Liver-enriched Transcription Factors as Prognostic Markers and Therapeutic Targets in Alcoholic Hepatitis

Bataller -UO 1（RFA AA-18-003） 摘要 肝细胞衰竭是酒精性肝炎（AH）患者的标志性发现。当前治疗 并不完全有效，需要有针对性的治疗。大多数研究都集中在 炎症的作用。相反，肝细胞衰竭的潜在机制和 AH中随后不良再生反应是未知的。我们的研究表明，严重的AH 其特征在于大量但无效的导管细胞积聚。的中心目标 该建议旨在确定肝细胞衰竭的分子机制， AH中随后的不良再生反应。最终目标是识别预后 用于精确医学的标记物和治疗靶标。我们已经产生了强大的 人类肝脏的初步数据显示：（1）早期形式的ALD进展为AH， 其特征在于HNF 4A功能的显著降低。(2)HNF 4A网络足迹 与AH患者的疾病严重程度密切相关。(3)TGFβ1和EGF是关键的上游 AH中的调节剂并调节培养的人肝细胞中的LETF。(4)基因签名 AH中的小管反应显示丰富的炎症、纤维化和增殖信号， HNF 4A活性降低。和（5）由LEFT靶基因编码的分泌蛋白是 在AH患者的血清中检测到。基于这些强有力的初步数据，我们的具体目标 （1）揭示TGFβ1对HNF 4A亚型调控的分子机制， 通过MS偶联表征HNF 4A P1依赖性亚型的相互作用组 免疫沉淀。此外，我们的目的是确定HNF 4A P2的DNA结合区域， 通过ChIPseq表达同种型，并产生将用于体外高表达的启动子报告系统。 新药的生产筛选。(2)为了研究的相关性和基因组图谱， AH中的小管增殖和去分化肝细胞，通过RNAseq 显微切割区域和基因签名与临床结果的相关性。我们有 开发了人类类器官的导管反应，代表了一种新的工具，研究效果 TGFβ1和EGFR配体在HNF 4A中的表达。（3）.鉴定新的血清蛋白生物标志物， AH通过糖蛋白选择偶联研究血清N-连接糖蛋白区室 并通过Snap-shot分析血清蛋白质组的翻译后修饰 蛋白质组阵列我们将联合收割机分析血清蛋白质组数据与转录组学研究相结合， 总的和显微解剖的肝组织，并将产生的签名与临床结果相关联 包括死亡率和对泼尼松龙和G-CSF的反应。

Bataller – UO1 (RFA AA-18-003) ABSTRACT Hepatocellular failure is a hallmark finding in patients with alcoholic hepatitis (AH). Current therapy is not fully effective and targeted therapies are needed. Most research has been focused on the role of inflammation. In contrast, the mechanisms underlying hepatocellular failure and the subsequent poor regenerative response in AH are unknown. Our group showed that severe AH is characterized by a massive, yet inefficient accumulation of ductular cells. The central aim of this proposal is to identify the molecular mechanisms underlying the hepatocellular failure and the subsequent poor regenerative response in AH. The ultimate goal is the identification of prognostic markers and therapeutic targets useful for precision medicine. We have generated strong preliminary data in human livers showing: (1) progression of early forms of ALD to AH is characterized by a profound decrease in the function of HNF4A. (2) HNF4A network footprint closely correlates with disease severity in AH patients. (3) TGFβ1 and EGF are key upstream modulators in AH and regulate LETF in cultured human hepatocytes. (4) The gene signature of the ductular reaction in AH shows enriched inflammatory, fibrogenic and proliferative signals and decreased HNF4A activity. And (5) Secreted proteins encoded by LEFT-target genes are detected in sera of patients with AH. Based on these strong preliminary data, our specific aims are (1) To uncover the molecular mechanism of HNF4A isoform regulation by TGFβ1, by characterizing the interactome of the HNF4A P1 dependent isoforms by MS coupled Immunoprecipitation. Moreover, we aim at identifying the DNA binding regions of HNF4A P2 isoform by ChIPseq, and generating a promoter reporter system that will be used for in vitro high- troughput screening of novel drugs. (2) To investigate the relevance and genomic profile of ductular proliferation and de-differentiated hepatocytes in AH by means of RNAseq of microdissected areas and correlation of gene signatures with clinical outcome. We have developed human organoids of ductular reaction that represents a novel tool to study the effect of TGFβ1 and EGFR ligands in HNF4A. And (3). To identify new serum protein biomarkers of AH by investigating serum N-linked glycoprotein compartment by glycoprotein selection coupled to MS and by analyzing post-translational modifications of the serum proteome by Snap-shot proteomic array. We will combine analysis of serum proteomic data with transcriptomic studies of total and microdissected liver tissue and correlate the resulting signatures with clinical outcomes including mortality and response to prednisolone and G-CSF.