18F-Labeled PET Probe Targeting Enhancer of Zeste Homologue 2 (EZH2) for Cancer Imaging

Article abstract of DOI:10.1021/acsmedchemlett.8b00613

The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3), one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET) imaging agent targeting E

Full text of DOI:10.1021/acsmedchemlett.8b00613

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
18F‑Labeled PET Probe Targeting Enhancer of Zeste Homologue 2
(EZH2) for Cancer Imaging
Lihai Yu,† Nikola Despotovic,‡ Michael S. Kovacs,§,∥,∇ Christopher L. Pin,*,‡,⊥,○
and Leonard G. Luyt*,†,§,#
†London Regional Cancer Program, 800 Commissioners Road East, London, Ontario N6A 5W9, Canada
§
∥
⊥
#
‡Department of Physiology and Pharmacology, Medical Imaging, Medical Biophysics, Paediatrics, and Chemistry, The
University of Western Ontario, London, Ontario N6A 3K7, Canada
∇Medical Imaging and ○Children’s Health Research Institute, Lawson Health Research Institute, London, Ontario N6A 4V2,
Canada
S
* Supporting Information
ABSTRACT: The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3),
one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET)
imaging agent targeting EZH2 has the potential to provide a method of stratifying patients for epigenetic therapies. In this
study, we designed and synthesized a series of fluoroethyl analogs based upon the structure of EZH2 inhibitors UNC1999 and
EPZ6438. Among the candidate compounds, 20b exhibited a high binding affinity to EZH2 (IC50 = 6 nM) with selectivity
versus EZH1 (IC50 = 200 nM) by SAM competition assay, and furthermore, EZH2 inhibition was demonstrated in the
pancreatic cancer cell line PANC-1 (IC50 = 9.8 nM). [18F]20b was synthesized successfully and showed 5-fold higher uptake in
PANC-1 cells than in MCF-7 cells. MicroPET imaging in a PANC-1 cell xenograft mouse model indicates that [18F]20b has
specific binding to EZH2, which was identified by ex vivo Western blot analysis of the tumor tissue.
KEYWORDS: Enhancer of zeste homologue 2 (EZH2), fluorine-18, positron emission tomography (PET), pancreatic cancer,
epigenetics
ectoderm development (EED). EZH2 is expressed at very low
pigenetics is the study of factors that promotes heritable
Echanges in gene expression without mutation to the levels in nonproliferating tissue yet increases during cancer
progression, with high levels of EZH2 demonstrated in
different cancer tissues such as prostate cancer, breast cancer,
and pancreatic cancer.1−3 In addition, gain of function mutants
for EZH2 have been identified in a number of cancers
including lymphomas. Importantly, increased EZH2 expression
is not observed in all tumors, even from the same cancer type,
suggesting some stratification of patients should be performed
before therapeutic targeting of this protein.
primary DNA sequence. Epigenetic changes typically involve
alterations to the DNA, the histones that make up the
nucleosome core around which DNA is wrapped, or
noncoding RNA transcripts that interact with mRNA or
DNA. Epigenetic processes play essential roles in development
and disease, and in the past decade, aberrant expression and
activity of epigenetic regulating proteins have been described
for many diseases including most cancers. Based on these
observations, inhibition of cancer-relevant histone modifica-
tions has become a potential strategy for mono- or
combinatorial cancer therapy. One particularly attractive
epigenetic target is enhancer of zeste homologue 2 (EZH2),
which is part the polycomb repressive complex 2 (PRC2)
along with suppressor of zeste 12 (SUZ12) and embryonic
EZH2 carries the catalytic activity for PRC24,5 required for
trimethylation of lysine 27 on histone 3 (H3K27me3), which is
Received: December 6, 2018
Accepted: February 21, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00613
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Figure 1. Structures of previous published EZH2 inhibitors.
mis-regulated in various cancers. Through this epigenetic
modification, PRC2 promotes gene repression. The SET
domain in the EZH2 sequence provides the active methyl
group from the methyl group donor S-adenosyl-L-methionine
(SAM) for H3K27me3. The gain of function EZH2 mutations
observed in some cancers correlate with increased H3K27me3.
