 × 105). The inhibitor was
given by intraperitoneal (ip) injection with the amount of EPZ6438 by
250 mg/kg and JQ-1 by 50 mg/kg. Each group has 20 mice
Page 9 of 10
Zhang et al. Cell Biosci (2017) 7:56
Abbreviations
DIPG: diffuse intrinsic pontine glioma; PRCs: polycomb repressive complexes;
NSCs: neural stem cells.
Authors’ contributions
Did experiments and analyzed the data: YZ, WD, JZ, LW; design the study
and draft the manuscript: XW, HS. All authors read and approved the final
manuscript.
Author details
1 Department of Radiology, The 5th Affiliated Hospital of Sun Yat-sen Univer-
sity, No. 52 Meihua Dong Road, Zhuhai 519000, Guangdong Province, People’s
Republic of China. 2 Neurosurgery Department, The 1st Affiliated Hospital
of Sun Yat-sen University, No. 58 Zhongshan No. 2 Road, Guangzhou 510030,
Guangdong Province, People’s Republic of China. 3 Department of Radiol-
ogy, The 3rd Affiliated Hospital of Sun Yat-sen University, No. 600 Tianhe
Road, Guangzhou 510630, Guangdong Province, People’s Republic of China.
4 Department of Interventional Medicine, The 5th Affiliated Hospital of Sun
Yat-sen University, No. 52 Meihua Dong Road, Zhuhai 519000, Guangdong
Province, People’s Republic of China.
Acknowledgements
None.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article.
Consent for publication
All participants have given consent for publication.
Ethics approval and consent to participate
All protocols, described below, were approved by the Animal Care and Use
Committee of Sun Yat-sen University.
Funding
This project was supported by Science and Technology Program of Zhuhai
City, 20171009E30011; National Natural Science Foundation of China (NSFC),
81620108017.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Received: 1 August 2017 Accepted: 20 October 2017
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... Recent data suggests that EZH2 activity plays a role in the growth of H3 K27M-mutant DIPG cells in in vivo mouse models [41]. EZH2 is involved in many cellular processes including cell cycle progression, proliferation and apoptosis [41,42]. The mutant H3 K27M binds to EZH2, which interferes with the methyltransferase activity resulting in hypermethylation and increased tumor formation [41,42]. ...
... EZH2 is involved in many cellular processes including cell cycle progression, proliferation and apoptosis [41,42]. The mutant H3 K27M binds to EZH2, which interferes with the methyltransferase activity resulting in hypermethylation and increased tumor formation [41,42]. EZH2 inhibitors have shown promising results in preclinical models in combination with other targets for treatment of DIPG, but more research is needed to better translate this data into human applicability [42]. ...
... The mutant H3 K27M binds to EZH2, which interferes with the methyltransferase activity resulting in hypermethylation and increased tumor formation [41,42]. EZH2 inhibitors have shown promising results in preclinical models in combination with other targets for treatment of DIPG, but more research is needed to better translate this data into human applicability [42]. ...
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Diffuse intrinsic pontine glioma (DIPG) is a type of intrinsic brainstem glial tumor that occurs primarily in the pediatric population. DIPG is initially diagnosed based on clinical symptoms and the characteristic location on imaging. Histologically, these tumors are characterized by a heterogenous population of cells with multiple genetic mutations and high infiltrative capacity. The most common mutation seen in this group is a lysine to methionine point mutation seen at position 27 (K27M) within histone 3 (H3). Tumors with the H3 K27M mutation, are considered grade 4 and are now categorized within the H3 K27-altered diffuse midline glioma category by World Health Organization classification. Due to its critical location and aggressive nature, DIPG is resistant to the most eradicative treatment and is universally fatal; however, modern advances in the surgical techniques resulting in safe biopsy of the lesion have significantly improved our understanding of this disease at the molecular level. Genomic analysis has shown several mutations that play a role in the pathophysiology of the disease and can be targeted therapeutically. In this review, we will elaborate on DIPG from general aspects and the evolving molecular landscape. We will also review innovative therapeutic options that have been trialed along with new promising treatments on the horizon.
... Drugs targeting proteins regulating epigenetic processes (epi-drugs) have gained increasing attention as treatment for hematological and solid malignancies, including DMG. [2][3][4] One key element in organization of the genome are histones and several histone mutations have been discovered that deeply influence transcriptional activity. 5 For example, reduction of histone methylation by Lysine to Methionine mutations at specific histones (K-to-M at H3K36, H3F3A, or HIST1H3B) has profound effects on cell growth through transcriptional modifications, a phenomenon conserved in all living systems, from plants 6 to eukaryotic cell, including cancer cells. ...
... Combinatorial therapies are generally more effective than single treatments in complex malignant disease such as adult or pediatric high-grade glioma. 2,4,[29][30][31] In adult glioma, combined treatment with a dopamine receptor antagonist, quetiapine, and radiotherapy, also induces genes in the cholesterin biosynthesis pathway thereby rendering glioma cells vulnerable to atorvastatin. 29 The combination of a statin and GSK126 seems to be a promising approach, affecting biological processes critical for tumor progression 32 at micromolar doses at which either drug alone does not alter cell growth. ...
