Yang Liu, Jing Yue, Xia Xia, Yin Xiushan
Applied Biology Laboratory, College of Pharmaceutical and Biological Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China.
J Oncol. 2023 Dec 14;2023:9904143. doi: 10.1155/2023/9904143. eCollection 2023.
The bromodomain-containing 4 (BRD4) is a member of the bromodomain and extra terminal domain (BET) family, which is an important epigenetic reader. It is currently a promising oncology target. In some tumors, BET bromodomain inhibitors have demonstrated promising results. Proteolysis-targeting methods (PROTAC), which rapidly and effectively degrade BRD4, have displayed considerable potential in the treatment of tumors in recent years. The purpose of this study is to examine the potential impact of BRD4 PROTAC compounds ARV-825 on oncogene BRD4-NUT fused protein in NUT carcinoma.
The effectiveness of ARV-825 was evaluated at the cellular level using the cell counting kit 8 test, wound healing, cell transfection, western blotting analysis, and RNA sequencing. The effectiveness of ARV-825 was also examined in vivo using a xenograft model.
The BRD4-NUT fusion gene was overexpressed in 3T3 cells, and the pathogenic fusion gene was simulated. The results showed that the overexpression of BRD4-NUT could promote the proliferation and migration of 3T3 cells, but the expression of BRD4 protein was degraded after the addition of the novel cereblon-based PROTAC compound ARV-825 against BRD4, resulting in inhibition of BRD4-NUT 3T3 cell proliferation and migration. Further RNA-seq analysis showed that overexpression of BRD4-NUT was accompanied by increased expression of gene (e.g., Myc, E2F, TRAFs, Wnt, Gadd45g, and Sox6) with significantly enriched pathway (e.g., small cell lung cancer, NF-kappa B signaling pathway, and breast cancer), promoted cell cycle from G 1 phase to S phase, and increased cell proliferation and migration, activated the antiapoptosisi signal, led to abnormal cell growth, and ultimately led to tumorigenesis. The addition of ARV-825 effectively rescued this process and effectively inhibited cell vitality, proliferation, and migration. In vivo studies demonstrated that treatment with ARV-825 greatly suppressed tumor growth without causing harmful side effects and downregulated the BRD4-NUT expression level.
Through the induction of BRD4 protein degradation, ARV-825 can successfully limit BRD4-NUT 3T3 cell proliferation in vitro and in vivo. These findings suggested that the BRD4 inhibitor ARV-825 would be an effective therapeutic strategy for treating NUT carcinoma that with the genetic feature of BRD4-NUT fusion event.
含溴结构域蛋白4（BRD4）是溴结构域和额外末端结构域（BET）家族的成员，是一种重要的表观遗传阅读器。它目前是一个有前景的肿瘤学靶点。在一些肿瘤中，BET溴结构域抑制剂已显示出有前景的结果。靶向蛋白水解方法（PROTAC）能快速有效地降解BRD4，近年来在肿瘤治疗中显示出巨大潜力。本研究的目的是探讨BRD4 PROTAC化合物ARV-825对NUT癌中致癌基因BRD4-NUT融合蛋白的潜在影响。
使用细胞计数试剂盒8检测、伤口愈合实验、细胞转染、蛋白质免疫印迹分析和RNA测序在细胞水平评估ARV-825的有效性。还使用异种移植模型在体内检测ARV-825的有效性。
BRD4-NUT融合基因在3T3细胞中过表达，并模拟了致病融合基因。结果表明，BRD4-NUT的过表达可促进3T3细胞的增殖和迁移，但加入新型基于cereblon的针对BRD4的PROTAC化合物ARV-825后，BRD4蛋白表达被降解，导致BRD4-NUT 3T3细胞增殖和迁移受到抑制。进一步的RNA测序分析表明，BRD4-NUT的过表达伴随着基因（如Myc、E2F、TRAFs、Wnt、Gadd45g和Sox6）表达增加，且通路（如小细胞肺癌、NF-κB信号通路和乳腺癌）显著富集，促进细胞周期从G1期进入S期，增加细胞增殖和迁移，激活抗凋亡信号，导致细胞生长异常，最终导致肿瘤发生。加入ARV-825有效挽救了这一过程，并有效抑制了细胞活力、增殖和迁移。体内研究表明，ARV-825治疗可显著抑制肿瘤生长，且无有害副作用，并下调BRD4-NUT的表达水平。
通过诱导BRD4蛋白降解，ARV-825可在体外和体内成功限制BRD4-NUT 3T3细胞增殖。这些发现表明，BRD4抑制剂ARV-825将是治疗具有BRD4-NUT融合事件遗传特征的NUT癌的有效治疗策略。