Therefore, inhibition of the SET domain in EZH2 is a
promising strategy for the cancer treatment, and several SAM-
competitive EZH2 inhibitors with low IC50 value have been
reported (Figure 1). GSK-A (Figure 1A) was first identified as
an EZH2-binding chemical scaffold through a high throughput
screening assay.6 GSK-A contains both pyridone and 1H-
pyrazolo[3,4-b]pyridine rings. Based on modifications to GSK-
A’s structure, a series of successful inhibitors with high affinity
to EZH2 (nanomolar range) and high selectivity against EZH1
have been developed, including EPZ005684, UNC1999,
GSK343, and E1 (Figure 1D).7−10 We chose to target
EZH2-specific inhibitors for developing diagnostic molecules
since EZH2 has been more commonly linked to cancer
progression than EZH1 and is the predominant methyltrans-
ferase in PRC2. In addition, while EZH2 expression is typically
low in nonproliferative cells and tissues, EZH1 expression is
readily detectable in adult tissues including skeletal muscle and
salivary glands, which may increase imaging background. With
the replacement of a pyridine ring with an indole ring, GSK126
(Figure 1B) was first developed in 2012, exhibiting high affinity
to EZH2 (Ki = 0.5−3 nM) and selectivity compared to EZH1
(Ki = 89 nM).11 The binding affinity was maintained by
removing 1-(pyridin-2-yl)piperazine group from the indole
ring and adding a series of six-membered rings on N-indole in
CPI169, CPI360, and CPI1205 (Figure 1C).12−14 GSK126
proceeded into clinical trials for lymphomas but ultimately was
terminated.15 The substitution of a methyl group with a
methoxy group in the sensitive pyridine moiety maintains
EZH2 inhibition activity. Based on a similar modification used
for GSK126, another series of indazole analogs (Figure 1D)
was successfully developed with a high retention of bioactivity
to EZH2. By replacing 1H-pyrazolo[3,4-b]pyridine ring by
aniline, EPZ6438 (Figure 1E) exhibited a 10-fold higher
affinity to EZH2 (Ki = 2.5 nM) than the similarly structured
EPZ005684 and 35-fold selectivity against EZH1.16 EPZ6438
(Tazemetostat) has also proceeded into clinical trials for non-
Hodgkin’s lymphoma, and these studies are ongoing.17 In
addition, a series of promising compounds, including
EPZ011989, ZLD10A, and ZLD1122, were recently de-
scribed.18−20
While much progress has been made in developing EZH2
inhibitors for therapy, there are no reports of using these
compounds for cancer diagnosis. The success in developing in
vivo molecular imaging agents for detecting histone deacety-
lases (HDAC), for example, using the PET agent FAHA,
suggests that epigenetic reprogramming proteins can be
targeted for imaging purposes.21 Positron emission tomog-
raphy (PET), like CT or MRI, is a noninvasive imaging
technique widely used to assist clinical diagnosis with the
advantage of exceptionally good sensitivity and the ability for
quantification. The goal of this study was to develop novel 18F-
labeled PET radiotracers specifically targeting EZH2 to allow
for clinical cancer diagnosis and patient stratification.
Since both EPZ6438 and UNC1999 exhibit high binding
affinity and selectivity for EZH2, we initially focused on
modifying these inhibitors in order to develop PET imaging
B
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Figure 2. Structures of EZH2 inhibitor analogs designed and synthesized in this study: (A) UNC1999 series and (B) EPZ6438 series.
agents. Based on SAR studies regarding EZH2 inhibitors, we
maintained the pharmacophore of pyridone, indazole, or
aniline rings to retain biologically relevant features.22
Considering the chemical framework of EPZ6438 and
UNC1999, we designed modifications for side substitution
on the indazole or aniline rings and/or removal of one alkyl
group in position 4 of pyridone. Since [18F]fluoroethyltosylate
is a widely used intermediate in the development of PET
tracers and can be coupled to secondary amines in radiosyn-
thesis, fluoroethyl groups were introduced into piperidine in
our target compounds. This provides a synopsis of the
rationale behind the development of the corresponding
radiotracers.
Table 1. Bioactivity of Compounds to EZH2 and Selectivity
versus EZH1
compound
IC50 (EZH2)
0.8 μM
IC50 (EZH1)
c Log P
9a
1.98
2.57
1.84
1.86
0.47
1.06
0.61
1.20
1.16
1.75
9b
10a
11a
20a
0.1 μM
1.3 μM
8.5 μM
30 nM
9 μM
>10 μM
20b
21a
21b
6 nM
36 nM
7 nM
200 nM
280 nM
22a
74 nM
22b
8 nM
450 nM
Based on this design strategy, 12 new compounds were
synthesized as shown in Figure 2, with experimental details and
synthetic schemes found in the Supporting Information. We
initially assessed their binding to EZH2 using a cell-free
competition assay for SAM that also suggests biological
activity. The binding results compare the ability of these new
compounds to inhibit PRC2 binding to SAM and are
summarized in Table 1, including calculated Log P values.
