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#293 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 3000 4000 5000 6000 7000 s iR N A Contact Cancer Research UK Cancer Therapeutics Unit The Institute of Cancer Research, London [email protected] 1000 2000 5000 6000 7000 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 ) 4 2 2 (# (# E L P O O L O #3 #2 #4 - SR A737 + 0 .2 3 µ M S R A 7 3 7 PO LA1 PO LE PO LE2 #3 #2 #4 - SR A737 + 2 .4 u M S R A 7 3 7 P 20 PO LA1 PO LE PO LE2 #3 #2 #4 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 40 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. * 6 5 NS * * 4 U n tre a te d S R A 7 3 7 [G I 5 0 ] 3 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 0 A5 4 9 C a lu - 6 N C I- H 1 9 7 5 **** 4 3 * *** 2 ** 1 0 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 * 60 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 * 0 W E E 1 s iR N A #2 20 A lls t a r s n e g a t iv e #3 W E E 1 s iR N A PO LE2 40 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 60 80 0 .1 n M 20 * W E E 1 s iR N A 40 * A lls t a r s n e g a t iv e 60 80 M ock *** n≥3 100 U n tre a te d * % c e ll v ia b ility * 80 -2 0 120 n=3 100 A549 Phosphorylation of RPA32 is a reported biomarker of replication stress. RKO D * 6. Replication stress response Untreated P ) ) 3 A A E 2 E L O L 4 (# (# (# ) ) 2 3 100 0 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 3000 4000 s iR N A 100 HCT116 U n tre a te d 20 N C I- H 4 6 0 6 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 60 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. 1000 * * 80 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. 3 100 -2 0 5 4 0 0 .1 C n≥3 M ock 3. Hit identification 10 0 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. 6 O L N C I- H 1 9 7 5 120 14 U n tre a te d 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. A 7 E C o n c e n tr a tio n o f S R A 7 3 7 ( µ M ) L 100 (# 10 1 1 A 0 .1 rs 0 .0 1 D e a th s iR N A 40 ) 0 60 1 0 U n tre a te d Figure 1: Structure of SRA737 (PNT737) 2. Aim A 549 1 P O L E 2 s iR N A 6 rs U n tre a te d 20 * P O L E s iR N A 80 8 ta D e a th s iR N A P O L A 1 s iR N A *** lls 40 2 A lls ta rs 100 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 **** lls N 5 120 P F3C NS A N A549 **** M O **** 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