American Association for Cancer Research
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Supplementary Figures 1-12 from CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells

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journal contribution
posted on 2023-03-31, 03:22 authored by Rebecca F. Rogers, Michael I. Walton, Daniel L. Cherry, Ian Collins, Paul A. Clarke, Michelle D. Garrett, Paul Workman

Supplementary Figures - SF1: GI50 determinations for CHK1 inhibitor SRA737 in A549 and SW620 cells. SF2: Knockdown of POLA1, POLE or POLE2 sensitises A549 and SW620 cells to the CHK1 inhibitor MK-8776. SF3: Knockdown of CHK1 sensitises A549 cells to the DNA polymerase inhibitor aphidicolin. SF4: POLA1, POLE or POLE2 knockdown is synthetically lethal with the CHK1 inhibitor SRA737 in additional NSCLC and colorectal cancer cell lines. SF5: Western blot analysis of POLA1, POLE and POLE2 knockdown in additional NSCLC cell lines. Western blot analysis of POLA1, POLE and POLE2 knockdown in additional colorectal cancer cell lines. SF7: SRA737 and gemcitabine synergistically inhibit the proliferation of 4 NSCLC cell lines. SF8: An additive interaction between SRA737 and aphidicolin in the HBEC3-KT cell line. SF9: Effect of CHK1 or B-family DNA polymerase pharmacological inhibition alone or in combination on RPA32 phosphorylation in A549 cells. SF10: Effect of CHK1 or B-family DNA polymerase pharmacological inhibition alone or in combination on �H2AX foci in A549 cells. SF 11: Correlation between POLA1, POLE and POLE2 basal protein expression in NSCLC and colorectal cancer cell lines. SF12: Lack of correlation between POLA, POLE and POLE2 basal protein expression and SRA737 or aphidicolin combination indices in NSCLC and colorectal cancer cell lines.

Funding

Institute of Cancer Research

Cancer Research UK

Wellcome Trust

University of Kent

Kent Cancer Trust

EB Hutchinson Charitable Trust

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ARTICLE ABSTRACT

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non–small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

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