Six compounds (9a, 9b, 10a, 10b, 11a, and 11b) share the
same core structure with UNC1999 (Figure 2A). Pyridone has
been accepted as an active moiety to occupy the SAM-binding
pocket in EZH2 protein and is thus retained in this series.
Compound 9b displays the lowest IC50 value (0.1 μM) for
EZH2 inhibition among the modified UNC-1999 compounds
and a high selectivity versus EZH1, with 90-fold difference in
IC50 value. Compared to compound 9b, compound 9a was
found to have a higher IC50 (0.8 μM), which demonstrates that
the replacement of methyl group by hydrogen in position 4 on
the pyridine ring decreased the compound’s interaction with
EZH2, which is consistent with SAR analysis published by
Yang.22 Extending the fluorine-piperidine substituent one
methylene away from the core (10a) resulted in a loss of
affinity, as did relocation of the isopropyl substituent on the
indazole (11a).
GSK343
EPZ6438
5−9 nM
15 nM (2.5 nM16
1200 nM
)
Six fluorine-containing EPZ6438 analogs were prepared as
indicated in Figure 2B. The oxygen atom in tetrahydropyran or
morpholine of EPZ6438 was replaced by N-fluoroethyl group
to obtain compounds 20b and 21b, respectively. Both 20b and
21b exhibit highly competitive activity against EZH2 at
nanomolar levels similar to the EPZ6438 reference (IC50
=
15 nM). Replacement of the benzyl group in the pyridine ring,
22b, maintains high binding. As a result, the modification on
the boundary site of EPZ6438 with a fluoroethyl group does
not affect EPZ6438 affinity to the SAM-competitive binding
site. The IC50 values of 20b, 21b, and 22b are more than 30-
fold higher to EZH1 than to EZH2, which indicates these three
compounds have high selectivity for EZH2. Finally, the
replacement of 4,6-dimethylpyridin-2(1H)-one with 6-methyl-
pyridin-2(1H)-one in 20a, 21a, and 22a decreased biological
activity based on competition assays. This is similar to the
effect seen between 9a and 9b based upon the presence of a
methyl group.
To examine cell uptake and maintenance of biological
activity in cells, we examined the effects of these compounds
on global H3K27me3 enrichment in histones isolated from
C
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Figure 3. Effect of UNC1999 and EPZ6438 compounds on EZH2 activity in PANC-1 cells. Representative Western blot analysis for H3K27me3 or
H3K4me3 on histone extracts from PANC-1 cells treated with UNC1999, EPZ6438, or modified compounds for 3 days. All blots were stripped
and reprobed to assess the loading control, histone 3 (H3). Cell culture experiments were performed twice (n = 2) for H3K27me3 and once (n =
1) for H3K4me3. Bars represent mean standard deviation. No statistical analysis was performed on this data.
Figure 4. Increasing concentrations of EPZ6438, 20b, 21b, 22a, and 22b on H3K27me3 in PANC-1 cells. (A) Representative Western blot analysis
for H3K27me3 on histone extracts from PANC-1 cells treated with increasing amounts of 21b, 22a, and 22b for 3 days. All blots were stripped and
reprobed to assess the loading control, histone 3 (H3). Inhibition curves for (B) parental EPZ6438, and compounds (C) 21b, (D) 22b, and (E)
control 22a. The % H3K27me3 represents the amount of H3K27me3:H3 relative to each blot’s DMSO control. IC50 values, if applicable, are listed
on their corresponding curve. There was no significant difference (p = 0.5795) between EPZ6438 and 21a’s abilities to inhibit EZH2 activity.
Similar Western blot analysis (F) and inhibition curves (G) for lead compound 20b.
