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Screening the druggable genome for synthetic lethal interactions with the CHK1
inhibitor PNT737 (SRA737)
Rebecca Rogers, Mike Walton, Paul Clarke, Ian Collins, Michelle D. Garrett and Paul Workman
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Contact
Cancer Research UK Cancer Therapeutics Unit
The Institute of Cancer Research, London
[email protected]
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1 Walton, M.I., et al. Oncotarget, 2016. 7(3): p. 2329-42
2 Osborne, J.D., et al. J. Med. Chem, 2016. 59(11): p.5221-37
3 NCT02797964 and NCT02797977
4 Chou, T.C. and Talalay, P. Adv Enz Regul, 1984. 22: p. 27-55
5 Chou, T.C., Cancer Res, 2010. 70(2): p.440-6
M.F. et al. Fam Cancer, 2015. 14(3): p. 437-48
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- SR A737
+ 0 .2 3 µ M S R A 7 3 7
PO LA1
PO LE
PO LE2
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- SR A737
+ 2 .4 u M S R A 7 3 7
P
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PO LA1
PO LE
PO LE2
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D e a th
10nM
3nM
1nM
0 .3 n M
-2 0
0 .3 n M
D e a th
0 .3 n M
0 .1 n M
0 .3 n M
0 .1 n M
0 .3 n M
0 .1 n M
M ock
D e a th
0 .3 n M
0 .1 n M
0 .3 n M
0 .1 n M
0 .3 n M
0 .1 n M
PO LE2
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Figure 4 : Hit validation in additional human NSCLC and colon cancer cell lines. (A-D) Cells were transfected with either 25 nM Allstars
negative control, WEE1 siRNA, death siRNA or individual Qiagen POLA1, POLE or POLE2 siRNA at a range of concentrations for 48 h. Mock
cells treated with 0.2 % HiPerFect alone. Cells were then treated with optimum non-lethal concentration of SRA737 for 82 h, n≥3. Statistical
analysis used unpaired students t-test *p<0.05, ***p<0.001.
5. Combination studies
Aphidicolin inhibits the B-family DNA polymerases. POLA1, POLE and POLE2
were also validated as hits using the selective tool compound aphidicolin.
1 .5
1 .0
0 .5
N S C L C c e ll lin e s
H T 29
H C T 116
S W 620
N C I- H 2 3
0 .0
C o lo n c a n c e r
c e ll lin e s
Combination indices (CI) for SRA737 and aphidicolin were calculated using the
Chou and Talalay method. CI < 1 indicates synergy, >1 antagonism and =1
additivity.4,5
The combination was robustly synergistic (CI≤0.8) in 8 out of 9 cancer cell lines
tested.
Figure 5: Summary of CI’s in panel of NSCLC and colon cancer cell lines. Cells were plated in 96 well plates and incubated for 36 h at 37 ºC.
Plates were then treated with SRA737, aphidicolin or a combination of both at a 1:1 ratio of the GI50s (previously determined) and incubated for 4
doubling times at 37 ºC. CI values; mean ± SD n≥3.
- + - +
POLE2
siRNA
- +
-
+
POLE
POLE2
RPA32
C-PARP
Figure 6: Replication stress biomarkers increase with combination of siRNA and SRA737 treatment. Cells were transfected with 0.1 nM
(A549) or 1 nM (SW620) POLA1 #3, POLE #2 or POLE2 #4 siRNA for 48 h. Allstars negative control at 25 nM, mock treated with lipid only. Cells
were then treated with 0.4 or 0.8 µM SRA737, A549 and SW620 respectively, for 24 h. GAPDH used as loading control. n=2.
7. DNA damage: γH2AX immunofluorescence
Combination of SRA737 and aphidicolin increases DNA damage compared to single agent.
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S R A 7 3 7 [G I 5 0 ]
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A p h id ic o lin [G I 5 0 ]
2
C o m b in a tio n (1 :1 [G I 5 0 ] )
G e m c ita b in e (2 0 0 n M )
1
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A5 4 9
C a lu - 6
N C I- H 1 9 7 5
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1
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S W 620
RKO
H C T 116
Figure 7: γH2AX intensity in cancer cells treated with SRA737, aphidicolin or combination. Cells were plated in 96 well plates, incubated
for 48 h, then treated for 24 h. Cells were fixed and permeabilised and incubated with γH2AX primary antibody (1:500). Cells were imaged on the
IN Cell and foci were analysed on IN Cell analyser workstation 3.7.2. (mean ± SD, n≥3).
Acknowledgments
Thanks to Mark Stubbs for advice and help with the Echo dispenser. Thanks also to Tom Matthews for the supply of
SRA737.
RR thanks the Wellcome trust for a PhD scholarship (102359/Z/13/Z) and The University of London scholarship fund.
+ -
POLE
siRNA
-
POLA1
siRNA
Untreated
- +
Allstars
negative
POLE2
siRNA
- + + -
Mock
POLE
siRNA
+ - +
POLA1
siRNA
Mock
Allstars
negative
Mock
-
GAPDH
- SR A737
+ 0 .2 5 µ M S R A 7 3 7
-
PNT737
POLA1
SW620
(c o m p a re d to u n tre a te d )
- SR A737
PO LE
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PhosphoRPA32 levels, seen as band
shift of total-RPA32, were higher in
cells treated with a combination of
POLA1, POLE and POLE2 siRNA and
SRA737, in comparison to cells treated
with the siRNA or drug alone.
