Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are currently used to treat BRCA1/2 mutant cancers. Although PARPi sensitivity has been attributed to homologous recombination (HR) defects, other roles of HR factors have also been linked to response to PARPi, including replication fork protection. In this study, we investigated PARPi sensitivity in ovarian cancer patient-derived xenograft (PDX) models in relation to HR proficiency and replication fork protection. Analysis of BRCA1/2 status showed that in our cohort of 31 ovarian cancer PDX models 22.6% harbored a BRCA1/2 alteration (7/31), and 48.3% (15/31) were genomically unstable as measured by copy number alteration analysis. In vivo, PARPi olaparib response was measured in 15 selected PDX models. Functional assessment of HR using ex vivo irradiation-induced RAD51 foci formation identified all olaparib-sensitive PDX models, including four models without BRCA1/2 alterations. In contrast, replication fork protection or replication speed in ex vivo tumor tissue did not correlate with olaparib response. Targeted panel sequencing in olaparib-sensitive models lacking BRCA1/2 alterations revealed a MUS81 variant as a possible mechanism underlying PARPi sensitivity. Combined, we show that ex vivo RAD51 analysis effectively predicts in vivo olaparib response and revealed a subset of PARPi-sensitive, HR-deficient ovarian cancer PDX models, lacking a BRCA1/2 alteration.

• Publication type
• Journal Article MeSH

The DNA damage checkpoints provide an anti-cancer barrier in diverse tumour types, however this concept has remained unexplored in prostate cancer (CaP). Furthermore, targeting DNA repair defects by PARP1 inhibitors (PARPi) as a cancer treatment strategy is emerging yet requires suitable predictive biomarkers. To address these issues, we performed immunohistochemical analysis of multiple markers of DNA damage signalling, oxidative stress, DNA repair and cell cycle control pathways during progression of human prostate disease from benign hyperplasia, through intraepithelial neoplasia to CaP, complemented by genetic analyses of TMPRSS2-ERG rearrangement and NQO1, an anti-oxidant factor and p53 protector. The DNA damage checkpoint barrier (γH2AX, pATM, p53) mechanism was activated during CaP tumorigenesis, albeit less and with delayed culmination compared to other cancers, possibly reflecting lower replication stress (slow proliferation despite cases of Rb loss and cyclin D1 overexpression) and progressive loss of ATM activator NKX3.1. Oxidative stress (8-oxoguanine lesions) and NQO1 increased during disease progression. NQO1 genotypes of 390 men did not indicate predisposition to CaP, yet loss of NQO1 in CaP suggested potential progression-opposing tumour suppressor role. TMPRSS2-ERG rearrangement and PTEN loss, events sensitizing to PARPi, occurred frequently along with heterogeneous loss of DNA repair factors 53BP1, JMJD1C and Rev7 (all studied here for the first time in CaP) whose defects may cause resistance to PARPi. Overall, our results reveal an unorthodox DNA damage checkpoint barrier scenario in CaP tumorigenesis, and provide novel insights into oxidative stress and DNA repair, with implications for biomarker guidance of future targeted therapy of CaP.

• Keywords
• DNA damage response barrier, NQO1 and oxidative stress, PARP inhibitor biomarkers, Prostate tumorigenesis, TMPRSS2-ERG, p53 and NKX3.1 tumour suppressors,
• MeSH
• PTEN Phosphohydrolase analysis   genetics   MeSH
• Oncogene Proteins, Fusion analysis   genetics   MeSH
• Genotype MeSH
• Middle Aged MeSH
• Humans MeSH
• NAD(P)H Dehydrogenase (Quinone) analysis   genetics   MeSH
• Prostatic Neoplasms genetics   metabolism   pathology   MeSH
• DNA Repair MeSH
• Oxidative Stress * MeSH
• DNA Damage * MeSH
• Disease Progression MeSH
• Prostate metabolism   pathology   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• PTEN Phosphohydrolase MeSH
• Oncogene Proteins, Fusion MeSH
• NAD(P)H Dehydrogenase (Quinone) MeSH
• NQO1 protein, human MeSH Browser
• PTEN protein, human MeSH Browser
• TMPRSS2-ERG fusion protein, human MeSH Browser

Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine.

• MeSH
• Biosensing Techniques * MeSH
• Microscopy, Confocal MeSH
• Cell Cycle Checkpoints drug effects   radiation effects   MeSH
• Lasers adverse effects   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• DNA Repair MeSH
• DNA Damage * drug effects   radiation effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH