Abstract 1816: BRD4 inhibitor I-BET151 sensitizes glioblastoma to radiotherapy by suppressing super-enhancer-driven COL1A1

Abstract Glioblastoma (GBM) is a highly aggressive and fatal brain tumor typically treated with high-dose radiation (RT) and chemotherapy. However, the limited RT efficacy has spurred interest in radiosensitizing strategies. BRD4, a BET protein, promotes tumor progression by regulating oncogenes through super-enhancers (SEs). Consequently, BRD4 inhibitors, such as I-BET151, are being investigated as potential therapeutic agents. This study investigates the synergistic efficacy of I-BET151 in combination with RT for GBM therapy. Analysis of public databases revealed elevated BRD4 expression in recurrent GBM compared to newly diagnosed cases, correlating with poor prognosis. Immunofluorescence analysis showed increased BRD4 expression in human (T98G and U87) and murine (GL261) GBM cells 48- and 96- hours post-RT (5 Gy). Pretreatment with I-BET151 (1µM or higher) sensitized GBM cells to RT (2.5 Gy or higher), enhancing RT-induced suppression of clonogenic survival, reducing proliferation and invasion, and significantly increasing apoptosis [e.g., GL261: 63.3% ± 0.5% vs. 10.9% ± 0.6% with RT alone (p < 0.001), 11.6% ± 0.6% with I-BET151 (p < 0.001)]. Co-treatment also amplified RT-induced DNA damage (γH2AX foci) 24 hours post-RT. In a subcutaneous GBM GL261 model in C57BL/6 mice, I-BET151 (10 mg/kg/day, intraperitoneally) administered concurrently with fractionated RT (2 Gy daily for 5 days) significantly reduced tumor growth compared to either treatment alone [e.g., tumor volume at 4 weeks: 259 ± 92.9 mm3 vs. 574.8 ± 126.5 mm3 with RT alone (p < 0.001), 705.8 ± 168.3 mm3 with I-BET151 (p < 0.001)]. Tumor tissues from the combination group consistently showed the highest levels of apoptosis (TUNEL staining), and lowest Ki67 index, confirming improved therapeutic efficacy. Mechanistically, BRD4 drives activation of SEs marked by H3K27ac. ChIP-seq analysis for H3K27ac in GL261 cells revealed that RT significantly increased SEs, which were reduced with I-BET151 co-treatment. RNA-seq identified 75 RT-upregulated genes that were downregulated with I-BET151 co-treatment. Pathway analysis linked these BRD4-regulated, RT-responsive genes primarily to extracellular matrix pathways. Integrated ChIP-seq and RNA-seq analysis identified COL1A1 as a key BRD4-dependent SE target post RT. I-BET151 suppressed COL1A1 expression both in vitro and in vivo. Moreover, RNAi-mediated COL1A1 silencing reduced proliferation, increased apoptosis, and enhanced RT-induced DNA damage, underscoring its pivotal role in BRD4-mediated radioresistance. In conclusion, I-BET151 enhances radiosensitivity in GBM cells by blocking RT-induced SE-activation and COL1A1 expression, thereby amplifying DNA damage and apoptosis. Further validation in orthotopic GBM models is essential to advance the clinical translation of this promising combination therapy. Citation Format: Xichen Fan, Xuenan Li, Ziheng Wang, Weichen Duan, Yafei Wang, Jiajia Chen. BRD4 inhibitor I-BET151 sensitizes glioblastoma to radiotherapy by suppressing super-enhancer-driven COL1A1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1816.