Characterisation of nucleases contributing to the DNA damage driven immune response in cancer

Abstract

Breast cancer is one of the most commonly occurring cancers, affecting 1 in 7 women in their lifetime. The most aggressive subtype of breast cancer is known as triple negative breast cancer (TNBC), which is named due to its lack of targetable receptors. As a result, there is a significant need for improved treatment. Recent studies have shown an upregulation in immune signalling in TNBC, specifically, activation of the cGAS-STING pathway. Further understanding of this pathway could provide novel therapeutic targets or uncover biomarkers indicating likelihood of good prognosis following immunotherapy. TNBC often display DNA damage repair defects, resulting in the accumulation of cytoplasmic nucleic acids which are potent activators of the cGAS-STING pathway. We hypothesised that nucleases involved in DNA damage repair may therefore be implicated in the activation of cGAS-STING signalling and screened the impact of several nucleases on pathway activation.

Initial screening revealed that depletion of ERCC1 appeared to hyperactivate the cGAS-STING pathway in HeLa cells, however, varying results were observed when analysing the impact of ERCC1 depletion on activation of the pathway in breast cancer cell lines. It is hypothesised that this response is not exclusively cGAS-STING dependent but may rely on an increase in double stranded DNA breaks, however, further investigation is required.

It was also observed that depletion of EXO1 abrogated cGAS-STING pathway activation in both the HeLa cells and across several breast cell lines, suggesting that EXO1 plays a key role in activation of the pathway. It was noted that EXO1 depletion led to an increase in cytoplasmic DNA, but concurrently resulted in reduction of overall micronuclei numbers, cGAS positive micronuclei, phosphorylation of STAT1 and decreased cytokine transcription. Furthermore, total cGAS levels following EXO1 depletion was found to decrease, irrespective of doxorubicin treatment. Further investigation into the role of EXO1 in cGAS levels revealed that, upon depletion of EXO1, cGAS is degraded by the proteosome.

In summary, EXO1 is an important nuclease required for the stabilisation of cGAS and therefore could act as a biomarker which would indicate the ability of a tumour to respond to DNA damaging chemotherapy in combination with immune checkpoint blockade.

Thesis is embargoed until 31 July 2031.

Date of Award	Jul 2026
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy & Cancer Research UK
Supervisor	Kienan Savage (Supervisor) & Stuart McIntosh (Supervisor)