B300: An assessment of PARP-1 inhibition in combination with external beam x-radiation in the treatment of neuroblastoma
1Radiation Oncology, Institute of Cancer Sciences, Glasgow, UK,2Great Ormond Street Hospital, London, UK
Neuroblastoma is the most common extracranial solid malignancy in children. It is an embryonal tumour of the sympathetic nervous system. High-risk disease often requires intensive multi-modal treatment which may involve radiotherapy. The DNA repair enzyme poly(ADP-ribose) polymerase-1 (PARP-1) becomes activated following genotoxic insult. It is thought that PARP-1 inhibitors decrease the threshold for cell death when used in combination with radiotherapy by inhibiting the repair of radiation-induced DNA damage. PARP-1 inhibitors olaparib and rucaparib are third generation PARP-1 inhibitors currently undergoing Phase II/III clinical evaluation.
Flow cytometry was used to investigate the effects of PARP-1 inhibitors and X-radiation as single agents and in combination. Treatments were assessed by their ability to induce DNA damage (measured by increased ?H2AX fluorescence) and on their effect on cell cycle progression. Immunofluorescence microscopy confirmed the cellular location of ?H2AX.
Combination therapy resulted in a greater increase in ?H2AX-positive cells compared to treatment with either drug alone, or radiation alone. PARP-1 inhibitor concentrations ? 10 µM caused accumulation of cells in G2. Combination therapy resulted in a significant increase in DNA damage 2 hr post irradiation, which persisted 24 hr post irradiation, indicated by a significantly higher percentage of ?H2AX positive cells (P<0.05) compared to the untreated control. Immunofluorescence microscopy confirmed the nuclear location of ?H2AX foci, with increased foci formation correlating with increased radiation dose.
As single agents, PARP-1 inhibition and X-radiation caused an increase in the G2 population of cells. This may be indicative of a G2/M arrest, which has been associated with increased rates of apoptosis in tumour cells. Cells are also more radiosensitive in G2/M, prompting the possibility of scheduling combination therapy. Combination therapy resulted in greater DNA damage which was both additive and dose-dependent. Our findings suggest that combination therapy in high-risk neuroblastoma patients may be beneficial.