EGR1 induction compromises the therapeutic efficacy of BET depletion in triple-negative breast cancer (RNA-Seq)

Bromodomain and extraterminal domain (BET) proteins are epigenetic readers of acetylated lysine residues, which regulate gene expression and are involved in tumorigenesis. Over the past two decades, BET-bromodomain (BD) inhibitors and BET-targeting PROTAC degraders have been developed and clinically tested. BET protein depletion with PROTAC degrader is generally recognized to provide stronger outcomes compared to just inhibiting the binding of protein to acetylated lysine residues with competitive inhibitors. In this study, we found that the bivalent BET-BD inhibitor MS645 exhibited much more potent anti-proliferative activity than BET degraders, including ARV771, AT1, MZ1 and dBET1 in triple-negative breast cancer cells. MS645 treatment dissociated BET proteins from chromatin and decreased E2F1-3 expression, thus inhibited cell growth of TNBC. While ARV771 treatment didn't affect E2F1-3 expression though it displaced BET proteins from chromatin as well. We found knockdown of BRD4 or BRD4 depletion by ARV771 treatment dramatically induced EGR1 expression. EGR1 interacts with Septin complex and maintained its nuclear distribution. Septin complex is recruited by EGR1 to E2F1-3 gene loci to activate E2F1-3 transcription, thus maintained cell cycle progression. Overall design: To investigate the differentially expressed genes in HCC1806 cells upon MS645 and ARV771 treatment, HCC1806 cells were treated with MS645 or ARV771 for 8h and RNA were isolated for RNA-sequencing

溴结构域和额外末端结构域（Bromodomain and extraterminal domain, BET）蛋白是乙酰化赖氨酸残基的表观遗传阅读器，可调控基因表达并参与肿瘤发生。过去二十年间，靶向BET的溴结构域（BD）抑制剂及靶向BET的蛋白水解靶向嵌合体（PROTAC）降解剂已被开发并进入临床测试。相较于仅通过竞争性抑制剂阻断蛋白与乙酰化赖氨酸残基的结合，使用PROTAC降解剂敲除BET蛋白通常被认为能带来更优异的干预效果。本研究发现，在三阴性乳腺癌（triple-negative breast cancer, TNBC）细胞中，双价BET-BD抑制剂MS645展现出比包括ARV771、AT1、MZ1及dBET1在内的BET降解剂更强的抗增殖活性。MS645处理可将BET蛋白从染色质上解离，并降低E2F1-3的表达，从而抑制三阴性乳腺癌细胞的增殖；尽管ARV771处理同样可将BET蛋白从染色质上解离，但并未对E2F1-3的表达产生影响。我们发现，通过ARV771处理敲除BRD4或使BRD4耗竭，可显著诱导EGR1的表达。EGR1可与中隔蛋白（Septin）复合物结合，并维持其在细胞核内的分布；EGR1将中隔蛋白复合物招募至E2F1-3基因位点以激活其转录，进而维持细胞周期进程。实验整体设计：为探究经MS645与ARV771处理后HCC1806细胞中的差异表达基因，将HCC1806细胞用MS645或ARV771处理8小时后提取RNA，用于RNA测序（RNA-sequencing）。