(c o m p a re d to u n tre a te d )
#4
PO LA1
-2 0
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W E E 1 s iR N A
#2
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A lls t a r s n e g a t iv e
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W E E 1 s iR N A
PO LE2
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0
A lls t a r s n e g a t iv e
PO LE
D e a th
1 nM
0 .3 n M
1 nM
0 .3 n M
0 .3 n M
0 .1 n M
W E E 1 s iR N A
A lls t a r s n e g a t iv e
M ock
U n tre a te d
PO LA1
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0 .1 n M
20
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W E E 1 s iR N A
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A lls t a r s n e g a t iv e
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M ock
***
n≥3
100
U n tre a te d
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% c e ll v ia b ility
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-2 0
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n=3
100
A549
Phosphorylation of RPA32 is a reported
biomarker of replication stress.
RKO
D
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6. Replication stress response
Untreated
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SK LU 1
Figure 2: Summary
of siRNA screen
hits. Data presented
as
difference
in
robust
Z-score
between treated and
untreated conditions;
the
greater
the
difference in robust
Z-score, the greater
the difference in cell
viability. n≥3
References
6 Hansen,
P
P
P O LA1
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s iR N A
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HCT116
U n tre a te d
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N C I- H 4 6 0
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3
0
-3
-6
10
120
% c e ll v ia b ility
***
40
C a lu - 6
P O LE2
s iR N A
1
γ H 2 A X in te n s ity /c e ll fo ld in c r e a s e
P O LE2
P O LE
8
4
*
2
γ H 2 A X in te n s ity /c e ll fo ld in c r e a s e
s iR N A
2000
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N C I- H 1 9 7 5
P O LE
SW 620
12
( + /- S R A 7 3 7 )
4
D iffe r e n c e in r o b u s t Z - s c o r e
( + /- S R A 7 3 7 )
D iffe r e n c e in r o b u s t Z - s c o r e
All three belong to the B- family of DNA polymerases (α, δ and ε) which function at the replication fork; Pol δ
and ε have also been implicated in other synthetic DNA processes.
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A5 4 9
We identified POLA1, POLE and POLE2 siRNA as hits in both A549 lung and SW620 colon human cancer
cell lines.
3
0
-3
-6
*
+ 0 .3 3 µ M S R A 7 3 7
C I ( E x c lu s iv e )
We used the Dharmacon druggable human genome siRNA library to conduct a synthetic lethal screen with
cell lines with low sensitivity to SRA737; non-small cell lung cancer (NSCLC) cell line A549 and colon
cancer cell line SW620.
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-2 0
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0 .1
C
n≥3
M ock
3. Hit identification
10
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C o n c e n tr a tio n o f S R A 7 3 7 ( µ M )
120
n≥4
0
P O LA1
20
0 .0 1
C a lu - 6
B
% c e ll v ia b ility
% c e ll v ia b ility
To identify those gene products whose loss is synthetically lethal with CHK1 inhibition in cancer cells in order
to identify patient populations likely to be sensitive to single agent CHK1 inhibition or to identify novel
combinations utilising CHK1 inhibitors.
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N C I- H 1 9 7 5
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It will now be important to establish if subsets of colon and endometrial cancers with mutations in their
POLE proofreading domain are sensitive to CHK1 inhibitors.
Figure 3: Hit validation in A549 and SW620. SRA737 dose-response curves in A549 (A) and SW620 (C) cells transfected with Allstars negative
siRNA, POLA1, POLE or POLE2 siRNA (SRB) (n=3). SRA737 GI50 determinations calculated from dose-response curves in A549 (B) and
SW620 (D) cells.
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C o n c e n tr a tio n o f S R A 7 3 7 ( µ M )
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A
0 .1
rs
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D e a th s iR N A
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60
1
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U n tre a te d
Figure 1: Structure of SRA737 (PNT737)
2. Aim
A 549
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P O L E 2 s iR N A
6
rs
U n tre a te d
20
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P O L E s iR N A
80
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ta
D e a th s iR N A
P O L A 1 s iR N A
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lls
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2
A lls ta rs
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ck
P O L E 2 s iR N A
120
NS
o
60
3
M ock
M
P O L E s iR N A
***
***
10
140
S R A 7 3 7 G I 5 0 (µ M )
80
C e ll v ia b ility (% M o c k )
P O L A 1 s iR N A
ta
N
H
A lls ta rs
100
SW 620
4
O
N
N
M ock
Our data support the case for the use of the clinically relevant combination of SRA737 and gemcitabine, as
gemcitabine is metabolised it is incorporated into DNA, inhibiting the B-family DNA polymerases.6
D
C
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lls
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120
P
F3C
NS
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A549
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B
A
HN
We have recently described the discovery of a highly selective and orally
bioavailable CHK1 inhibitor, SRA737 (previously PNT737/CCT245737),
that not only has potent anti-tumour activity in combination with standard-ofcare genotoxic agents but also as a single agent in defined tumour types.1,2
SRA737 is currently being evaluated in two clinical trials in patients with
advanced cancer.3
Inhibition of B-family DNA polymerases is synthetically lethal with CHK1 inhibition in cancer cell lines
C e ll v ia b ility (% M o c k )
(R)
POLA1, POLE and POLE2 were validated as hits in A549 and SW620 and additional NSCLC and colon
cancer cell lines using individual Qiagen siRNAs.
ck
HN
CHK1 inhibition sensitises cancer cells to genotoxic agents and recent
studies have indicated that CHK1 inhibitors could be used as single agents
to treat cancers with high levels of replication stress.
Conclusions
o
Check point kinase 1 (CHK1) is a key regulator of the cell cycle, DNA damage repair and DNA replication.
4. Hit validation
S R A 7 3 7 G I 5 0 (µ M )
1. Introduction
CRT Pioneer fund