Targeting the DNA damage response in hormone-driven cancers

Change log
Kolyvas, Emily 

Genomic instability is a hallmark of cancers including breast cancer (BC) and prostate cancer (PCa). Defects in the DNA damage response (DDR) as well as replication stress are responsible, in part, for driving genomic instability and accumulation of DNA damage. While the resulting alterations can be beneficial for tumour development and progression, it also presents a vulnerability for pharmacologic inhibition. This has been demonstrated previously by PARP inhibitor sensitivity in BRCA-deficient cancers. This has led to the discovery and development of several DDR pathway inhibitors. Understanding the biology and genomic context that could lead to tumour responses to these inhibitors will be instrumental for identifying the patient populations that will benefit most.

In this thesis, the DDR is explored as a therapeutic target in hormone-driven cancers. Hormone signalling has been shown to drive DNA repair, and is therefore a worthwhile model to study mechanisms of sensitisation to DNA damaging therapeutics.

The androgen receptor (AR) has been linked to radio-resistance in PCa and BC by regulating the expression of DNA repair genes. The interaction between AR and DNA repair cofactors, such as DNA-PKcs, is under active exploration as a potential target for sensitising AR-driven cancers to radiotherapy (RT). Chapter 3 aimed to investigate the intersection between AR and DDR signalling pathways and explore the potential of targeting AR and DNA-PKcs for radio-sensitisation. Results indicate that single-agent treatment with inhibition of AR or DNA-PKcs led to sensitisation, and combination treatment had an additive effect. Proteomics data to determine AR cofactors suggested an interaction between DNA repair proteins including DNA-PKcs and AR. Gene expression analyses following combination treatments revealed various signalling pathways implicated in sensitising these models to radiation. Further analysis will be instrumental in validating these pathways and associated TFs with a mechanism for radio-sensitisation in BC and PCa.

Furthermore, in Chapter 4, PTEN loss was assessed in BC and PCa as a sensitiser to DNA damage and DDR inhibitors. Nuclear PTEN has been implicated in maintenance of genome stability, and this was tested in PTEN knock-out cell line models. The results show that PTEN loss results in increased sensitivity to ionising radiation along with impaired recovery from exogenously induced replication stress. When screened against a panel of DDR inhibitors, PTEN loss proved to be a predictor of response to inhibitors that sense and respond to replication stress.

Interestingly, when assessing DDR inhibitor response in BC PDXs, nuclear PTEN protein expression, but not mRNA expression, is a predictor of response. This suggests that nuclear PTEN could be a useful biomarker of response to these inhibitors. Additionally, RNA sequencing of a PTEN knock-out model suggested increased immune activation. This could suggest a role for combination of DDR inhibitors with immune checkpoint blockade in this model.

To test these hypotheses, a CRISPR knock-out of PTEN in a BC patient derived xenograft (PDX) was developed and characterised. Given the known advantages of PDXs for preclinical drug development, generation of a CRISPR knock-out in a PDX is a valuable tool for studying specific effects of single gene loss. Results showed that a stable knock-out was developed that was maintained with passaging and with metastasis, providing a useful model for downstream analysis of DDR effects of PTEN loss.

Caldas, Carlos
androgen receptor, breast cancer, DNA damage repair, DNA-PKcs, patient-derived xenograft, prostate cancer, PTEN, radiation therapy, replication stress
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Cancer Research UK (S_4070)