Small Molecule Therapeutics Poly-ADP-Ribose Polymerase as a Therapeutic Target in Pediatric Diffuse Intrinsic Pontine Glioma and Pediatric High-Grade Astrocytoma Yevgen Chornenkyy 1,2 , Sameer Agnihotri 1 , Man Yu 1 , Pawel Buczkowicz 1 , Patricia Rakopoulos 1,2 , Brian Golbourn 1,2 , Livia Garzia 1 , Robert Siddaway 1 , Stephie Leung 1,2 , James T. Rutka 1,3 , Michael D. Taylor 1,3 , Peter B. Dirks 1,3 , and Cynthia Hawkins 1,2,4 Abstract Pediatric high-grade astrocytomas (pHGA) and diffuse intrin- sic pontine gliomas (DIPG) are devastating malignancies for which no effective therapies exist. We investigated the therapeu- tic potential of PARP1 inhibition in preclinical models of pHGA and DIPG. PARP1 levels were characterized in pHGA and DIPG patient samples and tumor-derived cell lines. The effects of PARP inhibitors veliparib, olaparib, and niraparib as monotherapy or as radiosensitizers on cell viability, DNA damage, and PARP1 activity were evaluated in a panel of pHGA and DIPG cell lines. Survival benefit of niraparib was examined in an orthotopic xenograft model of pHGA. About 85% of pHGAs and 76% of DIPG tissue microarray samples expressed PARP1. Six of 8 primary cell lines highly expressed PARP1. Interestingly, across multiple cell lines, some PARP1 protein expression was required for response to PARP inhibition; however, there was no corre- lation between protein level or PARP1 activity and sensitivity to PARP inhibitors. Niraparib was the most effective at reducing cell viability and proliferation (MTT and Ki67). Niraparib induced DNA damage (g H2AX foci) and induced growth arrest. Pretreatment of pHGA cells with a sublethal dose of niraparib (1 mmol/L) before 2 Gy of ionizing radiation (IR) decreased the rate of DNA damage repair, colony growth, and relative cell number. Niraparib (50 mg/kg) inhibited PARP1 activity in vivo and extended survival of mice with orthotopic pHGA xenografts, when administered before IR (20 Gy, fractionated), relative to control mice (40 vs. 25 days). Our data provide in vitro and in vivo evidence that niraparib may be an effective radiosensitizer for pHGA and DIPG. Mol Cancer Ther; 14(11); 2560–8. Ó2015 AACR. Introduction Brain tumors account for 20% of all neoplasms in children and are the largest group of solid tumors in childhood (1). Among these tumors, pediatric supratentorial high-grade astrocytomas (pHGA) and diffuse intrinsic pontine gliomas (DIPG) are incur- able pediatric malignancies for which no effective therapies exist. The prognosis is poor: the 5-year survival rate for pHGAs is 15% to 30% and the 2-year survival rate for DIPGs is <10% (2–7). Clinical investigations into the effects of adjuvant radiotherapy or temozolomide on patient survival have shown that temozo- lomide is ineffective in DIPG. However, radiotherapy, the current standard of care, can increase overall survival of pHGA by 6.26 months (8, 9). Unfortunately, in both pHGA and DIPG, radio- therapy is predominantly palliative offering some symptom con- trol for a limited period of time (10). Both temozolomide and radiotherapy also damage the developing cortex, deep brain structures, and posterior fossa, leading to adverse sequelae in patients, with higher risk when applied at a younger age (11–16). Chemo- and radiosensitizers may offer the opportunity to improve the therapeutic index by increasing efficacy of radiother- apy or chemotherapeutic drugs while reducing toxicity and dam- age to developing brain (17, 18). PARP enzymes catalyze the addition of PAR polymers onto acceptor proteins and are essential for single-strand break (SSB) DNA repair (19). Upon DNA damage, PARP1 binds to DNA via its N-terminal zinc finger motif and automodifies itself with long chains of PAR. This modification negatively charges PARP1 and allows it to dissociate from DNA while serving to recruit addi- tional downstream DNA repair components (19, 20). PARP1 has been shown to be involved in delaying the replication fork progression in homologous recombination (HR)-proficient DNA damaged cells and in alternative pathways of nonhomologous end joining (NHEJ; refs. 21–23). PARP inhibitors are synthetically lethal in BRCA1/2-mutated tumors with DNA repair deficiencies and have been shown to be effective when combined with DNA- damaging agents in isogenic BRCA1/2-deleted cell lines and BRCA1-deficient mouse models (24–27). High levels of PARP1 expression are found in pHGA and correlate with reduced overall survival (17). Furthermore, in a small cohort of DIPG cases, we identified PARP1 expression combined with LOH in other DNA repair genes (5). The effects 1 Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada. 2 Department of Labora- tory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. 3 Division of Neurosurgery, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada. 4 Division of Pathology, The Hospital for Sick Children, Uni- versity of Toronto, Toronto, Ontario, Canada. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Cynthia Hawkins, Department of Paediatric Laboratory Medicine, University of Toronto, Scientist, Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. Phone: 416-813-5938; Fax: 416-813-5974; E-mail: cynthia.hawkins@sickkids.ca doi: 10.1158/1535-7163.MCT-15-0282 Ó2015 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 14(11) November 2015 2560 Downloaded from http://aacrjournals.org/mct/article-pdf/14/11/2560/2332878/2560.pdf by guest on 10 June 2022