Objective

Cells lacking DNA dependent protein kinase (DNA-PK) fail to repair DSBs by non-homologous end-joining (NHEJ) and inhibitors of DNA-PK are known to sensitise cells to ionising radiation (IR).  However, the target inhibition requirements to maximize the lethality of this effect are unclear.  Using a high content imaging platform that allows us to multiplex multiple biomarker measurements at a range of timepoints, we have generated a rich data set data to describe the kinetics and mechanisms of combination of a range of DNA-PK inhibitors with IR.  This tracks the response from proximal biomarkers of target inhibition, through impact on the DNA damage response, to phenotypic effects on the cell cycle and cell death. Tthe platform has enabled us to compare continuous target exposure with compound washout.  Results generated from this approach has enabled us to generate and parameterise a PK-PD-efficacy model that is being validated in xenograft models.  This work demonstrates the value of high content biology to develop a temporal understanding of mechanism and target exposure requirements. Mechanistic modelling of this data in turn informs experimental designs to understand PK-PD-efficacy relationships and guide candidate drug selection.