PANC-1 cells. PANC-1 cells are originally derived from a
pancreatic ductal adenocarcinoma patient and exhibit high
levels of EZH2 expression and H3K27me3. The relative ratio
of H3K27me3-to-H3 densiometry (H3K27me3:H3) was used
as a marker of EZH2 activity. PANC-1 cells exposed to 1 μM
UNC1999 (0.089 0.2%), 9b (0.21 9%), or 10b (0.11
2%) displayed a reduction in EZH2 activity relative to DMSO-
the non-EZH2 target H3K4me3. Similar analysis of the
parental EPZ6438 and related compounds revealed maintained
EZH2 inhibition for compounds 21b (0.11 0.001), 20b (0.1
0.02), and 22b (0.1 0.002) similar to EPZ6438 (0.08
0.008). These levels of EZH2 activity are significantly lower (p
< 0.0001) in PANC-1 cells compared to DMSO control (0.84
0.1).
treated cells (0.85
Other UNC1999-derived compounds showed a loss of EZH2
inhibition ability (Figure 3). No differences were observed for
9%) based on H3K27me3:H3 ratios.
From this data, compounds 20b, 21b, 22a, and 22b were
selected for dose-dependent analysis (Figure 4). EPZ6438 was
selected for use as a positive control; 20b, 21b, and 22b as
D
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Figure 5. (A) Reagents and conditions: a. K2.2.2, K2CO3, ACN, 115 °C, 10 min; b. K2CO3, ACN, 130 °C (MW), 10 min. (B) Cell uptake [18F]20b
(1 MBq) in 100 μL of PBS buffer, PANC-1, or MCF-7 cells (600,000 each vial) in 1 mL of incubation medium, n = 5, and EPZ6438 (100 μg) in 10
μL of DMSO. (C,D) Correlating EZH2 activity to PET signal intensity for mice with PANC-1 tumor. microPET image at 100−120 min in NOD/
SCID mice (n = 2) with PANC-1 cells tumor xenografts were administered a 10 MBq IV dose of [18F]20b and blocked by EPZ6438. (E,F) EZH2
expression in tumor tissue samples from PANC-1 tumors were analyzed by Western blots.
candidate compounds; and 22a as the negative control. While
UNC1999, 11b, and 10b displayed comparable EZH2
inhibition at 1 μM, they were not chosen for further study
since the cell-free binding experiments (Table 1) suggested
that they have much weaker binding affinities than their
EPZ6438 counterparts. PANC-1 cells exposed to increasing
concentrations of EPZ6438 (n = 3), 21b (n = 3), 22b (n = 2),
and 20b (n = 3) for 3 days were found to have IC50 values of
fluoroethyltosylate prosthetic group, with a radiochemical
yield of 34% (decay corr.) after purification by semipreparative
HPLC. [18F]Fluoroethyltosylate was coupled to the precursor
compound 24, which contains a secondary amine, in
acetonitrile at 130 °C for 10 min. Purification by HPLC
provided [18F]20b in a yield of 25% (decay corr.). The total
yield for two steps is 8.5% (decay corr.) with nonradioactive
compound 20b used as a reference compound to confirm the
presence of [18F]20b (Figure S1, Supporting Information).
The specific activity is 10−25 GBq/μmol and radiopurity
>95%. The partition coefficient (log P = 1.93) was measured in
n-octanol/phosphate buffer. [18F]20b was used to perform the
following cell study.
EZH2 is overexpressed in various cancers, including
pancreatic and breast cancers. Human pancreatic carcinoma
cell (PANC-1) and human breast adenocarcinoma cell (MCF-
7) are typical cell lines for pancreatic cancer and breast cancer.
To determine if radiolabeled 20b targets EZH2 at the tracer
level in live cells, we investigated the uptake of [18F]20b to
target EZH2 in PANC-1 and MCF-7 cells. Since [18F]20b is an
analog of EPZ6438 and both have a similar affinity for EZH2,
we used a large excess of EPZ6438 for a blocking study to test
[18F]20b’s specific binding affinity to EZH2 in PANC-1 and
MCF-7 cells (Figure 5B). [18F]20b exhibited 5-fold higher
uptake in PANC-1 cells compared to MCF-7 cells.
Coincubation of the radiotracer [18F]20b with 100 μg of
EPZ6438 reduced radioactivity accumulation in both PANC-1
and MCF-7 cells. [18F]20b uptake decreased 90% lower in
16 14, 24 22, 23 8, and 9.8
4.0 nM, respectively
(Figure 4) indicating no significant difference (p = 0.5795)
between the EZH2 inhibitory potential of EPZ6438 and the
modified versions of this compound in PANC-1 cells. The IC50
for 22a (n = 2) could not be calculated based on this
concentration range. Combined, this data confirms that
compounds 20b, 21b, and 22b are taken up by tumor cells
and maintain the ability to inhibit EZH2-mediated
H3K27me3. Considering the biological data in Table 1 and
the finding that compound 20b has a lower Log P value than
21b and 22b, combined with evidence of superior EZH2
inhibition, we decided to proceed with the development of a
radiosynthetic route for radiolabeling 20b with the PET
radioisotope 18F to determine its potential as an in vivo imaging
agent.
