Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.

中文翻译：

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BRD4在启动子上的双峰结合和在Pol II暂停位点处的药物诱导的置换与I-BET敏感性相关。

转录失调是诸如癌症之类的疾病的主要驱动力。溴结构域和末端外（BET）蛋白（BRD2，BRD3，BRD4和BRDT）是通过特异性结合乙酰化赖氨酸残基来维持适当基因转录所必需的染色质读取器。使用BET抑制剂（I-BETs）有针对性地从染色质中置换BET蛋白是一种有前途的疗法，尤其是对于急性髓细胞性白血病（AML），并且对耐药机制的评估对于优化这些药物的临床疗效至关重要。为了揭示内在性I-BET耐药性的机制，我们在敏感性和耐药性体外白血病模型中，定量了用I-BET151剂量反应治疗后BRD2，BRD3和BRD4的染色质结合和置换，并使用抗体和化合物亲和力捕获方法在全基因组范围内绘制BET蛋白/ I-BET相互作用图谱，然后进行深度测序。BET蛋白对I-BET的敏感性的全基因组图谱揭示了侧翼转录起始位点（TSS）结合的双峰模式，其中染色质的药物介导置换主要影响TSS下游的BRD4并延长了RNA Pol II的暂停。在基因表达的RNA Pol II暂停位点的BRD4结合和药物介导的位移与基因表达的相关性揭示了敏感和耐药肿瘤细胞对I-BET的差异行为，并在敏感编码和非编码基因的启动子处鉴定出BRD4签名。我们提供的证据表明，通过BRD4的置换，I-BET诱导的启动子上的Pol II暂停转移是内在I-BET敏感性的决定因素。