Abstract 5375: Effective temozolomide sensitization by the ATM inhibitor KU 55933 is lost with the emergence of TMZ resistance in glioblastoma cell lines

Abstract

Abstract Cellular DNA repair mechanisms limit the success of temozolomide (TMZ) therapy in the treatment of glioblastoma (GBM) by countering TMZ induced DNA damage and contributing towards TMZ resistance. Hence DNA repair inhibition has the potential to overcome TMZ resistance in GBM. KU-55933 is a small molecule inhibitor of Ataxia telangiectasia (A-T) mutated protein (ATM), which plays a critical role in DNA repair. In this study, the chemosensitizing effects of KU-55933 were studied in combination with TMZ in two glioblastoma cell lines, U251 and U87. Treatment with a combination of TMZ and KU-55933, compared to TMZ alone, resulted in a significant increase in cell killing [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p<0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p<0.001), respectively]. To evaluate whether KU-55933 would enhance the efficacy of TMZ in resistant lines, the U251TMZ and U87TMZ lines were generated by subjecting U251 and U87 cells to in vitro TMZ selection. Interestingly, in these TMZ resistant lines, a combination of TMZ and KU-55933 did not result in a significant increase in cell killing compared to treatment with TMZ alone [U251TMZ survival: 0.84 ± 0.03 vs. 0.87 ± 0.01 (p>0.1), respectively, and U87TMZ survival: 0.62 ± 0.03 vs 0.63 ± 0.09 (p>0.1), respectively]. To investigate the potential mechanism of resistance to TMZ sensitization by KU-55933, the effect of KU-55933 on TMZ induced G2 arrest was analyzed by flow cytometry. Consistent with the clonogenic survival data, a combination of TMZ and KU-55933 significantly increased the number of cells arrested in the G2 phase in both parental lines, compared to treatment with TMZ alone [U251 G2 fraction: 61.8 ± 1.1% vs. 35 ± 0.8% (p<0.001), respectively, and U87 G2 fraction 25 ± 0.2% vs.18.6 ± 0.4% (p<0.001), respectively]. Co-treatment of the TMZ resistant lines with KU-55933 and TMZ also increased the fraction of cells arrested G2, as compared to treatment with TMZ alone, although the extent of the G2 arrest was markedly attenuated relative to the parental cells [U251TMZ G2 fraction: 20 ± 0.6% vs. 12 ± 0.4% (p<0.001), respectively, and U87TMZ G2 fraction 14 ± 3.14% vs. 9 ± 0.5%, (p>0.1), respectively]. Preliminary evaluation of double stranded break (DSB) repair by immunofluorescent monitoring of γH2AX foci formation 72 hours after treatment demonstrated a 2-fold increase in the number of unrepaired DSBs in U251 cells treated with a combination of TMZ and KU-55933, as compared to TMZ alone, (p<0.001) while no increase in foci formation was observed in the U251TMZ cells treated with combined therapy as compared to TMZ alone (p>0.1). In conclusion, KU-55933 sensitizes only inherently TMZ sensitive lines to TMZ and these effects may be linked to inhibition of ATM-mediated DNA damage repair following TMZ treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5375.