The DNA damage response in advanced ovarian cancer: functional analysis combined with machine learning identifies signatures that correlate with chemotherapy sensitivity and patient outcome

British Journal of Cancer ARTICLE www.nature.com/bjc OPEN Translational Therapeutics The DNA damage response in advanced ovarian cancer: functional analysis combined with machine learning identifies signatures that correlate with chemotherapy sensitivity and patient outcome Thomas D. J. Walker1,7, Zahra F. Faraahi2,7, Marcus J. Price1, Amy Hawarden1, Caitlin A. Waddell1, Bryn Russell1, Dominique M. Jones3, Aiste McCormick4, N. Gavrielides1, S. Tyagi1, Laura C. Woodhouse5, Bethany Whalley1, Connor Roberts1, Emma J. Crosbie1,6 and ✉ Richard J. Edmondson 1,6 1234567890();,: © The Author(s) 2023 BACKGROUND: Ovarian cancers are hallmarked by chromosomal instability. New therapies deliver improved patient outcomes in relevant phenotypes, however therapy resistance and poor long-term survival signal requirements for better patient preselection. An impaired DNA damage response (DDR) is a major chemosensitivity determinant. Comprising five pathways, DDR redundancy is complex and rarely studied alongside chemoresistance influence from mitochondrial dysfunction. We developed functional assays to monitor DDR and mitochondrial states and trialled this suite on patient explants. METHODS: We profiled DDR and mitochondrial signatures in cultures from 16 primary-setting ovarian cancer patients receiving platinum chemotherapy. Explant signature relationships to patient progression-free (PFS) and overall survival (OS) were assessed by multiple statistical and machine-learning methods. RESULTS: DR dysregulation was wide-ranging. Defective HR (HRD) and NHEJ were near-mutually exclusive. HRD patients (44%) had increased SSB abrogation. HR competence was associated with perturbed mitochondria (78% vs 57% HRD) while every relapse patient harboured dysfunctional mitochondria. DDR signatures classified explant platinum cytotoxicity and mitochondrial dysregulation. Importantly, explant signatures classified patient PFS and OS. CONCLUSIONS: Whilst individual pathway scores are mechanistically insufficient to describe resistance, holistic DDR and mitochondrial states accurately predict patient survival. Our assay suite demonstrates promise for translational chemosensitivity prediction. British Journal of Cancer; https://doi.org/10.1038/s41416-023-02168-3 INTRODUCTION An impaired DNA damage response (DDR) is fundamental to the development of the genome instability that defines cancer [1]. The DDR is complex with pathway overlap but can be represented as five pathways; two for double-strand breaks (DSB) and three for single-strand breaks (SSB). The role of homologous recombination (HR) pathway is the best-described DDR pathway. Tasked with the high-fidelity homology-based repair of DNA DSB, HR repair aligns to cell cycle to ensure DNA is repaired prior to mitosis or S phase [2, 3]. Nonhomologous end joining (NHEJ) represents the second DSB DNA repair pathway in the cell. NHEJ can directly ligate broken DNA and can accommodate non-compatible sequences with noncomplementary break overhangs (reviewed in ref. [4]). NHEJ operates throughout the cell cycle thus, whilst error-prone, is the predominant DSB repair DDR pathway in human cells. Three DDR pathways each govern distinct forms of SSB DNA repair: The base-excision repair (BER) pathway responds to nonhelix-distorting single-base lesions such as oxidised bases, deaminated bases, and alkylated bases that are caused by reactive oxygen species (ROS) or ionising radiation assault [5]. Nucleotide excision repair (NER) repairs helix-distorting bulky lesions and 1 Division of Cancer Sciences, Faculty of Biology, Medicine & Health, University of Manchester, M13 9WL Manchester, UK. 2Early Phase Drug Development, Labcorp, Harrogate HG3, UK. 3Manchester University Hospitals NHS Foundation Trust, Oxford Road, Manchester M13 9WL, UK. 4Department of Gynaecological Oncology, Glasgow Royal Infirmary, 16 Alexandra Parade, Glasgow G31 2ER, UK. 5Christie NHS Foundation Trust, Manchester, UK. 6Department of Obstetrics and Gynaecology, Manchester Academic Health Science Centre, Saint Mary’s Hospital, Oxford Road, Manchester M13 9WL, UK. 7These authors contributed equally: Thomas D. J. Walker, Zahra F. Faraahi. ✉email: Received: 8 June 2022 Revised: 12 January 2023 Accepted: 16 January 2023 T.D.J. Walker et al. 2 crosslinks [5, 6], while mismatch repair (MMR) responds to WatsonCrick mismatch base erroneous insertion, deletion, and misincorporations [7, 8]. However, the reality is that DDR pathway overlap and redundancy exists [9]. Moreover, the DDR needs to be seen in the context of other intracellular processes including mitochondrial (Mt) dysfunction and reactive oxygen species (ROS) both of which influence chemotherapy response [10]. Chemoresistance is multifaceted and driven by mechanistic and temporal factors connected to both the tumour microenvironment and the intrinsic ability of the ovarian cancer cell to resist chemotherapy (CT) [11]. Platinum CT is accepted to act by increasing intracellular ROS beyond a critical redox-homoeostasis threshold from which cancer cells cannot recover [10]. Chemoresistant cancer cells likely evolve rapid homoeostatic recovery or increased tolerances to accommodate the frequently-observed ROS abundance and oxidative stress increases [12] while perturbed ROS signalling enhances cell proliferation and survival [13]. As key producers and modulators of ROS [14], mitochondria are highly relevant to platinum CT efficacy. It is established that cancer cell Mt mutations act as oncogenomes [15] to influence oncogenesis [16] wherein chemoresistant cell Mt dysfunction associates with aggressive ovarian cancer subtypes, apoptotic resistance [17], and metastasis independent of the microenvironment [18]. Ovarian cancer is a heterogeneous set of diseases characterised by chromosomal instability and is an ideal model for profiling the DDR landscape [19]. The relevance of the DDR status to patient survival is exemplified by firstly the prognostic importance of BRCA gene mutation testing (as a means to presume HR functionality) currently operational in UK clinical practice, and secondly the success of PARP inhibitor therapy [20] to modulate patient tumour DDR response for increased progression-free longevity prior to patient relapse. In contrast to traditional cell line models, primary cultured patient cells offer a superior means to understand tumour heterogeneity (a) within a single tumour site, (b) across disparate sites within the same patient, and (c). between individual patients. Such facets permit deeper analytical integration of tumour DDR states for greater tumour diagnostic resolution. Although individual pathway aberrations have been described for HR [21, 22] and NHEJ [23, 24] in particular, to date a comprehensive assessment has not been carried out. In order to understand how DDR deficiencies relate to ovarian cancer therapy response and patient survival, we developed a suite of ex vivo assays to test DDR function in live cells isolated fro (...truncated)