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Journal ArticleDOI

554 Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells

01 Nov 2014-European Journal of Cancer (Elsevier BV)-Vol. 50, pp 179-180

AboutThis article is published in European Journal of Cancer.The article was published on 2014-11-01 and is currently open access. It has received 1 citation(s) till now. The article focuses on the topic(s): EHMT2 & Cancer epigenetics.

Topics: EHMT2 (63%), Cancer epigenetics (63%), Histone methyltransferase (62%), EZH2 (56%)

Summary (2 min read)

Jump to: [Introduction][Background][Results][Discussion] and [Conclusions]

Introduction

  • Many cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases, also known as Background.
  • The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing.
  • Indeed, expression of certain genes is only induced upon dual inhibition.
  • The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s.
  • The authors have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and they report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.

Background

  • EZH2 along with EED and SUZ12 are the indispensible core components of the Polycomb Repressive Complex (PRC2) responsible for maintenance of the repressive epigenetic mark H3K27me3: trimethylation of lysine 27 of histone 3 [1].
  • This article International License (http://creativecommo reproduction in any medium, provided you link to the Creative Commons license, and Dedication waiver (http://creativecommons article, unless otherwise stated.
  • Ated with gene amplification, has been well documented in a variety of cancers [2], [3].
  • Combining this evidence, it would again suggest that specifically targeting either EZH2 or EHMT2 alone may not be sufficient to reverse epigenetic silencing of genes, but rather combined inhibition may be required.
  • To this end, the authors have examined the effect of dual EZH2 and EHMT2 gene knockdown or enzyme inhibition in breast cancer cells.

Results

  • Each group has been compared to the untreated sample following normalisation to GAPDH.
  • All hit compounds showed a dose-dependent increase of KRT17, FBXO32, as well as JMJD3 mRNA.
  • Almost no enrichment was observed of this gene set in MDA-MB-231 cells after treatment with any of the compounds (HKMTI-1-005, GSK343 and UNC0638) (Fig. 4a), suggesting that EZH2 has cell-type-specific targets.
  • Together, these data strongly support that the hit compound HKMT-I-005 reduces levels of H3K27me3 and H3K9me3 at concentrations of compound that are less or equivalent to the growth inhibition IC50 concentration for MDA- MB-231 (Table 2).

Discussion

  • It is widely accepted that the installation, maintenance and functional output of epigenetic modifications occur in concert via combinatorial sets of modifications.
  • This suggests that the compounds are able to elicit a transcriptional response that is specific to a particular cell line, and thus, represent a means of tailoring the response to the targets that are specifically epigenetically repressed in the cancer cells to be treated.
  • SPINK1 expression is increased upon treatment with HKMT-I-005, HKMT-I-011 and HKMT-I-022 at multiple doses and time points.
  • Genome-wide expression analysis revealed that genes upregulated upon treatment with HKMTI-1-005 were more enriched for genes silenced by EZH2 than treatment with either the specific EHMT2 inhibitor UNC0638 or the specific EZH2 inhibitor GSK343.
  • The hit compounds reported herein represent starting points for the further optimisation of dual EZH2/ EHMT2 inhibitors.

Conclusions

  • Many cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases, including EZH2 and EHMT1/2.
  • The authors have shown that combined inhibition of EHMT1/2 and EZH2 increases growth inhibition in tumour cells over inhibition of only EHMT1/2 or EZH2 and results in re-expression of silenced genes.
  • The authors report the first dual EZH2-EHMT1/2 substrate competitive inhibitors and show that they may have greater activity in tumour cells that overexpress wild type EZH2.

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RES E A R C H Open Access
Dual EZH2 and EHMT2 histone
methylt rans ferase inhibition increas es
biological ef ficacy in breas t cancer cells
Edward Curry
1
, Ian Green
2
, Nadine Chapman-Rothe
1
, Elham Shamsaei
1
, Sarah Kandil
1
, Fanny L Cherblanc
3
,
Luke Payne
2
, Emma Bell
2
, Thota Ganesh
4
, Nitipol Srimongkolpithak
3
, Joachim Caron
3
, Fengling Li
5
,
Anthony G. Uren
6
, James P. Snyder
7
, Masoud Vedadi
5
, Matthew J. Fuchter
3*
and Robert Brown
1,8*
Abstract
Background: Many cancers show aberrant s ilencin g of gene expression and overexpression of histone
methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin
histone methylation mar ks H3K27me and H3K9me, respectively, which are associated with transcriptional
silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3
by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with
multiple repressive marks.
Results: We report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are
markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition,
rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We
sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay,
based
on the substrate competitive EHMT2 inhib itor BIX01294, we have id entified proof-of-concept compounds that
induce re-expression of a s ubset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation
verified a decrease in silencin g marks and an increase in permissive marks at the promoter and transcripti on start
site of re-expressed genes , while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3.
The co mpou nds inhibi t growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar
IC50s. Bi ochem ically, the compounds are substrate compet itive inhib itors against both EZH2 and EHMT1/2.
Conclusions: We have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting
biological responses of gene transcripti on and cancer cell growth inhibition compared to inhibition of single
HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.
Background
EZH2 along with EED and SUZ12 are the indispensible
core components of the Polycomb Repressive Complex
(PRC2) responsible for maintenance of the repressive
epigenetic mark H3K27me3: trimethylation of lysine
27 of histone 3 [1]. High expression of the histone
methyltransferase (HKMT) EZH2, in some cases associ-
ated with gene amplification, has been well documented
in a variety of cancers [2], [3]. EZH2 overexpression has
been linked to poor prognosis [4, 5] and shown to be a
marker of aggressive breast cancer [6], associated with
difficult-to-treat basal or triple negative breast cancer [7].
Gene knockdown of EZH2 reduces growth of a variety of
tumour cell types [5, 8, 9]. Several groups have reported
specific co-factor competitive EZH2 inhibitors [1016],
which have shown a strong capacity to reduce growth of
cells expressing mutated forms of EZH2 (such as certain
non-Hodgkins lymphoma [12]). However, removal of the
repressive mark H3K27me3 alone may not always be
* Correspondence: m.fuchter@imperial.ac.uk; b.brown@imperial.ac.uk
3
Department of Chemistry, Imperial College London, South Kensington
Campus, London SW7 2AZ, UK
1
Department of Surgery and Cancer, Ovarian Cancer Action Research Centre,
Imperial College London, Hammersmith Hospital Campus, London W12 ONN, UK
Full list of author information is available at the end of the article
© 2015 Curry et al.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Curry et al. Clinical Epigenetics (2015) 7:84
DOI 10.1186/s13148-015-0118-9

