Promiscuous targeting of bromodomains by Bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia [set 1]

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. 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 or JQ1) 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（bromodomain, BRDs）已成为癌症治疗领域极具吸引力的靶点。选择性强效的BET抑制剂（BET inhibitor）及其在多种肿瘤模型中展现出的显著活性，已快速推进至临床研究阶段，并推动了针对非BET型溴结构域的药物开发工作。然而，溴结构域蛋白复合物复杂的多结构域/亚基组装模式，使得对其药理学靶向作用后果的预测变得极为复杂。为解决这一难题，我们开发了一种广谱溴结构域抑制剂（bromosporine, BSP），其可通过纳摩尔级亲和力靶向包括BET家族在内的多种溴结构域蛋白，为识别溴结构域发挥调控功能的细胞过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测试了BSP的作用，正如预期般观察到了强劲的抗增殖活性。对短期抑制剂处理后BSP对转录组谱的调控模式进行比较后发现，其仅产生了BET抑制剂特有的转录特征，未出现可归因于其他溴结构域被抑制的显著额外转录变化。由此可见，通过非选择性靶向溴结构域，可确认BET家族而非其他溴结构域蛋白是依赖于环境的初级转录应答的核心调控因子。
细胞于处理前一日以2×10^5个/毫升的密度接种。实验处理设置终浓度为0.1%的二甲基亚砜（DMSO，货号D1435；Sigma），将细胞与DMSO或500 nM受试化合物（BSP或JQ1）共孵育6小时后提取总RNA。总RNA提取采用标准TRIzol（Invitrogen）操作流程，并通过RNeasy纯化柱（货号74106 plus；Qiagen）完成样本制备。RNA浓度采用Nanodrop分光光度计（型号ND1000；赛默飞世尔科技）进行定量，RNA完整性则通过BioAnalyzer生物分析仪（型号2100；安捷伦实验室，美国）进行评估。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。