BCL6转录激活促进KRAS突变肺癌抵抗BET抑制剂的分子机制研究

Mutation of oncogenic KRAS often occurs in human malignancies with the highest mortality rates. KRAS-mutant cancers represent more aggressive and are associated with resistance to therapy. However, the underlying mechanism remains elusive. Our preliminary results showed that bromodomain and extra terminal protein (BET) inhibitors remarkably up-regulated the expression of the BCL6 (B-cell lymphoma 6) transcriptional repressor in a set of KRAS-mutant lung cancer cell lines and lung tumor tissues. Blockade of BCL6 function either by gene knockout or by pharmacological inhibitors significantly potentiated the antitumor efficacy of BET inhibitors. Mechanism study further showed that the binding of BCL6 to its DNA sequence were altered by BET inhibitors. Therefore, we hypothesize that BET inhibition relieves BCL6-mediated self-repression and consequently leads to its transcriptional activation, which markedly promotes primary resistance to BET inhibitors in KRAS-mutant lung cancer. However, detailed mechanisms of the regulation of BCL6 self-repression and its downstream signaling pathways remain unclear. In this project, we will further pursue the above mechanism study by using the RNA-sequencing technology, signaling pathway analysis and pharmacological validation in mice. Our study will reveal novel signaling transduction underlying drug resistance in KRAS-mutant lung cancer, providing critical molecular basis for the treatment of this malignant tumor.

癌基因KRAS在人类常见致死性肿瘤中频发突变，KRAS突变肿瘤恶性程度强且表现出先天耐药性，但是具体机制仍不清晰。项目前期研究发现表观遗传药物BET bromodomains小分子抑制（BETi）能明显地激活KRAS突变肺癌细胞及原位癌组织中转录抑制因子BCL6的表达。基因敲除或小分子抑制剂干预BCL6功能可有效增强BETi的肿瘤杀伤能力。初步的机理研究显示，BETi引起BCL6与其自身DNA的结合序列发生改变。因此，我们推测BCL6反馈自抑制的解除引起BCL6的转录激活，进而诱发KRAS突变肺癌抵抗BETi的治疗。然而，在这一过程中BCL6反馈自抑制的调控以及BCL6转录激活后介导的下游信号途径仍有待研究。本项目计划利用转录组测序、细胞信号转导分析和体内药理学验证等多层次阐明上述科学问题，为临床KRAS突变肺癌的耐药性解析和治疗提供重要科学依据。

KRAS是非小细胞肺癌中突变比例最高的癌基因，然而目前临床上仍缺乏治疗KRAS突变肿瘤的有效方案。原癌基因BCL6最初在弥漫性大B细胞淋巴瘤中被鉴定出来，发挥转录抑制功能。有研究表明，BCL6在非小细胞肺癌中扩增，其抑制剂能引起非小细胞肺癌细胞基因组不稳定。然而，BCL6表达是否介导KRAS突变非小细胞肺癌的治疗抵抗仍不清楚。在本项目研究中，我们通过临床药物筛选，发现表观遗传药物BET抑制剂（BETi）能显著激活KRAS突变非小细胞肺癌细胞中BCL6的转录表达。我们利用基因干扰和特异性BRD4抑制剂，发现BET家族成员中只有BRD2或BRD3的抑制可以上调BCL6表达，而BRD4的抑制对BCL6的表达没有影响。ChIP-seq数据分析显示，BETi处理后BCL6的DNA结合谱发生明显变化，其中BCL6与其自身基因的结合从启动子区域转移到编码区域。我们通过包括ChIP-qPCR、ChIP-re-ChIP以及蛋白质免疫共沉淀等多种技术，发现BRD3和BCL6相互作用，共同结合到BCL6基因上抑制其自身表达，而BETi的加入可以解除该自反馈抑制环路。RNA-seq和ChIP-seq数据的整合分析揭示，转录激活的BCL6可以抑制死亡相关蛋白激酶2（DAPK2），最终活化mTOR信号通路。在体外细胞水平上，BETi和BCL6抑制剂有明显的协同效应，有效抑制KRAS突变肿瘤细胞的克隆形成和锚定非依赖性生长；在体内动物水平上，相比于对照组和单药组，药物联用组可以显著抑制模型鼠肿瘤的恶性生长。鉴于BETi上调BCL6后最终激活mTOR信号通路，我们进而将BETi与mTOR抑制剂进行联用，结果显示二者同样发挥协同抗癌效应。本项目研究鉴定出BCL6是参与KRAS突变非小细胞肺癌抵抗BETi治疗的重要调控因子，为临床上BETi的合理使用以及KRAS突变肺癌的靶向干预提供了新策略。