Abstract 5543: DNA damage response and anti-apoptotic proteins are predictors of histone deacetylase inhibitors-mediated radiosensitization of glioblastoma
Authors: Pont Lotte M. Berghauser , Kloezeman Jenneke , Naipail Kishan , Bent Martin van den , Gent Dik van , Dirven Clemens , Kanaar Roland , Lamfers Martine , Leenstra Sieger 
Published: May 2015

Background. Histone deacytelase inhibitors (HDACi) have radiosensitizing properties in established cancer cell lines. This study was conducted in the context of ‘precision medicine’, to determine the response of various HDACi as radiosensitizers in a variety of primary glioblastoma stem cell (GSC) cultures. The objective was to assess the response rate, the predictors of outcome and response markers related to DNA repair and apoptosis. Further, the optimal timing and sequence of treatments was investigated of combined treatment as well as effects of fractionation.

Methods. SAHA, LBH589, Valproic Acid (VPA), MS-275 and Scriptaid were tested alone and in combination with radiation on twenty-two primary serum-free GSC cultures. SAHA and LBH589 were studied in more detail, with regard to acetylated-H3, p21Cip1/WAF1, Bcl-2, LC3I/II, caspase-3/7, 53BP1, and Chek2/ATM activation in various primary GSC cultures with different sensitivity to treatment. A variety of timing schedules and dose fractions were tested, the latter compared with single radiation dose.

Results. The various HDACi sensitized (p&lt;0.05) a subset of primary GSC cultures to radiation: SAHA 50%, LBH589 50%, VPA and Scriptaid 40%, and MS/275 60%. The overlap between SAHA and LBH589 sensitive cultures was high with 9/11 sensitive cultures being responsive for both HDACi. The Chek2, ATM and Bcl-2 levels were higher in untreated sensitive GSC cultures compared to resistant cultures, whereas Bcl-XL was higher in resistant cultures. All sensitive cultures phosphorylated Chek2 after applying radiation, the resistant GSC cultures did not. Other observed differences after treatment, related to sensitivity were histone-H3 acetylation, caspase-3/7 activation, and enhanced DNA damage foci (53BP1 and γH.2AX) by SAHA or LBH589. Incubation 24h and 48h before radiation resulted in significantly better combination effects compared to other sequences (p&lt;0.05). Fractionizing of radiation did not negatively affect the responses (p&gt;0.05).

Conclusion. This study shows that HDACi as radiosensitizers have a wide range of effects in patient-derived GSC cultures, and tumour sensitivity is associated with protein levels of untreated GSC cultures of DNA damage response markers Chek2, ATM, and apoptotic markers Bcl-XL, and Bcl-2. The Chek2 activation upon radiation was associated with response in vitro. Candidates to measure treatment effects are enhanced DNA damage response foci, and caspase-3/7 activation. In the light of precision medicine, these markers need to be studied to assess their in vivo specificity. Furthermore, timing of the treatments is essential to obtain the sensitizing effects.

Citation Format: Lotte M. Berghauser Pont, Jenneke Kloezeman, Kishan Naipail, Martin van den Bent, Dik van Gent, Clemens Dirven, Roland Kanaar, Martine Lamfers, Sieger Leenstra. DNA damage response and anti-apoptotic proteins are predictors of histone deacetylase inhibitors-mediated radiosensitization of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2014-5543