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High-Content Approaches to Understanding Chk1 Inhibitor Pharmacology, Andy Massey, Vernalis Research, Cambridge

Vernalis Research

Checkpoint kinase 1 (Chk1) has important roles in DNA replication in the absence of DNA damage induced by cytotoxic chemotherapeutic agents, particularly in cancer cells, which demonstrate increased sensitivity to Chk1 inhibitors. At Vernalis Research, the Operetta system is being used for single-cell immunofluorescent and live-cell imaging, to further understand the mode of action through which the Chk1 inhibitor, V158411, as monotherapy induces DNA damage and cell death in cancer cells.

HT29 cells are treated with increasing concentrations of V158411, then fixed and imaged for γH2AX expression. A dose- and time-dependent induction of γH2AX can be seen, and γH2AX induction is an early event that correlates with DNA damage response activation. A high-throughput phenotypic assay has been developed for cell-cycle analysis.1

HT29 cells are fixed and stained with Hoechst 33342, morphological properties (standard and STAR) calculated and PhenoLOGIC used to separate single cells from cell clumps. DNA content, EdU incorporation and pHH3 (S10) expression levels can then be used to separate cells into the various phases of the cell cycle.

EdU and pHH3 markers

Use of EdU and pHH3 (S10) markers allows separation of G2 from M-phase cells

Chk1 inhibition induces DNA damage in S phase cells. γH2AX induction is an early event that correlates with DNA damage repair activation. Complete and sustained inhibition of Chk1 activity is necessary for a robust cellular response, and there is a correlation between the induction of DNA damage and inhibition of cellular proliferation. V158411 induces caspase-3/7 cleavage, with H2AX induction and caspase-3/7 cleavage found to be mutually exclusive events. This work was recently published in Oncotarget2.

The findings from these studies can be used to further drive the preclinical and clinical stages of drug development, generating insights into dosing regimens, sensitive patient populations, biomarker discovery, and the identification of potential novel drug combinations.

References:

  1. Massey AJ (2015). "Multiparametric Cell Cycle Analysis Using the Operetta High-Content Imager and Harmony Software with PhenoLOGIC". PLoS ONE 10(7): e0134306. doi:10.1371/journal.pone.0134306

  2. Wayne, J., Brooks, T., & Massey, A. (2016). "Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase". Oncotarget, 7(51), 85033-85048.

For Research Use Only. Not for Use in Diagnostic Procedures.