RDNA-04. POTENTIAL MECHANISM OF TEMOZOLOMIDE-MEDIATED RADIO-SENSITIZATION IN MGMT HYPERMETHYLATED GLIOBLASTOMA CELLS. (5th November 2018)

• Record Type: Journal Article
• Authors: Kitange, Gaspar; Smith, Emily; Gupta, Shiv; Sarkaria, Jann
• Is Part Of: Neuro-oncology. Volume 20(2018)Supplement 6
• Page Start: vi222
• Page End: vi222
• Publication Date: 2018-11-05
• Subjects: Brain Neoplasms -- Periodicals; Brain -- Tumors -- Periodicals; Brain -- Cancer -- Periodicals; Nervous system -- Cancer -- Periodicals; 616.99481
• DOI: 10.1093/neuonc/noy148.920
• Languages: English
• ISSNs: 1522-8517

Abstract

Abstract: Temozolomide (TMZ) mediated radio-sensitization is preferentially observed in MGMT hypermethylated glioblastoma (GBM) and is modulated by unknown underlying mechanisms. To gain insight into the potential mechanism of TMZ-induced radio-sensitization, we examined the effect of TMZ pre-exposure on the processing of radiation (RT)-induced double strand breaks (DSBs) in an isogenic model, the U251-vector versus U251-MGMT cells derived by the lentiviral MGMT transduction. Cells were treated with RT (2Gy) alone or after 2 days of pre-exposure to low concentrations of TMZ (20 µM) and survival was evaluated by clonogenic assay and DSBs by western blotting and immunofluorescence based γH2AX and RPA foci. TMZ pre-exposure significantly sensitized RT in U251-vector but not in U251-MGMT cells. In contrast, no significant difference was observed between RT alone and the concurrent RT/TMZ treatment. The radio-sensitization mediated by TMZ pre-exposure in U251-vector cells was accompanied with delayed DSB processing as evidenced by prolonged phosphorylation of KAP1(p-KAP1) and H2AX (γH2AX). Since DNA-PKcs plays a critical role in repair of RT-induced DSBs, we evaluated whether TMZ pre-exposure impacts the phosphorylation of DNA-PKcs in context of RT sensitization. Interestingly, we observed that TMZ alone induced phosphorylation of DNA-PKcs in both U251-vector and U251-MGMT cells and this effect was paradoxically suppressed by RT in time related manner, but in contrast, RT alone significantly increased p-DNA-PKcs. Further experiments confirmed that TMZ induces p-DNA-PKcs in vitro and in vivo and this effect was pharmacologically suppressed by ATR and ATM inhibitors. Importantly, RT-induced DSBs were decorated with p-RPA only in cells pre-exposed to TMZ suggesting single stranded DNA perhaps due to DSB end-resection. Together these results suggest that TMZ-mediated radio-sensitization is due to delayed repair of RT-induced DSBs and the ongoing studies are focused on establishing a link between TMZ-mediated radio-sensitization and TMZ-induced phosphorylation of DNA-PKcs.