Mechanism of Pancreatic Carcinogenesis

 描述(申请人提供)：胰腺导管腺癌(PDAC)是一种致命的疾病，目前还没有有效的治疗方法。因此，迫切需要了解PDAC病理生物学基础的分子机制，以便开发新的治疗方法来治疗这种令人沮丧的疾病。转录因子GLI1在胰腺癌中表达增加，并通过激活参与胰腺癌启动的关键基因的转录而在胰腺癌的发生中发挥重要作用。多系白血病1(MLL1)组蛋白甲基转移酶(HMT)是一种以其在白血病中的作用而闻名的蛋白质，MLL1基因的易位导致癌基因融合蛋白的形成。据报道，MLL1与许多活跃转录的基因相关，并调节某些基因的表达，如HOX基因。MLL1在PDAC中没有突变，以前也没有被认为与这种疾病的发病机制有关。然而，我们发现MLL1在人PDAC样本中过表达。值得注意的是，我们已经取得了新的发现，MLL1蛋白复合体和相关蛋白晶状体上皮衍生生长因子(LEDGF)在PDAC细胞中与GLI1相互作用，调节GLI1靶基因的激活，如转化生长因子1和白介素6Ra。我们假设MLL1复合体和GLI1在胰腺上皮细胞中相互作用，协调激活关键基因的转录，驱动癌前病变的发生和发展，导致PDAC。基于我们的发现，我们建议阐明GLI1和MLL1相互作用调节转录的机制，并建立这种HMT复合体在GLI1诱导的PDAC中的生物学意义。我们提出了三个目标：首先，我们将研究GLI1和MLL1复合蛋白之间的物理相互作用，以确定哪个MLL1复合蛋白(S)直接与GLI1相互作用。我们还将进行结合分析，以绘制GLI1的哪些区域对GLI1/MLL1相互作用重要。其次，我们将研究GLI1和MLL1复合体在基因启动子上相互作用以调节转录的机制。我们将确定以MLL1复合体为靶向GLI1靶基因的启动子招募机制。我们还将确定与GLI1-MLL1复合体与靶基因结合相关的组蛋白修饰或核小体组织的特定变化。此外，我们将使用全球方法，RNA-SEQ和CHIP-SEQ，来识别其他由PDAC细胞中的GLI1和MLL1复合体协调调控的靶基因。这些数据将被用来从机械上确定GLI1是否通常参与PDAC细胞中MLL1复合体的招募，以及MLL1和GLI1在启动子上的共存是否与组蛋白修饰的变化有关，例如与基因激活相关的组蛋白3、赖氨酸4三甲基化。最后，我们将利用一个已建立的PDAC遗传性小鼠模型来检验遗传和小分子抑制剂介导的MLL1功能缺失如何影响依赖GLI1的PDAC的启动。这些动物模型实验将在体内对我们的假设进行测试，并将评估靶向MLL1复合体以抑制PDAC中GLI1驱动的致癌作用的可行性。

 DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease for which there is currently no effective treatment. Therefore, there is an urgent need to understand the molecular mechanisms underlying the pathobiology of PDAC so that new therapeutic approaches can be developed to target this dismal disease. The transcription factor, GLI1, is increased in PDAC and plays important roles in pancreatic carcinogenesis by activating transcription of key genes involved in PDAC initiation. Multiple lineage leukemia 1 (MLL1) histone methyltransferase (HMT) is a protein well known for its role in leukemia, where translocations of the MLL1 gene lead to formation of oncogenic fusion proteins. MLL1 is reported to be associated with many actively transcribed genes and to regulate the expression of certain loci, e.g., HOX genes. MLL1 is not mutated in PDAC and has not been previously implicated in the pathogenesis of this disease. However, we have found that MLL1 is overexpressed in human PDAC samples. Significantly, we have made the novel discovery that the MLL1 protein complex and an associated protein, lens epithelial derived growth factor (LEDGF) interact with GLI1 in PDAC cells to regulate the activation of GLI1 target genes, e.g., TGFß1 and IL6Ra. We hypothesize that the MLL1 complex and GLI1 interact in pancreatic epithelial cells to coordinately activate transcription of key genes driving the development and progression of preneoplastic lesions leading to PDAC. Based on our findings, we propose to elucidate the mechanisms by which GLI1 and MLL1 interact to regulate transcription and establish the biological significance of this HMT complex in GLI1-induced PDAC. We propose three Aims: First, we will examine the physical interactions between GLI1 and MLL1 complex proteins to determine which MLL1 complex protein(s) interacts directly with GLI1. We will also perform binding assays to map which regions of GLI1 are important for GLI1/MLL1 interactions. Second, we will investigate the mechanisms by which GLI1 and the MLL1 complex interact at gene promoters to regulate transcription. We will determine the promoter recruitment mechanism targeting the MLL1 complex to GLI1 target genes. We will also determine the specific alterations in histone modifications or nucleosome organization associated with binding of GLI1-MLL1 complex to the target genes. In addition, we will use global approaches, RNA-seq and ChIP-seq, to identify additional target genes that are coordinately regulated by GLI1 and the MLL1 complex in PDAC cells. These data will be used to mechanistically ascertain if GLI1 is generally involved in recruiting the MLL1 complex in PDAC cells and if the co-occurrence of MLL1 and GLI1 at promoters is globally associated with changes in histone modifications, e.g., histone 3, lysine 4 trimethylation, associated with gene activation. Finally, we will utilize a wel-established genetic mouse model of PDAC to examine how genetic and small molecule inhibitor-mediated loss of MLL1 function affects the GLI1-dependent initiation of PDAC. These animal model experiments will provide a test for our hypothesis in vivo and will evaluate the feasibility of targeting the MLL1 complex to inhibit GLI1-driven carcinogenesis in PDAC.