sufficient for reversal of gene silencing. Indeed, it has been
shown that highly specific EZH2 inhibitors require a mu-
tant EZH2 status to inhibit cell growth, being less effective
in cells solely expressing wild type EZH2 [5, 8, 9]. Elimin-
ation of further repressive methylation marks by inhibition
of additional HKMTs may be required to fully realise the
epigenetic potential of HKMT inhibitors.
EHMT2 (also known as G9a) and the highly homolo-
gous EHMT1 (also known as GLP) are HKMTs partly
responsible for mono- and di-methylation of lysine nine of
histone 3 (H3K9me1 and H3K9me2, respectively); re-
pressive chromatin marks found on the promoter re-
gions of genes that are often aberrantly silenced in
cancer [17]. EHMT2 is overexpressed and amplified in
various cancers including leukaemia, prostate carcin-
oma, and lung cancer, with gene knockdown of EHMT2
inhibiting cancer cell growth in these tumour types [18,
19]. BIX-01294 (see Fig. 2) was pre viously identified as
an inhibitor of the HKMTs EHMT2 and EHMT1, a nd
subsequent medicinal chemistry studie s around the 2,
4-diamino-6, 7-dimethoxyquinazoline template of BIX-
01294 have yielded a number of follow-up EHMT2
inhibitors [2025].
In addition to its role in methylating H3K9, EHMT2
has been shown to be able to methylate H3K27 [26,
27]. It has been suggested that this c ould provide cells
with a me chanism to compensate in part for a loss of
EZH2 [28]. The picture is further complicate d by re cent
evidence that EHMT2 and EZH2 (via the PRC2 com-
plex) interact physically and s hare targets for epigenetic
silencing [29]. Combining this evidence, it would again
suggest that spe cifically targeting either EZH2 or
EHMT2 alone may not be sufficient to reverse epigen-
etic silencing of genes , but rather combined inhibition
may b e required. To this end, we have examined the
effect of dual EZH2 and EHMT2 gene knockdown or
enzyme inhibition in breast cancer cells. Consistent
with the requirement for removal of both repressive
H3K9 and H3K27 methylation marks, we show that
dual inhibition of EHMT2 and EZH2 pharmacologically
or by SiRNA is necessary for reactivation of certain
genes and induces greater inhibition of cell growth than
targeting either HKMT alone in triple negative breast
cancer MDA-MB-231 cells. Further, we have identified
proof-of-concept compounds which are dual (substrate
competitive) EZH2-EHMT1/2 inhibitors.
Results
Combined inhibition of EZH2 and EHMT2 is more
effective at inducing gene re-expression and inhibiting
tumour cell growth than single HKMT inhibition
SiRNA knockdown in the MDA-MB-231 breast tumour
cell line was used to examine the effect of combined in-
hibition of EZH2 and EHMT2 expression on epigenetic
regulation at select target genes, compared to knockdown
of either gene alone (Fig. 1a). Knockdown of EZH2 with
two independent SiRNAs induced 24-fold increased
mRNA levels of KRT17 and FBXO32, genes which are
known to be silenced in an EZH2 d ependent manner [30].
Knockdown of EHMT2 (G9a) had limited effects on
mRNA levels of these target genes. However, double
knockdown of EZH2 and EHMT2 had dramatic effects on
the level of SPINK1 mRNA, a gene which was not upregu-
lated by silencing of EZH2 or EHMT2 individually. Thus,
for at least certain genes, dual reduction in EZH2 and
EHMT2 levels is necessary to observe marked changes in
target gene expression 48 h following knockdown.
The effects on gene expression of the selective EZH2
inhibitor GSK343 [10] and the selective EHMT2 inhibi-
tor UNC0638 [22] (Fig. 2) used alone or in combination
were also examined using the MDA-MB-231 triple
negative breast cancer cell line (Fig. 1b). When MDA-
MB-231 cells were treated with the EZH2 inhibitor
GSK343 at 115 μM for 48 h alone, there was little
change in the mRNA levels of KRT17, FBX032 and
SPINK1 and the H3K27 demethylase JMJD3 (Fig. 1b).
UNC0638 at 115 μM for 48 h alone showed dose-
dependent upregulation of FBX032 and JMJD3; however,
KRT17 and SPINK1 mRNA levels were not significantly
altered. However, the combination treatments with
GSK343 and UNC0638 showed marked increase in
mRNA levels of all the target genes, in contrast to the sin-
gle agent treatment. Consistent with dual EZH2/EHMT2
SiRNA knockdown, SPINK1 has the biggest change in
mRNA levels between the single and combination treat-
ments, having a 50-fold increase with the combination
treatment. Global levels of H3K27me3 and H3K9me3 in
MDA-MB-231 cells were examined following treatment
with GSK343 and UNC0638 as single agents and in
combination (Additional file 1: Figure S1 ). Together,
these data show a minor decrease in global levels of
these silencing marks following treatment with either
GSK343 or UNC0638 as single agent s across a range of
doses yet a strong dose-dependent decrease in the levels
of these marks when cells are treated with these com-
pounds in combination. This provides further compelling
evidence for the efficacy of dual HKMT inhibition in
reversal of epigenetic silencing in cancer cells.
Next, the effects on cell viability of GSK343 and
UNC0638 used alone or in combination were examined
(Fig. 1c). Treatment alone with GSK343 showed no
significant reduction in cell viability up to 15 μM, while
UNC0638 sole treatment caused a dose dependant reduc -
tion in cell viability, with a calculated IC
50
of 9 μM. When
the cells were treated with both compounds in combin-
ation, a marked increase in growth inhibition was observed
when compared to single agent treatment using UNC0638
or GSK343 (Fig. 1c). This is particularly apparent at a
Curry et al. Clinical Epigenetics (2015) 7:84 Page 2 of 12

