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

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.

Phosphatase and Tensin Homolog Is a Potential Target for Ovarian Cancer Sensitization to Cytotoxic Agents.

OBJECTIVES: The phosphatase and tensin homolog (PTEN) tumor suppressor protein has been found to be inactivated or mutated in various human malignancies and to play a role in cisplatin and poly(ADP-ribose) polymerase inhibitor sensitivity. In this study, we assessed the association of PTEN loss with homologous recombination (HR) deficiency and increased chemosensitivity. MATERIALS AND METHODS: The PTEN knockdown models were created using MISSION shRNA lentiviral transduction particles in cell lines derived from normal ovarian surface epithelium and a mixed endometrioid/clear-cell carcinoma. Sensitivity to common therapeutics was assessed using sulforhodamine B assay. Twenty-eight unselected primary epithelial ovarian cancer cultures derived from ascitic fluid collected at the time of surgery and matched genomic DNA were assessed for PTEN mutations using polymerase chain reaction amplification and Sanger sequencing and for mRNA expression using quantitative reverse transcription-polymerase chain reaction; HR was determined using γH2AX/RAD51 assay. The Cancer Genome Atlas data were analyzed using cBioPortal. RESULTS: In the carcinoma cell line, the PTEN knockdown enhanced sensitivity to cisplatin, rucaparib, doxorubicin, camptothecin, paclitaxel, and irradiation. In the primary ovarian cancer cultures, 2 point mutations were found (1105T>TG, 25L>L in 6 cultures and 1508G>GA, 159R>R in 4 cultures). The PTEN mRNA expression varied over 40-fold between the cultures, but did not correlate with HR status or in vitro sensitivity to cisplatin or rucaparib. The Cancer Genome Atlas data showed a rate of 8% alteration in PTEN and a trend toward improved survival in PTEN-mutated cases. CONCLUSIONS: These data indicate that although PTEN mutations in ovarian cancer are rare, PTEN inhibition results in therapeutic sensitization. Therefore, PTEN may be an important therapeutic target, in at least some cancers.

Advanced Ovarian Cancer Displays Functional Intratumor Heterogeneity That Correlates to Ex Vivo Drug Sensitivity.

INTRODUCTION: Epithelial ovarian cancer is recognized to be heterogeneous but is currently treated with a single treatment strategy. Successful patient stratification of emerging chemotherapy agents is dependent upon the availability of reliable biomarkers indicative of the entire tumor. AIM: The aim of this study was to evaluate intertumor and intratumor heterogeneity within a series of epithelial ovarian cancer using homologous recombination (HR) DNA repair status. METHODS: Primary cultures generated from ascites and solid tumor from multiple intra-abdominal sites were characterized by their morphology and expression of protein markers. Results were compared with Formalin fixed paraffin embedded tissue pathology.Homologous recombination function was determined by quantification of nuclear Rad51 foci. Growth inhibition (sulforhodamine B) assays were used to calculate the GI50 for cisplatin and rucaparib. RESULTS: Ascites with matched solid tumor were cultured from 25 patients.Concordance in functional HR status between ascites and solid tumor subcultures was seen in only 13 (52%) of 25 patients. Heterogeneity in HR status was seen even in patients with homogeneous histological subtype. Homologous recombination defective cultures were significantly more sensitive to cisplatin and rucaparib.Additionally, intertumor and intratumor heterogeneity was seen between the expression of epithelial and ovarian markers (EpCAM, cytokeratin, CA125, MOC-31, and vimentin). There was no relationship between heterogeneity of HR functional status and antigen expression. CONCLUSIONS: Intertumor and intratumor functional HR heterogeneity exists that cannot be detected using histological classification. This has implications for biomarker-directed treatment.

DNA damage repair in ovarian cancer: unlocking the heterogeneity.

Treatment for advanced ovarian cancer is rarely curative; three quarters of patients with advanced disease relapse and ultimately die with resistant disease. Improving patient outcomes will require the introduction of new treatments and better patient selection. Abrogations in the DNA damage response (DDR) may allow such stratifications.A defective DNA-damage response (DDR) is a defining hallmark of high grade serous ovarian cancer (HGSOC). Indeed, current evidence indicates that all HGSOCs harbour a defect in at least one major DDR pathway. However, defective DDR is not mediated through a single mechanism but rather results from a variety of (epi)genetic lesions affecting one or more of the five major DNA repair pathways. Understanding the relationship between these pathways and how these are abrogated will be necessary in order to facilitate appropriate selection of both existing and novel agents.Here we review the current understanding of the DDR with regard to ovarian, and particularly high grade serous, cancer, with reference to existing and emerging treatments as appropriate.

Ovarian Cancers Harbor Defects in Nonhomologous End Joining Resulting in Resistance to Rucaparib.

Purpose: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focused on homologous recombination (HR), DNA double-strand breaks are repaired primarily by nonhomologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance.Experimental Design: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay.Results: NHEJ was defective (D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P = 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, induced resistance to rucaparib (P = 0.014) and HR function recovery in a BRCA1-defective cell line.Conclusions: This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin Cancer Res; 23(8); 2050-60. ©2016 AACR.

Functional characterisation of a novel ovarian cancer cell line, NUOC-1.

BACKGROUND: Cell lines provide a powerful model to study cancer and here we describe a new spontaneously immortalised epithelial ovarian cancer cell line (NUOC-1) derived from the ascites collected at a time of primary debulking surgery for a mixed endometrioid / clear cell / High Grade Serous (HGS) histology. RESULTS: This spontaneously immortalised cell line was found to maintain morphology and epithelial markers throughout long-term culture. NUOC-1 cells grow as an adherent monolayer with a doubling time of 58 hours. The cells are TP53 wildtype, positive for PTEN, HER2 and HER3 expression but negative for oestrogen, progesterone and androgen receptor expression. NUOC-1 cells are competent in homologous recombination and non-homologous end joining, but base excision repair defective. Karyotype analysis demonstrated a complex tetraploid karyotype. SNP array analysis of parent and derived subpopulations (NUOC-1-A1 and NUOC-1-A2) cells demonstrated heterogeneous cell populations with numerous copy number alterations and a pro-amplification phenotype. The characteristics of this new cell line lends it to be an excellent model for investigation of a number of the identified targets. MATERIALS AND METHODS: The cell line has been characterised for growth, drug sensitivity, expression of common ovarian markers and mutations, clonogenic potential and ability to form xenografts in SCID mice. Copy number changes and clonal evolution were assessed by SNP arrays.

Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition.

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure.Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs.Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine.

The use of ovarian cancer cells from patients undergoing surgery to generate primary cultures capable of undergoing functional analysis.

The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.