CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells a C Rebecca F
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Published OnlineFirst March 11, 2020; DOI: 10.1158/0008-5472.CAN-19-1372 CANCER RESEARCH | TRANSLATIONAL SCIENCE CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells A C Rebecca F. Rogers1, Michael I. Walton1, Daniel L. Cherry2, Ian Collins1, Paul A. Clarke1, Michelle D. Garrett2, and Paul Workman1 ABSTRACT ◥ Checkpoint kinase 1 (CHK1) is a key mediator of the DNA Replication stress, DNA damage, and apoptosis were increased in damage response that regulates cell-cycle progression, DNA human cancer cells following depletion of the B-family DNA damage repair, and DNA replication. Small-molecule CHK1 polymerases combined with SRA737 treatment. Moreover, phar- inhibitors sensitize cancer cells to genotoxic agents and have macologic blockade of B-family DNA polymerases using aphi- shown single-agent preclinical activity in cancers with high levels dicolin or CD437 combined with CHK1 inhibitors led to syn- of replication stress. However, the underlying genetic determi- ergistic inhibition of cancer cell proliferation. Furthermore, low nants of CHK1 inhibitor sensitivity remain unclear. We used the levels of POLA1, POLE, and POLE2 protein expression in developmental clinical drug SRA737 in an unbiased large-scale NSCLC and colorectal cancer cells correlated with single-agent siRNA screen to identify novel mediators of CHK1 inhibitor CHK1 inhibitor sensitivity and may constitute biomarkers of this sensitivity and uncover potential combination therapies and phenotype. These findings provide a potential basis for combin- biomarkers for patient selection. We identified subunits of the ing CHK1 and B-family polymerase inhibitors in cancer therapy. B-family of DNA polymerases (POLA1, POLE,andPOLE2) whose silencing sensitized the human A549 non–small cell lung Significance: These findings demonstrate how the therapeutic cancer (NSCLC) and SW620 colorectal cancer cell lines to benefit of CHK1 inhibitors may potentially be enhanced and could SRA737. B-family polymerases were validated using multiple have implications for patient selection and future development of siRNAs in a panel of NSCLC and colorectal cancer cell lines. new combination therapies. Introduction DNA polymerases become uncoupled from DNA helicases, which continue to unwind the DNA. This DNA unwinding generates DNA is routinely subject to exogenous or endogenous sources of stretches of ssDNA that are protected by binding of replication protein damage (1). Therefore, cells have evolved complex surveillance A (RPA; 8). RPA recruits a number of replication stress response mechanisms, known as cell-cycle checkpoints, to tightly regulate the proteins and is reliant on the ATR–CHK1 pathway to initially stabilize cell cycle and maintain genomic integrity (2, 3). In normal cells, these replication forks and delay the onset of mitosis until replication is checkpoints are activated in response to DNA damage, so that cell- resumed and completed (7, 9). Checkpoint kinase 1 (CHK1) is a serine/ cycle progression can be coordinated with the detection and repair of threonine kinase and a critical component of several cell-cycle check- damaged DNA to prevent genomic instability. In cancer cells, these points acting through multiple mechanisms, including replication fork cell-cycle checkpoints are frequently deregulated, leading to high levels stabilization and repair (10, 11). Cancer cells with defects in cell-cycle of replication stress and genomic instability, an enabling characteristic control, including those with high levels of replication stress, may of cancer (4, 5). therefore be more dependent on cell-cycle checkpoints and specifically Replication stress is often described as the slowing or stalling of CHK1 for survival (12). Such cancer-specific dependency could be replication fork progression during DNA synthesis and is caused by a exploited for therapeutic gain, making CHK1 an attractive anticancer number of different factors (6, 7). As a result of replication fork stalling, target. Preclinical validation of CHK1 as a cancer drug target, using siRNA and chemical tools, has led to the development of a number of CHK1 1 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, inhibitors (13), including the highly selective, potent, and orally 2 London, United Kingdom. School of Biosciences, Stacey Building, University of bioavailable clinical drug candidate SRA737 (14, 15), which is under- Kent, Canterbury, Kent, United Kingdom. going evaluation in phase I clinical trials (www.clinicaltrials.gov; Note: Supplementary data for this article are available at Cancer Research NCT02797977 and NCT02797964). CHK1 inhibitors have