5 μM concentration of both compounds, where alone they
have no significant effect on reducing cell viability, while in
combination, they markedly reduce cell viability to >50 %
(p < 0.01).
Analogues of an EHMT2-specific inhibitor can upregulate
EZH2 silenced genes
Both EZH2 and EHMT1/2 belong to the SET-domain
superfamily [31], the catalytic SET-domain being respon-
sible for the methylation of the targeted lysine residues.
BIX-01294 has previously been shown, both structurally
and biochemically, to bind to the substrate (histone)-
binding pocket of EHMT1/2 [32]. Since protein recogni-
tion motifs for histone binding at repressive sites are
similar [33] and EHMT2 ha s been shown to be able to
methylate H3K27, in addition to its more common
H3K9 target [27], it is likely that there are common as-
pects to the histone substrate binding pockets of the
repressive HKMTs EZH2 and EHMT1/2. We therefore
felt it would be feasible to use quinazoline template of
BIX-01294 in the discovery of dual (substrate competi-
tive) EZH2-EHMT1/2 inhibitors.
A compound library based on the selective BIX-01294
EHMT2 inhibitor was synthesised and characterised
analogously to previously reported methods [2022, 24,
25, 32]. In light of the reported selectivity of this chem-
ical scaffold towards EHMT1/2, the library was primarily
examined for compounds show ing additional EZH2 in-
hibitory activity, as defined by re-expression of KRT17
and FBXO32, genes which are known to be silenced in
an EZH2 dependent manner [30]. The majority of com-
pounds had little or no effect on both KRT17 and
FBXO32 RNA levels. However, we identified three com-
pounds which upregulate KRT17 and FBXO32 RNA
levels. The data for these compounds along with a com-
parison of the related EHMT2 inhibitors BIX-01294 and
Fig. 1 MTT and mRNA levels in MDA-MB-231 cells after pharmacological inhibition and siRNA knockdown of EZH2 and EHMT2(G9a), individually
and in combination. a Expression levels of KRT17, FBX032, JMJD3, EZH2, SPINK1 and EHMT2 were measured by qRT-PCR in the MDA-MB-231 cell
line 48 h after transfection with siRNAs targeting EZH2 and EHMT2, both individually and in combination. All measurements were normalised to
the fold-change (relative to GAPDH) in the mock transfection control. Error bars represent the mean ± SD of experiment performed in technical
triplicate. b Expression levels of KRT17, FBX032, JMJD3 and SPINK1 were measured by qRT-PCR in the MDA-MB-231 cell line treated for 48 h with
GSK343, UNC0638, and UNC0638 (at 7.5 μM) with increasing doses of GSK343. Each group has been compared to the untreated sample following
normalisation to GAPDH. Error bars represent the mean ± SD of experiment performed in technical triplicate. c MTT assay for cell viability of MDA-MB-
231 cells after treatment. MDA-MB-231 cells were seeded in 96 well plates. After 24 h, increasing doses of GSK343, UNC0638 or combination treatments
(1, 2.5, 5, 7.5, 10 and 15 μM) were added to cells. Control was media with 0.5 % DMSO. Cell viability was measured by MTT assay after a 48-h treatment
and a 24-h proliferation period. Error bars represent the mean ± SEM of five independent repeats
Curry et al. Clinical Epigenetics (2015) 7:84 Page 3 of 12

