Abstract B27: The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair
Authors: Chatterjee Payel , Choudhary Gaurav , Heston Warren D. , Klein Eric A. , Almasan Alex 
Published: May 2013

Abstract
Exposure to genotoxic agents, such as irradiation produces DNA damage and its toxicity is augmented when the DNA repair is impaired. Poly ADP ribose polymerase (PARP) inhibitors, such as rucaparib (CO-338; formerly known as AG014699 and PF-01367338) have been also found to be most effective in cells deficient in DNA repair. Radiation dose and scheduling experiments were performed in the prostate cancer cell line C4-2 in conditions mimicking brachytherapy with low dose radiation (Ir-192) as compared to external beam radiotherapy (EBRT). These studies have established the effectiveness of rucaparib to radiosensitize prostate cancer cells when the two clinical modalities are used: EBRT or brachytherapy, with a clear benefit of lower doses administered over a longer period of time, that caused DNA damage due to DNA replication fork collapse when rucaparib was present. Clonogenic survival assay were then performed on a panel of prostate cancer cell lines (LNCaP, C4-2, PC3, DU145, MDaPc2B, CWR22rv1, and VCaP) following radiation and rucaparib, used either alone or together. The combination index (CI) calculated for these various combinations revealed synergistic interactions, which was strongest for LNCaP cells. In addition to apoptosis, senescence was an important response, particularly in PTEN-negative tumors, as it was not observed in Du145 cells that have a wild-type PTEN allele. Absence of PTEN therefore allows activation of senescence, which contributes to decreased clonogenic survival. Increased radiosensitivity in presence of rucaparib was associated with persistent DNA breaks as determined by gamma-H2AX and p53BP1 foci. Interestingly, VCaP cells showed such foci constitutively, indicative of persistent DNA damage that was not present if the TMPRSS2/ERG gene fusion was depleted by siRNA. This response correlated with the ability of the gene fusion protein to bind DNA-PKcs on the chromatin and, as a result, inhibited its kinase activity. DNA-PKcs functional deficiency caused by TMPRSS2/ERG destabilized critical nonhomologous end-joining (NHEJ) components, such as Ku70 and XRCC4. When the TMPRSS2/ERG gene fusion protein was depleted by siRNA the DNA-PKcs kinase activity was restored and the DNA damage response (constitutive or induced) diminished, as measured by gamma-H2AX and p53BP1 foci. Therefore, the presence of the TMPRSS2/ERG fusion gene protein, by inhibiting NHEJ DNA repair, sensitized prostate cancer cells to TREATMENT WITH radiation and/or rucaparib. These results support the clinical testing of rucaparib in combination with radiotherapy. Late stage metastatic tumors, which harbor a gene fusion and are defective in PTEN could benefit most from a combination therapy using brachytherapy and rucaparib in a neoadjuvant setting.
Citation Format: Payel Chatterjee, Gaurav Choudhary, Warren D. Heston, Eric A. Klein, Alex Almasan. The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B27.