Promiscuous targeting of bromodomains by Bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia [set2]. Homo sapiens

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET inhibitors and their significant activity in diverse tumor models has rapidly translated into clinical studies and has motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of bromodomain protein complexes complicates predictions of consequences of their pharmacological targeting. To address this issue we developed a promiscuous bromodomain inhibitor (bromosporine, BSP) that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle we studied the effect of BSP in leukemic cell-lines known to be sensitive to BET inhibition and found as expected strong anti-proliferative activity. Comparison of the modulation of transcriptional profiles by BSP at short inhibitor exposure resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, non-selective targeting of BRDs identified BETs, but not other BRDs, as master regulators of a context dependent primary transcription response. Overall design: Cells were seeded the day prior to treatment at 2x10^5 per ml. Treatments were performed so that a final concentration of 0.1 % DMSO (Cat.#D1435; Sigma) was achieved and cells were incubated with DMSO or 500 nM test compound (BSP, JQ1, LP99, GSK2801, iCBP112 or OF1) for 6 h prior to isolation of RNA. Total RNA was isolated using a standard TRIzol (Invitrogen) protocol and prepared using RNeasy columns (Cat.#74106 plus; Qiagen). RNA was quantified using a Nanodrop spectrophotometer (model ND1000; Thermo Fisher) and integrity assessed on a BioAnalyzer (2100; Agilent Laboratories, USA). All samples had a RNA Integrity Number (RIN) ≥ 9. Biological triplicates were performed for each condition tested.

溴结构域（Bromodomains, BRDs）已成为癌症治疗中极具吸引力的靶点。选择性强效BET抑制剂的开发及其在多种肿瘤模型中的显著活性，已快速推进至临床研究阶段，并推动了针对非BET型BRDs的药物研发工作。然而，溴结构域蛋白复合物复杂的多结构域/亚基架构，使得对其药理学靶向作用后果的预测变得困难重重。为解决这一难题，我们研发了一种广谱溴结构域抑制剂——溴孢子素（bromosporine, BSP），该化合物以纳摩尔级亲和力广泛靶向各类BRDs（包括BET家族蛋白），为识别BRDs发挥调控功能的细胞进程与疾病提供了研究工具。作为原理验证，我们在已知对BET抑制敏感的白血病细胞系中考察了BSP的作用效果，正如预期般观察到了显著的抗增殖活性。对比短时间抑制剂暴露下BSP与其他抑制剂对转录谱的调控模式，发现BSP仅呈现出BET抑制剂的特征性转录变化，未出现可归因于其他BRDs抑制的显著额外转录改变。由此可见，对BRDs的非选择性靶向仅证实了BET家族蛋白是依赖于情境的初级转录应答的核心调控因子，而非其他BRDs。整体实验设计：实验前一日将细胞以2×10^5个/ml的密度接种培养。所有处理体系均添加终浓度为0.1%的二甲基亚砜（DMSO，货号D1435；Sigma），随后将细胞置于含DMSO或500 nM受试化合物（BSP、JQ1、LP99、GSK2801、iCBP112或OF1）的培养基中孵育6小时，之后提取RNA。总RNA提取采用标准TRIzol（Invitrogen）操作流程，并通过RNeasy柱式纯化试剂盒（货号74106 plus；Qiagen）完成样本制备。使用Nanodrop分光光度计（型号ND1000；赛默飞世尔科技）对RNA进行定量，并通过生物分析仪（2100；安捷伦实验室，美国）评估RNA完整性，所有样本的RNA完整性指数（RNA Integrity Number, RIN）均≥9。每个测试条件均设置3次生物学重复。