UNC0638 and a representative number of negative com-
pounds are shown in Table 1 (for chemical structures see
Fig. 2 and the Additional file 2 for hit characterisation
data). All hit compoundsHKMTI-1-005, HKMTI-1-011,
HKMTI-1-022showed upregulation of KRT17, FBXO32,
and JMJD3 mRNA at a 10 μM dose. The reported
EHMT2-specific inhibitors BIX-01294 and UNC0638,
while being closely related to our hit s from a chemical
structure perspective, elicit different effe cts on expres -
sion of the target genes. BIX-01294 (Table 1, entry 4)
does not upregulate KRT17 but does upregulate
FBXO32. This is compatible with the observation that
FBXO32 i s regulated via multiple mechanisms , poten-
tially r esponding to a variety of factors [34]. An analo -
gous effect is observed for UN C0638 (Table 1, entry 5).
The specific EZH2 inhibitor GSK343 has no effect
whatsoever on all the target genes studied ( Table 1,
entry6)whenexaminedupto 72 h following treatment
and at concentrations up to 10 μM.
To further evaluate the three hit compounds identi-
fied, we treated MDA-MB-231 cells for 48 and 72 h at
various concentrations of compounds and examined
gene expression effects (Fig. 3a). All hit compounds
showed a dose-dependent increase of KRT17, FBXO32,
as well as JMJD3 mRNA. Higher doses of certain com-
pounds started to cause cell death, and because of this,
at these doses, the expression of KRT17 was often below
the detection limit of low-expressed genes.
Chromatin immunoprecipitation (ChIP) experiments
were carried out on treated MDA-MB-231 cells to
verify that the detected gene upregulation is indeed due
to chromatin remodelling (Fig. 3b). We tested the silen-
cing marks H3K9me3 a nd H3K27me3 as well as the
activating mark s H3K4me3, H3K4me2, H3K27ac and
H3K9ac. All three compounds showed a clear decrease
in repressive chromatin mark s (H3K27me3, H3K9me3),
and at least in some instances, an increase in permissive
marks, at two target genes (Fig. 3b). This is consistent with
the compounds having dual HKMT inhibitory activity in
removing both H3K9me and H3K27me marks, while
allowing activating marks to be established at these loci.
Genome-wide changes in gene expression
Agilent microarrays were used to perform gene expression
profiling in MDA-MB-231 breast cancer cells after 24 h of
treatment with the hit compound HKMTI-1-005, the
EZH2 inhibitor GSK343 [10] and EHMT2 inhibitor
UNC0638 [22]. To validate the finding of the initial ex-
pression data for the hit compounds, a second microarray
experiment was performed on the same platform using
HKMTI-1-005-treated MDA-MB-231 cells after 24 h of
treatment. To assess the extent to which our selected
Table 1 RT-PCR data for a single dose of a panel of HKMT
inhibitor compounds
Entry Compound KRT17 FBXO32 JMJD3 EZH2
1 Hit HKMTI-1-005 4.05 3.65 3.12 0.63
2 Hit HKMTI-1-022 4.28 29.4 11.56 0.21
3 Hit HKMTI-1-11 6.95 33.25 6.25 0.22
4 G9ai BIX01294 1.06 3.34 2.7 0.87
5 G9ai UNC0638
a
1.1 5.5 3.4 0.4
6 EZH2i GSK343 0.9 1.2 1.0 1.0
7 Negative HKMTI-1-002 0.66 1.12 1.57 0.86
8 Negative HKMTI-1-012 1.32 1.06 0.9 1.38
9 Negative HKMTI-1-013 0.78 0.93 0.87 0.13
RNA levels for target genes are normalised against the housekeeping gene
GAPDH, and shown is the fold increase compared to the mock treated sample
a
UNC0638 treatment at 7.5 μM, all other compounds given at 10 μM
Fig. 2 Chemical structure of histone lysine methyltransferase inhibitors
Curry et al. Clinical Epigenetics (2015) 7:84 Page 4 of 12

analoguesderived from the selective EHMT1/2 inhibi-
tor BIX-01294had gained EZH2 inhibitory activity,
lists of genes activated or represse d following siRNA
knockdown of EZH2 in MDA-MB-231 cells were identi-
fied [35] a nd shown in A dditional file 3: Table S4. These
lists of target genes were investigated in the context of
genome-wi de changes in gene expression following
treatment with the compounds. HKMTI-1-005 showed
very significant enrichment for upregulation of EZH2 si-
lenced genes ( Fig. 4a) in both the initial array (p =
4.53x10
43
) and the validation array (p = 1.99x10
49
).
GSK343 and UNC0638 also both showed a significant
upregulation of EZH2 t arget genes (Fig. 4a) thoug h to a
lesser extent than HKMTI-1-005. Indeed, analysis of
the difference in systematic upregulation showed that
HKMTI-1-005 upregulated EZH2-silenced ge nes sig-
nificantly more tha n either GSK343 (p =5.8x10
5
)or
UNC0638, (p =1.7x10
4
).
The same enrichment test s were repeated using target
gene set s identified in an EZH2 siRNA knockdown
study in another breast cancer cell line, MCF-7 [30].
Almost no enrichment wa s observed of this gene set in
MDA-MB-231 cells after treatment with any o f t he
compounds (HKMTI-1-005, GSK343 a nd UN C0638)
(Fig. 4a), suggesting that EZH2 has cell-type-spe cific
targets. To investigate this further, we undertook a
meta-analysis to identify consensus target genes based
on 18 independent E ZH2 siRNA studies (d etails of the
meta-analysis are provided in Methods also see
Additional fi le 4: Table S5). Encouragingly, treatment
of MDA-MB-231 cells with HKMTI-1-005 resulted in
highly significant upregulation of these consensus
EZH2-repressed genes (Fig. 4a). This suggests that key
EZH 2 ta r g e t ge n e s that are conser ved across a wide
range of cell lines are re-expressed upon treatment with
our dual HKMT inhibitor. Furthermore, this identifies
Fig. 3 Effects of hit compounds on RNA levels and histone marks. a Sybr green real-time PCR mRNA level measurement of EZH2 target genes
and executing enzymes following a 48-h compound treatment at different concentrations of MDA-MB-231 cells. Measurements marked with an * are
below detection limit, most likely due to cell death. All RT-PCR experiments were performed in triplicate, normalised to GAPDH and displayed as fold
difference to the untreated sample. Error bars represent the mean ± SD of experiment performed in technical triplicate. b Sybr green real-time PCR
measurement of the FBXO32 transcription start site and KRT17 promoter region following chromatin immunoprecipitation, using antibodies to the
histone marks shown, of MDA-MB-231 cells treated with three selected compounds at 5 μM for 72 h. Shown are representative examples of triplicate
ChIP experiments which consistently showed the same changes. The fold differen ce to the untreated sample is shown. Each IP-value has been
det ermin ed as the relative increase to the no-antibody control and then normalised t o GAPDH levels
Curry et al. Clinical Epigenetics (2015) 7:84 Page 5 of 12

