Abstract 293: Screening the druggable genome for synthetic lethal interactions with the CHK1 inhibitor PNT737
Authors: Rebecca Rogers, Mike I. Walton, Paul Clarke, Ian Collins, Michelle D. Garrett, Paul Workman
Journal: Cancer Research (2017)

Abstract Check point kinase 1 (CHK1) is a key regulator of the cell cycle, DNA damage repair and DNA replication. 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. We have recently described the discovery of a highly selective and orally bioavailable CHK1 inhibitor, PNT737, that not only has potent antitumour activity in combination with standard-of-care genotoxic agents but also as a single agent in defined tumour types. Here we sought to identify gene products whose loss would be synthetically lethal with CHK1 inhibition, with the aim of identifying patient populations likely to be sensitive to single agent CHK1 inhibition or to novel combinations utilising CHK1 inhibitors. To do this, we performed a large siRNA screen of the druggable genome (~6500 genes) in A549 (NSCLC) and SW620 (colon cancer) cell lines, with and without PNT737 treatment, and determined effects on cell viability by SRB. POLA1, POLE and POLE2 (B-family DNA polymerases) were identified as significant hits causing synthetic lethality with PNT737 in both cancer cell lines. Treatment with additional siRNA sequences subsequently validated these genes in both the original two cell lines and extra NSCLC and colon cancer cell lines. Interestingly, a number of biomarkers for replication stress, pRPA2 and pCHK1, were increased in cells treated with POLA1, POLE and POLE2 siRNA in combination with PNT737, in comparison to cells treated with the siRNA or drug alone. Further studies conducted with PNT737 and the B-family DNA polymerase inhibitor aphidicolin showed that these agents had a synergistic effect on inhibiting cell viability on 8 out of 9 NSCLC and colon cancer cell lines tested. In addition, immunofluorescence analysis revealed that there was an increase in the level of γH2AX, a marker of DNA damage, in 4 out of 5 cell lines that exhibited synergy when treated with a combination of aphidicolin and PNT737, as compared to cells treated with either agent alone. Our data indicate that the combination of a reduction in POLA1, POLE or POLE2 activity (by siRNA transfection or aphidicolin treatment) and CHK1 activity (PNT737 treatment) increases replication stress and DNA damage in NSCLC and colon cancer cells. Encouragingly, our data support the case for the use of the clinically relevant combination of PNT737 and gemcitabine, as gemcitabine is metabolised it is incorporated into DNA, inhibiting the B-family DNA polymerases. Furthermore, 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. Citation Format: Rebecca Rogers, Mike I. Walton, Paul Clarke, Ian Collins, Michelle D. Garrett, Paul Workman. Screening the druggable genome for synthetic lethal interactions with the CHK1 inhibitor PNT737 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 293. doi:10.1158/1538-7445.AM2017-293