Abstract 3303: Radioresistance in glioma stem cells driven by Rad51 dependent homologous recombination repair
Authors: Henry King, Helen Payne, Tim Brend, Anjana Patel, Alex Wright, Teklu Englu, Lucy Stead, Heiko Wurdak, Susan C. Short
Journal: Cancer Research (2015)

Abstract A cell population with stem cell like characteristics is thought to underlie treatment resistance and local recurrence in glioma. In this work we investigate the role of the DNA repair protein RAD51 in these cells and the impact of inhibiting homologous recombination repair on radiation resistance in vitro and in vivo. We used a model of inducible differentiation of patient derived glioma cells to investigate repair protein levels, repair foci formation and kinetics in clonogenic, stem-like populations and their differentiated counterparts. We examined co-expression of stem cell markers with RAD51 protein at whole population level using western blotting, immunocytochemistry and RT-PCR in cultured cells and immunohistochemistry in tumor material. Single cell expression was analysed using the Fluidigm C1 platform. We examined the effect of two specific inhibitors of RAD51 (B02, RI-1) on the same cell pairs in vitro and used the γH2AX assay to assess differences in repair kinetics. We used subcutaneous models of glioma to evaluate the effect of one of these agents (RI-1) on tumour growth delay with and without fractionated radiation doses in vivo. Primary glioma stem cells expressed high levels of RAD51 protein and exhibited high numbers of foci per nucleus following radiation exposure. Levels of both protein and repair foci fell after cells were transferred to differentiating conditions (serum+BMP4), as did expression of stem cell markers (NESTIN, SOX2). Single cell analysis showed a highly significant association between SOX2 and RAD51 expression (P&amp;lt; 0.0001). A similar association between RAD51 expression and stem cell marker expression was demonstrated in a series of glioblastoma specimens. When primary glioma cultures were treated with B02, RI-1 or siRNA targeting RAD51 in vitro, significant radiosensitisation was achieved with dose modifying factors ∼1.4 after single radiation doses. Delayed repair of DNA double strand breaks was also apparent, with a higher proportion of γH2AX foci unresolved in treated cells at 24 hours post irradiation. We also demonstrated significantly enhanced tumor growth delay in vivo when RI-1 was combined with fractionated radiation therapy (5Gy x 3) in a subcutaneous model. These data suggest that RAD51 dependent DNA repair by homologous recombination represents a specific target in the stem-like fraction of glioblastoma and inhibiting this pathway is a promising approach to radiosensitisation. Citation Format: Henry King, Helen Payne, Tim Brend, Anjana Patel, Alex Wright, Teklu Englu, Lucy Stead, Heiko Wurdak, Susan C. Short. Radioresistance in glioma stem cells driven by Rad51 dependent homologous recombination repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3303. doi:10.1158/1538-7445.AM2015-3303