THE DNA HYPOMETHYLATING AGENT SGI110 REVERSES THE PLATINUM RESISTANCE OF OVARIAN CANCER MODELS
3756
Joanne Munck, John Lyons, Neil Thompson, Nicola Wallis and Pietro Taverna.
Astex Pharmaceuticals, 436 Cambridge Science Park, Milton Road, Cambridge, CB4 0QA, United Kingdom
SGI110 confers LINE-1 demethylation in an OC cell line panel
Cells were treated for 3 consecutive days with 0.1 µM SGI110. Methylation of global LINE-1 elements were determined by pyrosequencing
metabolism and deactivation.
We confirm SGI110 activity across the panel of OC cell lines, as indicated by demethylation of global
LINE-1 elements.
SCID Beige mice bearing subcutaneous A2780 tumours. 2 mg/kg SGI110 was administered subcutaneously,
SGI110 sensitises A2780, OAW28 and Ovcar8 cell lines to cisplatin
once daily on days 1-5. A single dose of cisplatin was administered intraperitoneally on day 8, at 2 mg/kg or 4
% Cell Proliferation
SGI110 confers MLH1 demethylation and
re-expression in A2780 xenografts
% Cell Proliferation
* p<0.05 vs single agent SGI110.
% Cell Proliferation
mg/kg. Both agents were dosed either as single agent or in combination. Data represent mean ± SD (n=8).
Ovcar8 and OAW28 cells have relatively
high levels of ZIC1 methylation. SGI110
-20
confers ZIC1 demethylation.
-30
-40
-50
59
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SK 3
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O
A
27
80
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A
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0.
3
ci
sp
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m
g/
kg
SGI110 sensitised A2780, Ovcar8 and OAW28 cells to cisplatin in vitro. Despite SGI110 activity in
59M, Ovcar3 and SKOV3 cells (LINE-1 demethylation, shown above), SGI110 did not sensitise these
A2780 Tumours
MLH1 RNA levels
0
3
0.3
0.0
SGI-110 ( M)
7
cell lines to cisplatin. This suggests that 59M, Ovcar3 and SKOV3 cells do not have an epigenetic
Epigenetic silencing of MLH1 silencing is not responsible for the
cisplatin resistance of Ovcar8 and OAW28 cells
4
SGI-110 ( M)
1
associated with SGI110-induced MLH1 promoter demethylation and MLH1 reexpression.
© Astex Pharmaceuticals Inc.
OVCAR8
Day 7
resistance to the A2780 cells.
0.1 0.3 [SGI110] µM
ci
sp
l
m
g/
kg
SCID Beige mice bearing subcutaneous A2780 tumours were dosed on days 1-5 with single agent SGI110 (s.c)
Actin
0
0.1 0.3
OAW28
Day 4
Day 6
Day 4
MLH1
2
SGI110 sensitised A2780 cells to cisplatin in vitro. This sensitisation was
0 0.1 0.3 1
4
0
+/- 3-day pre-treatment with SGI110. ii. MLH1 promoter methylation determined by pyrosequencing. iii. RT-PCR analysis of
ci
sp
l
Cells were treated for 3 consecutive days with SGI110. i. Alamar Blue Proliferation Assay. Cells exposed to cisplatin for 4 days
MLH1. iv. Western blot of MLH1 expression.
Day 4
1
0
0.3
1 [SGI110] µM
0
0.1 0.3
Poster presented at AACR Annual Meeting, San Diego. Apr 5-9 2014
• We identify epigenetic silencing of ZIC1 as a potential cisplatin resistance mechanism in Ovcar8 and OAW28
Day 7
1
0.1 0.3 1 [SGI110] µM
MLH1
Actin
MLH1
Actin
Cells were treated for 3 consecutive days with SGI110 and harvested on day 4, 6 or 7 as indicated.
MLH1 is not epigenetically silenced in Ovcar8 or OAW28 cells. Therefore, the cisplatin resistance
or on day 1 with single agent cisplatin (i.p). Tumours were excised on day 8. Tumour MLH1 methylation and RNA
expression levels were determined by pyrosequencing and RT-PCR, respectively.
• SGI110 reverses the known cisplatin resistance mechanism (epigenetic silencing of MLH1) of A2780 cells by
conferring MLH1 promoter demethylation and re-expression.
A2780
2
SUMMARY AND CONCLUSIONS
• SGI110 sensitises OAW28 and Ovcar8 cells to cisplatin. These models have distinct mechanisms of cisplatin
3
C
on
tr
ol
1.0
Actin
mechanism of cisplatin resistance.
5
m
g/
kg
MLH1
6
SG
I
3 [SGI110] µM
2
1
m
g/
kg
0 0.03 0.3
Fold change mRNA
(iv)
Fold change MLH1 RNA
(iii)
0.3
0
Alamar Blue Proliferation Assay. Cells were exposed to cisplatin for 4 days +/- 3-day pre-treatment with SGI110.
0
-10
-60
0 SGI110 = 29.5 M
0.1 M SGI110 = 6.8 M
0.3 M SGI110 = 3.3 M
0.1
10
O
-50
4
100
2
10
0
1
Cisplatin ( M)
0.1
0.1
ci
sp
l
SG
I
2
-40
m
g/
kg
-30
m
g/
kg
-20
25
0.0
% Cell Proliferation
20
-10
50
0
0.01
40
0.