The radiosynthesis of [18F]20b was accomplished by a two-
step approach (Figure 5A). Aqueous [18F]F− was dried under
the presence of Kryptofix2.2.2. and K2CO3 by azeotropic
drying. Ethylene di(p-toluenesulfonate) was reacted with dried
[18F]F−/Kryptofix2.2.2 complex resulting in the [18F]-
E
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PANC-1 cells treated by EPZ6438 with a 50% reduction in
MCF-7 cells. These results suggest that EZH2 is expressed to
higher levels in PANC-1 cells compared to MCF-7 cells and
confirms [18F]20b exhibits specificity for EZH2 in a live cell
situation. Therefore, we next examined [18F]20b’s ability to
identify EZH2 accumulation in animals.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: 519-685-8600 ext. 53302. E-mail: lluyt@uwo.ca.
*Tel: 519-685-8500 ext. 53073. Fax: 519-685-8156. E-mail:
cpin@uwo.ca.
ORCID
As a pilot experiment for PET imaging, 3 × 106 PANC-1
cells were transplanted into NOD/SCID mice and imaging
performed once tumors reached 0.8−1 cm in diameter. At this
time, mice were imaged for 2 h using PET scanning. PET
images show moderate tumor uptake (Figure 5C,D) when
mice were administrated with 10 MBq [18F]20b. While high
background was observed in both mice, pretreatment with
EPZ6438 resulted in decreased [18F]20b uptake in the tumor
suggesting that [18F]20b selectively binds EZH2 protein in
vivo. Surprisingly, [18F]20b activity differed significantly
between mice. To determine if this reflected differences in
EZH2 activity, histones were isolated from PANC-1-derived
tumors following imaging and assessed for global H3K27me3
levels (ex vivo). As suggested by the imaging results, the tumor
displaying higher [18F]20b uptake had significantly more
H3K27me3 present, indicating higher EZH2 activity (Figure
5E, F). The correlation between tumor signal intensity and
EZH2 activity suggests that in vivo EZH2 activity can be
noninvasively imaged and quantified with [18F]20b. However,
the high level of background uptake for this PET agent
indicates that further modifications to the imaging agent are
required to make this approach clinically viable. Although the
LogP value is reasonable for a small molecule, the high liver
uptake suggests that a further lowering of LogP may be helpful,
as well as studies to investigate in vivo stability. For example,
the addition of a PEG chain to 20b could decrease background
in the PET images by reducing its lipophilicity.
Conclusion. A fluorine containing analogue of EPZ6438,
[18F]20b, was discovered to exhibit high binding affinity to
EZH2 protein in vitro, inhibit H3K27me3 methylation in cells,
and selectively label EZH2 protein in PANC-1-derived
xenograft tumors. Currently, [18F]20b is not suitable for
clinical diagnosis due to high background uptake of the
imaging agent but will need to be further modified to reduce
background. However, PET imaging and Western blot analysis
demonstrates that [18F]20b or derivatives of this compound
optimized for pharmacokinetic behavior may be used to stratify
the expression of EZH2 in patients and noninvasively
determine epigenetic methylation status (H3K27me3) in
vivo. New PET tracers based on further chemical modifications
to the parent EPZ6438 will be investigated in the future.
Leonard G. Luyt: 0000-0002-0941-4731
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was supported by the Cancer Research Society with
funding through an operating grant titled “Targeting enhancer
of Zeste 2 (EZH2) for non-invasive cancer imaging”.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors wish to thank Dr. Ting-Yim Lee and his staff for
providing access to small animal imaging equipment.
ABBREVIATIONS
■
EZH2, enhancer of zeste homologue 2; PET, positron-
emission tomography
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00613.
Procedures for the synthesis of all candidate compounds
with chemical structural characterization, methods for
binding affinity screening using SAM-competing assay
and cell inhibition study, synthetic schemes for all
compounds, and HPLC chromatograms for 20b and
[18F]20b (PDF)
F
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