Citations
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DissertationDOI
30 Jun 2017
TL;DR: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases.
Abstract: Background: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although the effects as a single agent are limited. This investigation determined whether the Histone Deacetylase (HDAC) inhibitor SAHA, or inhibitors of the histone methyltransferases G9a and EZH2 (BIX 01294 and GSK343) and Nuclear export inhibitor (NEI) LMB, enhance TRAIL-induced apoptosis and overcome TRAIL resistance in both suspension culture, and 3D cell culture as a model of solid disseminated MM lesions that form in bone. Methods: The effects of TRAIL sensitizers and/or TRAIL treatment were investigated in both suspension cultures and in an alginate-based 3D culture model. Apoptosis was detected by assessment of nuclear morphology using Hoechst 33342/PI staining. TRAIL-resistant cells were generated by acute exposure of TRAIL sensitive cells to TRAIL followed by the selection of TRAIL-resistant cells (TRAILR). Apoptotic effects in quiescent cells (labelled as PKH26Hi) were also determined. Subsequently, an investigation was undertaken to identify potential mechanisms of action of these agents when used alone and in combination with TRAIL. Results: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM2, RPMI 8226, NCI-H 929, U266, JJN3 human MM cell lines and ADC-1 plasma cell leukaemia cells. All epigenetic modifiers and NEI synergistically enhanced TRAIL responses in several lines and responses were potentiated in 3D culture. Interestingly, TRAILR cells were sensitive to BIX 01294 and LMB; however, TRAIL responses in cells that had been selected for TRAILR were not further enhanced by SAHA and GSK343. Quiescent PKH26Hi cells were resistant to dual therapy. Mechanistically, TRAIL and TRAIL sensitizers induced apoptosis via both extrinsic and intrinsic pathways in addition to decreasing the expression of oxidative enzyme catalase. Conclusions: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases. These agents may be a therapeutic option in combination with TRAIL and may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance, and not in quiescent cells.

3 citations


Cites background from "554 Dual EZH2 and EHMT2 histone met..."

  • ...Cotreatment with the Euchromatic Histone Lysine Methyltransferase 2 (EHMT2) inhibitor UNC0638 significantly inhibited the growth of breast cancer cells (Brown et al., 2014), suggesting that EZH2 inhibitors GSK343 could be a potential therapeutic option for cancer (Xiong et al., 2016)....

    [...]


