BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma
Jun Watanabe, … , Oren J. Becher, Rintaro Hashizume
Jun Watanabe, … , Oren J. Becher, Rintaro Hashizume
Published May 21, 2024
Citation Information: J Clin Invest. 2024;134(13):e174794. https://doi.org/10.1172/JCI174794.
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Research Article Oncology

BET bromodomain inhibition potentiates radiosensitivity in models of H3K27-altered diffuse midline glioma

Abstract

Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.

Authors

Jun Watanabe, Matthew R. Clutter, Michael J. Gullette, Takahiro Sasaki, Eita Uchida, Savneet Kaur, Yan Mo, Kouki Abe, Yukitomo Ishi, Nozomu Takata, Manabu Natsumeda, Samantha Gadd, Zhiguo Zhang, Oren J. Becher, Rintaro Hashizume

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Figure 4

BET bromodomain inhibition increased radioresponse and apoptosis in DMG cells.

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BET bromodomain inhibition increased radioresponse and apoptosis in DMG ...
(A) Clonogenic survival for K27M-mutant DMG cells (SF8628, DIPG007, GEMM-DMG) treated with AZD5153 (50 nM for SF8628 and DIPG007, 10 nM for GEMM-DMG) for 12 hours before being subjected to IR. Survival fractions, shown as mean ± SEM based on averages from triplicate samples, were normalized to plating efficiency. DEF was calculated at 10% survival level. (B) Effects of AZD5153 and IR on cell proliferation in BrdU incorporation assay. Cells were treated with 500 nM AZD5153 in the presence or absence of 4 Gy IR for 48 hours, pulsed with 10 μM BrdU for 1 hour, then analyzed by flow cytometry. Left: Cell sorting scatter plots for vehicle control– (0.5% DMSO), AZD5153-, and IR-treated cells are shown. Right: Graphs show S-phase composition. One-way ANOVA comparisons between each treatment (n = 3), ****P < 0.0001; control vs. AZD5153: ***P = 0.0005 (SF8628), **P = 0.0065 (GEMM-DMG); AZD5153 vs. AZD5153 + IR: **P = 0.0020 (SF8628), ***P = 0.0002 (GEMM-DMG); control vs. IR: *P = 0.0177 (SF8628), IR vs. AZD5153 + IR: *P = 0.0147 (DIPG007). (C) Annexin V analysis of AZD5153 apoptosis effects. Cells were treated with vehicle control (0.05% DMSO) or 1 μM AZD5153 concurrently with and without 6 Gy IR. Cells were collected after 48 hours and treated with Alexa Fluor 488–Annexin V and flow sorted. Bar graphs represent Annexin V–positive cell numbers. One-way ANOVA comparisons of each treatment (n = 3), ****P < 0.0001; control vs. AZD5153: *P = 0.0115 (SF8628), **P = 0.0076 (DIPG007), **P = 0.0030 (GEMM-DMG); control vs IR: *P = 0.0124 (SF8628), **P = 0.0014 (DIPG007), *P = 0.0245 (GEMM-DMG); AZD5153 vs. AZD5153 + IR: **P = 0.0027 (SF8628), ***P = 0.0029 (DIPG007), *P = 0.0177 (GEMM-DMG); IR vs. AZD5153 + IR: **P = 0.0027 (SF8628), **P = 0.0029 (DIPG007), **P= 0.0022 (GEMM-DMG).