1
0
75
Ovcar3
30-fold
0
ZIC1 De-methylation
OAW28 cells
ZIC1 methylation
50
30
C
on
tr
ol
% Cell Proliferation
MLH1 De-Methylation
SGI-110 ( M)
Table 2 Effect of SGI110 on DOK2 and ZIC1 RNA levels across cell line panel
Ovcar8
OAW28
A2780
SKOV3
59M
DOK2
65-fold
41-fold
17-fold
110-fold
90-fold
ZIC1
11-fold
13-fold
2-fold
0
0
Methylation levels of ZIC1 in untreated cells and SGI110-treated OAW28 cells, as determined by pyrosequencing.
% Cell Proliferation
(ii)
A2780
100
0
SGI110 confers ZIC1 demethylation
% Cell Proliferation
(i)
10
been identified as a prognostic biomarker in OC (Huang et al., 2013)
% MLH1 demethylation
1. SGI110 REVERSES THE CISPLATIN-RESISTANCE OF
A2780 CELLS IN VITRO
20
was exclusively re-expressed in the cell lines that were sensitised to cisplatin by SGI110. ZIC1 has previously
panel of OC cell lines. We suggest that
OAW28 and Ovcar8 cells and demonstrate that it is reversed by SGI110.
30
expression did not correlate with SGI110-induced sensitisation to cisplatin across the OC cell panel (Table 2). ZIC1
A2780 OC model, by abrogating the epigenetic silencing of MLH1. We
epigenetic silencing of ZIC1 is a mechanism of cisplatin resistance in the
DOK2
ZIC1
40
SGI110 conferred re-expression of DOK2 and ZIC1 in both OAW28 and Ovcar8 cells (Table 1). DOK2 re-
• Here we demonstrate that SGI110 reverses the cisplatin-resistance of the
demonstrate SGI110 activity in a
OAW28
50
1
to modification by cytidine deaminase: a common pathway of decitabine
increase in RNA levels in response to 1 µM SGI110 (0 = no increase).
0
0.
1
0.
3
-60
A2
78
0
O
vc
ar
8
O
A
W
28
• SGI110 is a dinucleotide of decitabine and deoxyguanosine, which is resistant
-50
DOK2
ZIC1
UNG
OXSR1
CENPB
GTF2B
ESSRA
SGPL1
GABPA
TWIST1
MDH1
NR2E1
NR3C2
SOX9
TOB1
HIRIP3
FZD1
APRT
GSK3B
MLH1
Ovcar8
65-fold
11-fold
0
0
0
0
0
0
0
0
0
0
0
0
2-fold
0
0
0
0
0
Fold change RNA
resistant recurrent ovarian cancer patients (NCT01696032).
-40
OAW28
41-fold
13-fold
0
0
0
0
0
0
0
3-fold
0
7-fold
0
3-fold
0
0
0
0
0
0
0
0.
1
0.
3
1
currently in a Phase II clinical trial in combination with carboplatin, in platinum-
RT-PCR analysis of candidate biomarkers following 3-day treatment with SGI110. Table 1 and 2 show fold-
Table 1
1
• SGI110 is a novel second generation DNA hypomethylating agent, which is
reported by Lum et al., 2013.
-30
0
0.
1
0.
3
the epigenetic silencing of specific genes by DNA methylation.
We analysed the effect of SGI110 on the RNA levels of potential OC cisplatin resistance biomarkers previously
-20
Fold change RNA
There is evidence that the acquisition of platinum resistance is associated with
SGI110 induces ZIC1 and DOK2 expression in OAW28 and Ovcar8 cells
0
-10
0
0.
1
0.
3
1
SGI110 sensitises A2780 xenografts to cisplatin
to first-line chemotherapy, but >70% relapse, developing platinum-resistance.
% Zic1 Demethylation
surgery followed by platinum-based chemotherapy. Patients commonly respond
59
M
O
vc
ar
3
SK
O
V3
of less than 25%. The most common treatment strategy comprises debulking
3. SGI110 SENSITISES OVARIAN CANCER CELL LINES WITH DISTINCT MECHANISMS OF CISPLATIN RESISTANCE
2. SGI110 REVERSES THE CISPLATIN-RESISTANCE
OF A2780 CELLS IN VIVO
% LINE-1 demethylation
INTRODUCTION
• Patients with advanced stage ovarian cancer (OC) have a 5-year survival rate
observed in these cell lines is due to distinct epigenetic mechanisms to that of A2780 cells.
cells and demonstrate that SGI110 restores ZIC1 expression.
• We demonstrate that SGI110 reverses the cisplatin resistance of OC models by restoring expression of
epigenetically silenced genes. This supports our ongoing Phase II trial evaluating SGI110 in combination with
carboplatin, in platinum-resistant recurrent ovarian cancer patients (NCT01696032).
References
1.Lum et al., 2013. Gynecologic Oncology. 130.369-376
2.Huang et al., 2013. Epigenetics. 8;6.624-634
Poster can be downloaded from www.astx.com