Background: Deficiencies in the DNA damage response (DDR) sensitize cancer cells to radiotherapy, chemotherapy, and targeted agents such as PARP inhibitors. Identifying these deficiencies can reveal synthetic lethal (SL) interactions - where two individually non-lethal defects become lethal in combination - enabling precision oncology. Currently, DDR deficiencies are primarily assessed through genomic and transcriptomic profiling; however these indirect and do not always reflect functional impairment. Moreover, several DDR inhibitors in clinical development, such as ATM, ATR, WEE1 and CHK1 inhibitors, lack robust predictive biomarkers. These limitations highlight the need for functional assays to assess DDR function to guide personalised therapy.
Aim: To functionally characterize the DDR pathway using multiplex immunofluorescence, enabling the identification of targetable deficiencies and sensitivities to DDR-targeted therapies.
Materials and Methods: NSCLC cell line A549 was administered with a range of targeted DDR inhibitors to establish a ‘ground truth’ of pathway-specific deficiencies, then imaged 0-48 hours post irradiation. Cyclic immunofluorescence was implemented to image over 40 markers relating to DDR, cell state, fate and signalling markers.
Results: Single cell and single foci analyses enabled the reconstruction of DDR progression along a pseudotime, revealing the dynamics of pathway activation and resolution. Inhibitor-induced changes in DDR signalling were detectable, including activation of compensatory pathways, suggesting potential synthetic lethal interactions.
Conclusion: Deep multiplex immunofluorescence of DDR provides a functional readout of DDR activity, offering a powerful approach to map resistance mechanisms and identify novel phenotypes predictive of sensitivity to DDR-targeted therapies.