ESTRO 2024 - Abstract Book

S5416

Radiobiology - Tumour biology

ESTRO 2024

the 5-year survival rate is below 10%. The intrinsic radioresistance of GBM is one key feature leading to treatment failure, recurrence, and poor outcome.

BET (Bromodomain and Extra-Terminal motif) proteins (BRD2, BRD3, BRD4 and BRDT) regulate the gene expression of oncogenic transcription factors (e.g., MYC) and of proteins involved in DNA damage repair. BRD4 has been found to be upregulated in GBM and is negatively associated with the prognosis [1]. Different BET inhibitors (BETi) have been developed and some of them (e.g., trotabresib) are already used in clinical studies [2, 3]. Newer BETi (e.g., dBET6) are PROTACs (Proteolysis Targeting Chimeric Molecules) leading to the degradation of BET proteins [4].

In this study, we aim to target BET proteins by the BET inhibitors trotabresib and dBET6 to counteract the radioresistance of GBM cell lines.

Material/Methods:

The two human GBM cell lines LN18 and LN229 were used to investigate the effects of the BETi dBET6 and trotabresib. BETi were dissolved in DMSO and in all experiments PBS and the solvent DMSO (0.1 %) were used as controls.

Cell cycle distribution 2, 6, 24, and 48 hours after BETi treatment (10, 100, 1000 nM) was analyzed by propidium iodide staining and subsequent flow cytometry.

Colony formation assay (CFA) was used to investigate the clonogenic survival after the combined treatment with BETi (100, 250, 500, 750, 1000, 2500 nM) and radiation (0, 2, 4, 6, 8 Gy). Based on the cell cycle results, cells were treated with BETi 24 hours before radiation. The survival curves were fitted to the linear quadratic model and D 50 values and sensitizing enhancement ratios (SER) were calculated.

The effect of BETi (100, 1000, 2500 nM) on the expression of BET proteins (BRD2, BRD3 and BRD4) and MYC was determined by Western Blot.

Statistical analysis was performed by student’s t-test or ANOVA.

Results:

Cell cycle analysis revealed the strongest effects 24 hours after the addition of BETi; both, dBET6 (1 µM) and trotabresib (1 µM) significantly increased the G1 phase and reduced the radioresistant S-phase in LN18 and LN229 cells. Interestingly, the survival of LN18 cells was more affected by trotabresib whereas the survival of LN229 cells was stronger reduced by dBET6. This is also reflected by the IC50 values; 904 nM (LN229) and > 2500 nM (LN18) for dBET6, and 2418 nM (LN229) and 633 nM (LN18) for trotabresib. The clonogenic survival of irradiated LN18 cells was significantly reduced by 500 - 2500 nM dBET6 (SER: 1.55 – 2.37) and 250 - 2500 nM trotabresib (SER: 1.39 – 2.08) indicating radiosensitization by BETi. In contrast, in the more radiosensitive cell line LN229, only 500 nM and 2500 nM trotabresib (SER: 1.38 and 1.43) significantly increased the radiosensitivity.