Targeting HNF1A-mediated therapeutic resistance in pancreatic ductal adenocarcinoma

胰腺导管腺癌(Pda)是死亡率最高的癌症之一，其主要原因是其死亡率高达11%。 攻击性和对治疗的抵抗力。虽然几乎所有的PDA病例都是由基因突变引起的 KRAS基因，靶向KRAS或其效应物(如MEK和ERK)的努力遇到适应性抗性。 胰腺癌干细胞(PCSCs)，转录可塑性癌细胞的一个亚群，是 尤其是抗药性和致癌性，是侵略性和治疗的关键组成部分- 掌上电脑的耐受性。目前没有针对私营军保公司的战略，也缺乏信息。 关于他们的司机。我们之前发现HNF1a是一种胃肠系转录因子，是一种 新颖的PCSC状态主调节器。我们的初步数据表明，HNF1a可以有效地表达 被BET抑制剂(Beti)阻断，这是一类抑制表观遗传阅读器蛋白BRD4的药物。 有趣的是，HNF1a的重新表达挽救了Beti处理后的细胞周期进程和PCSC的特性 PDA细胞，这表明HNF1A是这些药物的一个新的关键靶点。我们还发现， HNF1a是一种新的抗靶向KRAS和下游MEK/ERK信号的驱动因子。重要的是， Beti与MEK和ERK抑制剂(Meki/Erki)联合使用会增加生长停滞和细胞死亡 以依赖于HNF1A的方式。我们假设HNF1A直接受BRD4调控，因此 用Beti靶向，并且用Beti抑制HNF1a的表达将使HNF1a依赖无效 PCSCs与KRAS消融适应性抵抗。在这项提案中，我们的目标是描述对 HNF1a及其在治疗反应和耐药中的作用在具体目标1中，我们将描述 Beti-靶标BRD4对HNF1a的调控具体目标1将结合BRD4的遗传操作，芯片- 聚合酶链式反应和记者实验证明BRD4对HNF1A的调控。HNF1a在BRD4中的重新表达 耗尽的细胞将1)确定HNF1A在BRD4介导的体外和体内细胞生长和存活中的作用， 以及2)使用RNA-SEQ/CHIP-SEQ来确定HNF1A对BRD4转录组的贡献 确定HNF1A如何决定对Beti的反应。在具体目标2中，我们将建立Beti作为一种手段 克服HNF1a介导的对KRAS消融的抵抗。特定目标2将使用带和不带PDA电池 异位表达HNF1a以检测HNF1a对Beti和KRAS途径抑制活性的贡献； 我们将利用体外和体内试验来研究这些药物如何影响PCSCs，并使用RNA-Seq/ChIP- SEQ以确定HNF1A如何促进对靶向KRAS信号的抵抗。两个目标都将利用Next- 产生尚未在PDA或联合疗法中探索的溴域选择性抑制剂。 上述研究的完成将极大地提高我们对PDA生物学的理解并揭示 新的治疗靶点。通过提高对KRAS抵抗力量的认识，抑制和扩大 将PDA的治疗谱纳入PCSCs，可以实现更有效的治疗PDA。

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers with a survival rate of 11% due to its aggressive nature and resistance to therapies. While nearly all PDA cases are driven by mutations in the KRAS gene, efforts to target KRAS or its effectors (e.g., MEK and ERK) are met with adaptive resistance. Pancreatic cancer stem cells (PCSCs), a subpopulation of transcriptionally-plastic cancer cells that are especially drug resistant and particularly tumorigenic, are a critical component of the aggressive and therapy- resistant nature of PDA. There are currently no strategies to target PCSCs, as well as a lack of information regarding their drivers. We previously identified HNF1A, a gastrointestinal-lineage transcription factor, as a novel master regulator of the PCSC state. Our preliminary data show that HNF1A expression can be potently blocked by BET-inhibitors (BETi), a class of drugs which inhibit the epigenetic reader protein BRD4. Interestingly, re-expression of HNF1A rescues cell cycle progression and PCSC-properties in BETi-treated PDA cells, suggesting that HNF1A is a novel and critical target for these drugs. We have also found that HNF1A is a novel driver of resistance to targeting KRAS and downstream MEK/ERK-signaling. Importantly, the use of BETi in combination with MEK- and ERK-inhibitors (MEKi/ERKi) increases growth arrest and cell death in an HNF1A-dependent manner. We hypothesize that HNF1A is directly regulated by BRD4, and is therefore targetable with BETi, and that the inhibition of HNF1A expression with BETi will nullify HNF1A-dependent PCSCs and adaptive resistance to KRAS-ablation. In this proposal, we aim to characterize the regulation of HNF1A and its role in therapeutic response and resistance. In Specific Aim 1, we will characterize the regulation of HNF1A by BETi-target BRD4. Specific Aim 1 will combine genetic manipulation of BRD4, ChIP- PCR, and reporter assays to demonstrate regulation of HNF1A by BRD4. Re-expression of HNF1A in BRD4- depleted cells will 1) define the role of HNF1A in BRD4-mediated cell growth and survival in vitro and in vivo, as well as 2) determine the contribution of HNF1A to the BRD4 transcriptome using RNA-seq/ChIP-seq to identify how HNF1A determines response to BETi. In Specific Aim 2, we will establish BETi as a means to overcome HNF1A-mediated resistance to KRAS-ablation. Specific Aim 2 will use PDA cells with and without ectopic HNF1A expression to examine the contribution of HNF1A to BETi and KRAS-pathway inhibitor activity; we will utilize in vitro and in vivo assays to examine how these drugs affect PCSCs and use RNA-seq/ChIP- seq to determine how HNF1A promotes resistance to targeting KRAS-signaling. Both aims will utilize next- generation bromodomain-selective inhibitors that have not been explored in PDA or combinatorial therapies. The completion of the above studies will dramatically improve our understanding of PDA biology and uncover novel therapeutic targets. By improving our understanding of resistance to KRAS suppression and expanding the therapeutic spectrum of PDA to include PCSCs, more effective treatment of PDA can be achieved.