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Background Diffuse Midline Glioma, H3K27M-mutant (DMG) is a rare, highly aggressive pediatric tumor affecting the brainstem, and is one of the deadliest cancers. Currently available treatment options such as chemotherapy and radiotherapy do only modestly prolong survival. In this pathology, H3K27 mutations deregulate Polycomb Repressive Complex 2 (PRC2), including enzymatic activity of EZH2, which is therefore under investigation as a therapeutic target. Methods We used a chemical EZH2 inhibitor, GSK126, small interfering RNAs and a CRISPR/Cas9 knockout approaches in a series of DMG tumor cell lines to investigate metabolic treatment responses by proteomic analysis. A combination strategy was elaborated and studied in primary and established DMG cells, spheroid 3D cultures and in vivo in a chick chorio-allantoic membrane DMG assay and an orthotopic intracranial DMG mouse model. Results GSK126 shows significant (p<0.05-0.001) inhibitory effects in in vitro cell proliferation assays and induces apoptosis. Chemical targeting of EZH2 induced expression of proteins implicated in cholesterol metabolism. Low-dose GSK126 treatment together with statins revealed strong growth inhibition in combinatorial treatments, but not in single treatments, both in DMG cells in vitro, in DMG spheroid cultures and in chick and mouse in vivo models (p<0.05). All statistical tests were two-sided. Conclusions Our results reveal an unexpected GSK126-inducible sensitivity to cholesterol biosynthesis inhibitors in highly aggressive pediatric glioma that warrants further evaluation as treatment strategy. This combinatorial therapy should have few side effects because of the low doses used to achieve significant anti-tumor activity.
... HDACi + H3K27 demethylase inhibitor Panobinostat + GSKJ4/1 HDACs + JMJD3 [16] HDACi + BETi Panobinostat + JQ1 HDACs + BRD4 [40] HDACi + CDK7i Panobinostat + THZ1 HDACs + CDK7/9 [40] HDACi + demethylating agent Panobinostat + 5-azacytidine HDACs + DNMTs [42] HDAC + Curaxin Panobinostat + CBL0137 HDACs + FACT [21] BETi + CDK7i JQ1 + THZ1 BRD4 + CDK7/9 [40] BETi + EZHi JQ1 + EPZ6468 BRD4 + EZH2 [43] BETi + HATi JQ1 + ICG-001 BRD4 + CBP [44] HDACi + H3K4me1 histone demethylase inhibitor Corin HDACs + LSD1 [45] Importantly, the inhibition of PRC2 by H3K27M is not equivalent to complete PRC2 loss of function, and despite a global reduction in H3K27me3 in DIPGs PRC2 and its product, H3K27me3, persist at hundreds of genomic loci. This occurs at CpG islands, high-affinity PRC2 binding sites thought to be "strong" PRC2 targets [12,13,17]. ...
... However, as mentioned above, DMG cells had shared resistance to both panobinostat and JQ1 due to their similar transcriptional targets [40]. The combination of JQ1 and EZH2 inhibitor EPZ6438 blocked proliferation, increased apoptosis and extended survival in a mouse model of DMGs [43]. Given the poor BBB penetration of EPZ6438 [46], it is surprising that this drug was able to reduce tumour burden. ...
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... For example, PRC2deficient malignant peripheral nerve sheath tumors are sensitive to inhibition of H3K27ac-reader bromodomain and extra-terminal motif (BET) proteins when combined with MAPK/ERK kinase (MEK) inhibition 16 . Similarly, H3K27M mutations in diffuse intrinsic pontine glioma predispose to sensitivity to both EZH2 inhibition and BET inhibition through the drug JQ1 [17][18][19] . Lastly, GSK-J4, which inhibits histone demethylases and stabilizes H3K27me3, inhibits chemo-resistant lung cancer cells and mammosphere-derived breast cancer stem cells, both of which harbor decreased H3K27me3 [20][21][22] . ...
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... Molecularly these tumors form a distinct cluster by DNA methylation profiling, and frequently have cooccurring mutations in TP53 and ATRX (Korshunov et al., 2016). Unlike H3K27, H3G34 is not subject to posttranslational modifications but instead the H3G34R/V mutation has been found to inhibit SETD2-mediated deposition of H3K36me3 (Zhang et al., 2017a). Unlike K27altered DMGs, the effects of H3G34R/V are only seen in cis (e.g., only mutant nucleosomes have H3K36me3 loss) and accordingly global levels of H3K36me3 are not significantly effected. ...
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... In a panel of diffuse midline glioma patient-derived lines, the EZH2 inhibitors, GSK343 and EPZ6438, slowed the proliferation of H3.3 K27M cell lines but not the H3 WT cells [126]. EPZ6438 significantly improved the survival of mice implanted with H3 K27M neuronal stem cells compared to untreated [161]. The thinking behind the efficacy of this approach is that blocking EZH2 removes the remaining H3K27me3, including that on the p16 gene. ...
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... Combination therapy was able to reverse this subset of SE-associated genes which were inadvertently increased with ICG-001 treatment. Another study used H3K27M/PDGFB expressing NSCs as a DIPG model, and showed that JQ1 synergized with the EZH2 inhibitor EPZ6438 (tazemetostat) in-vitro and in-vivo [80]. Taylor et al. showed that targeting the NOTCH pathway with the γ-secretase inhibitor MRK003 also synergized with JQ1 in 2/3 DIPG cell lines tested [81]. ...
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... tumors. However, there is no information on the status of p53 in these tumors and the neural stem cells used in this study were from the dorsal forebrain [51]. Thus, these studies appear to be inconclusive on whether EZH2 inhibitors would be efficacious in patients with DMGs. ...
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... EPZ6438, an EZH2 inhibitor, and JQ1, a BET inhibitor [61]. Although synergistic interactions were not evaluated, this study showed that the EPZ6438/JQ1 combination inhibited tumour growth to higher levels than each drug individually on DIPG cells from mice. ...
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