Innate Immune Signaling in Fibrolamellar Carcinoma

纤维板层癌(FLC)是一种罕见的肝癌，困扰着儿童和年轻人。当时 在诊断中，约60%的患者没有有效的治疗选择，癌症为IV期。对于剩余的 40%，肝移植是唯一的根治方法，只要能及时甚至找到合适的供者 然后，伴随着短期和长期并发症的重大风险。 几乎所有的FLC患者都有Chr的基因组改变。19导致DNAJB1基因外显子1融合 Hsp40家族与PKA的催化亚单位--PRKACA的外显子2-10有关。由此产生的DNAJB1-PRKACA融合 该基因编码一种保持催化活性的DNAJB1-PRKACA蛋白。使用CRISPR/CAS9技术 结合流体动力尾静脉注射，我们和其他人已经最终证明了同线DNAJB1- PrKACA融合基因在小鼠体内的表达8足以诱发具有FLC特征的肝癌。 FLC被认为起源于肝脏干细胞，但DNAJB1-PRKACA促进肿瘤发生的机制 转型还没有被很好地理解。为了在与发展相关的背景下进行功能研究，我们 利用CRISPR/Cas9技术在小鼠肝脏类器官中设计了同线DNAJB1-PRKACA。我们发现 表达DNAJB1-PRKACA的肝细胞器容易发生溶细胞死亡。接下来，我们表演了 广泛的转录组分析揭示了一个突出的天然免疫特征成分 NLRC4炎症性小体。此外，我们还确定了表达肝脏器官的DNAJB1-PRKACA表现出 切割的caspase-8水平显著升高，这与其有效的激活相一致。 在这项工作中，我们将首先测试NLRC4炎症体是否被DNAJB1-PRKACA激活 并确定其在溶细胞死亡中的作用。然后我们将确定是否需要NLRC4和/或Casp-8基因 用于通过工程DNAJB1-PRKACA融合基因在小鼠肝脏中诱导的肿瘤发生。后者会 是对FLC发病机制认识上的重大突破。

Fibrolammellar Carcinoma (FLC) is a rare form of liver cancer that afflicts children and young adults. At the time of diagnosis, the cancer is stage IV in ~60% of patients without effective treatment option. For the remaining 40%, liver transplantation is the only curative therapy, providing a suitable donor can be found in time and even then, with significant risks for short- and long-term complications. Virtually all patients with FLC have a genomic alteration on Chr. 19 resulting in fusion of exon 1 of DNAJB1 of the Hsp40 family to exons 2-10 of PRKACA, a catalytic subunit of PKA. The resulting DNAJB1-PRKACA fusion gene encodes a Dnajb1-Prkaca protein with preserved catalytic activity. Using CRISPR/Cas9 technology combined with hydrodynamic tail vein injection, we and others have shown conclusively that a syntenic Dnajb1- Prkaca fusion gene on mouse Chr. 8 is sufficient to elicit liver cancer with hallmarks of FLC. FLC is thought to arise from liver stem cells, but the mechanisms whereby Dnajb1-Prkaca promotes oncogenic transformation are not well understood. To permit functional studies in a developmentally relevant context, we used CRISPR/Cas9 technology to engineer syntenic Dnajb1-Prkaca in mouse liver organoids. We discovered that liver organoids expressing Dnajb1-Prkaca are prone to undergoing lytic cell death. Next, we performed extensive transcriptome analyses that revealed an innate immune signature prominently featuring constituents of the NLRC4 inflammasome. We moreover determined that Dnajb1-Prkaca expressing liver organoids exhibit significantly elevated levels of cleaved caspase-8 consistent with its potent activation. In this work, we will first test if the NLRC4 inflammasome is activated by Dnajb1-Prkaca in mouse liver organoids and establish its role in lytic cell death. We will then determine if the Nlrc4 and/or Casp-8 genes are necessary for tumorigenesis elicited by engineering of the Dnajb1-Prkaca fusion gene in the mouse liver. The latter would represent a major breakthrough in understanding the pathogenesis of FLC.