References
More filters

DissertationDOI
30 Jun 2017
TL;DR: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases.
Abstract: Background: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although the effects as a single agent are limited. This investigation determined whether the Histone Deacetylase (HDAC) inhibitor SAHA, or inhibitors of the histone methyltransferases G9a and EZH2 (BIX 01294 and GSK343) and Nuclear export inhibitor (NEI) LMB, enhance TRAIL-induced apoptosis and overcome TRAIL resistance in both suspension culture, and 3D cell culture as a model of solid disseminated MM lesions that form in bone. Methods: The effects of TRAIL sensitizers and/or TRAIL treatment were investigated in both suspension cultures and in an alginate-based 3D culture model. Apoptosis was detected by assessment of nuclear morphology using Hoechst 33342/PI staining. TRAIL-resistant cells were generated by acute exposure of TRAIL sensitive cells to TRAIL followed by the selection of TRAIL-resistant cells (TRAILR). Apoptotic effects in quiescent cells (labelled as PKH26Hi) were also determined. Subsequently, an investigation was undertaken to identify potential mechanisms of action of these agents when used alone and in combination with TRAIL. Results: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM2, RPMI 8226, NCI-H 929, U266, JJN3 human MM cell lines and ADC-1 plasma cell leukaemia cells. All epigenetic modifiers and NEI synergistically enhanced TRAIL responses in several lines and responses were potentiated in 3D culture. Interestingly, TRAILR cells were sensitive to BIX 01294 and LMB; however, TRAIL responses in cells that had been selected for TRAILR were not further enhanced by SAHA and GSK343. Quiescent PKH26Hi cells were resistant to dual therapy. Mechanistically, TRAIL and TRAIL sensitizers induced apoptosis via both extrinsic and intrinsic pathways in addition to decreasing the expression of oxidative enzyme catalase. Conclusions: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases. These agents may be a therapeutic option in combination with TRAIL and may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance, and not in quiescent cells.

3 citations


Frequently Asked Questions (2)
Q1. What contributions have the authors mentioned in the paper "Dual ezh2 and ehmt2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells" ?

The authors report that gene expression and inhibition of triple negative breast cancer cell growth ( MDA-MB-231 ) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. The authors sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. The authors have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and they report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells. Ac. uk Department of Chemistry, Imperial College London, South Kensington Campus, London SW7 2AZ, UK Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London W12 ONN, UK Full list of author information is available at the end of the article © 2015 Curry et al. This article International License ( http: //creativecommo reproduction in any medium, provided you link to the Creative Commons license, and Dedication waiver ( http: //creativecommons article, unless otherwise stated. However, removal of the repressive mark H3K27me3 alone may not always be is distributed under the terms of the Creative Commons Attribution 4. 0 ns. org/licenses/by/4. 0/ ), which permits unrestricted use, distribution, and give appropriate credit to the original author ( s ) and the source, provide a indicate if changes were made. BIX-01294 ( see Fig. 2 ) was previously identified as an inhibitor of the HKMTs EHMT2 and EHMT1, and subsequent medicinal chemistry studies around the 2, 4-diamino-6, 7-dimethoxyquinazoline template of BIX01294 have yielded a number of follow-up EHMT2 inhibitors [ 20–25 ]. It has been suggested that this could provide cells with a mechanism to compensate in part for a loss of EZH2 [ 28 ]. To this end, the authors have examined the effect of dual EZH2 and EHMT2 gene knockdown or enzyme inhibition in breast cancer cells. Consistent with the requirement for removal of both repressive H3K9 and H3K27 methylation marks, the authors show that dual inhibition of EHMT2 and EZH2 pharmacologically or by SiRNA is necessary for reactivation of certain genes and induces greater inhibition of cell growth than targeting either HKMT alone in triple negative breast cancer MDA-MB-231 cells. Elimination of further repressive methylation marks by inhibition of additional HKMTs may be required to fully realise the epigenetic potential of HKMT inhibitors. The picture is further complicated by recent evidence that EHMT2 and EZH2 ( via the PRC2 complex ) interact physically and share targets for epigenetic silencing [ 29 ]. Combining this evidence, it would again suggest that specifically targeting either EZH2 or EHMT2 alone may not be sufficient to reverse epigenetic silencing of genes, but rather combined inhibition may be required. Further, the authors have identified proof-of-concept compounds which are dual ( substrate competitive ) EZH2-EHMT1/2 inhibitors. 

While this scaffold has been extensively pursued for selective EHMT1/2 inhibition, further studies are needed to confirm whether it is possible to simultaneously increase potency against both EZH2 and EHMT1/2 and whether it is possible to engineer EHMT1/2 activity out of this scaffold to identify a selective substrate competitive EZH2 inhibitor.