been Online (http://cancerres.aacrjournals.org/). shown to potentiate a number of chemotherapeutic agents (16), in Corresponding Authors: Michelle D. Garrett, University of Kent, Giles Lane, particular gemcitabine (17, 18), and demonstrate single-agent activity Canterbury, CT2 7NJ, United Kingdom. Phone: 44-0-1227-816-140; E-mail: in cancer subtypes with high levels of replication stress (17, 19, 20). [email protected]; and Paul Workman, Cancer Research UK Cancer Ther- Despite these observations, the underlying genetic determinants of apeutics Unit, The Institute of Cancer Research, London, SM2 5NG, United Kingdom. Phone: 44-0-20-7153-5209; E-mail: [email protected] sensitivity to clinically relevant CHK1 inhibitors remain unclear and there are currently no clinically validated biomarkers for patient Cancer Res 2020;80:1735–47 selection. The identification of genes that are synthetically lethal doi: 10.1158/0008-5472.CAN-19-1372 in combination with CHK1 inhibition could lead to novel drug Ó2020 American Association for Cancer Research. combination studies and potential predictive biomarkers of CHK1 AACRJournals.org | 1735 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst March 11, 2020; DOI: 10.1158/0008-5472.CAN-19-1372 Rogers et al. inhibitor sensitivity. This may allow the identification of sensitive primary antibodies: POLA1 (ab31777), POLE (ab134941), POLE2 patient populations and aid the clinical application and evaluation of (ab57298), CHK1 pS345 (CST-2348), CHK1 (SC-8408), RPA32 CHK1 inhibitors. (ab125681), cleaved PARP (CST-9541), and GAPDH (ab8245). Synthetic lethality occurs when the simultaneous loss of function Membranes were then washed and incubated with horseradish of two gene products results in cell death but the loss of either alone peroxidase–conjugated secondary antibodies (mouse/rabbit) for does not (21). Using the clinical drug candidate SRA737, we 1 hour. Proteins were visualized using enhanced chemilumines- performed a large-scale synthetic lethal siRNA screen in human cence (Pierce Biotechnology) and hyperfilm (GE Healthcare). cancer cells to identify those gene products whose loss is synthet- ically lethal with CHK1 inhibition. Here, we show that siRNAs Screening with an siRNA library targeting POLA1, POLE,andPOLE2, which encode subunits The Dharmacon druggable genome siRNA library (Dharmacon, GE belonging to the B-family of DNA polymerases, are synthetically Healthcare Life Sciences) consists of siRNAs (four pooled siRNAs per lethal in combination with CHK1 inhibition in multiple human gene), which target the expression of 7,593 genes that encode known non–small cell lung cancer (NSCLC) and colorectal cancer cell lines, drug targets or potentially druggable proteins. Of these 7,593 genes, suggesting a new potential treatment approach. Moreover, combi- 6,371 (84%) were screened, which included the kinase, phosphatase, natorial depletion of the B-family DNA polymerases and small- and drug targets subsets. Dharmacon druggable human genome molecule CHK1 inhibition resulted in increased replication stress, siRNA library master plates (384-well) containing lyophilized siRNA DNA damage, and cell death. Pharmacologic blockade of both B- were resuspended in RNAse-free water to a final concentration of family DNA polymerases and CHK1 led to synergistic inhibition of 5 mmol/L. Using the automated Echo 550 Liquid Handler (Labcyte), cancer cell proliferation. Furthermore, we show that low POLA1 0.5 mL of siRNA was then transferred from a master plate to six 96-well and POLE protein expression may represent biomarkers for single- daughter plates, to give a final assay concentration of 25 nmol/L. agent CHK1 inhibitor sensitivity. siRNAs were introduced into cells by reverse transfection; 50 mLof HiPerFect in Opti-MEM (Invitrogen) was added to each well at a final assay concentration of 0.3% and incubated for 20 minutes. Cells Materials and Methods (50 mL), at optimum seeding density, were then added to each well, Cell lines incubated for 48 hours at 37C and treated with SRA737 at the Human cell lines were obtained from the ATCC, except SKLU1, maximum sublethal dose (0.4 mmol/L or 0.8 mmol/L for A549 and which was obtained from the Health Protection Agency Culture SW620, respectively) or DMSO vehicle (0.004% or 0.008% for A549 Collections (United Kingdom). Lines were validated by DNA profiling and SW620, respectively) in 100 mL medium followed by incubation and confirmed free of Mycoplasma species by PCR using the Venor- for 84 hours at 37C. Plates were fixed with 10% trichloroacetic acid GeM Mycoplasma PCR Detection Kit (Minerva Labs). All cell lines and stained with 0.4% sulphorhodamine-B (SRB) in 1% acetic acid. were purchased more than a year prior to the experiments and