Identifying therapeutic options for intrahepatic cholangiocarcinoma

在美国，每年约有10，000名患者被新诊断为胆管癌，并且他们的5年生存率为100%。 存活率低于10%。肝内胆管细胞癌细胞来源的异质性及其分子生物学特征 (ICC)突出了对生物标志物的需求，以分层肿瘤和产生靶向治疗。除了胆管上皮细胞 在人ICC的细胞来源中，肝细胞（HC）被认为是ICC的细胞来源。这是显而易见的快速HC驱动 通过共表达活化的AKT（myrAKT）和Notch胞内结构域（NICD）在小鼠中诱导ICC 使用睡美人转座子系统的HC。 我们使用大型ICC患者队列进行的初步分析表明，AKT-NICD的基因特征 （AN）驱动的鼠ICC与约30%的人ICC显著相关，支持该ICC的临床相关性。 模型Sox 9或雅普的缺失延迟了ICC的形成，而是诱导了AN驱动的SOX 9-/雅普+或SOX 9 +/雅普- ICC中，它们也已在人类ICC中被鉴定。这些数据表明，Notch独立调节 HC驱动的ICC中的Sox 9和雅普。重要的是，我们发现雅普和Sox 9的共抑制完全阻止了Notch-1的表达。 依赖ICC的形成。然而，由雅普和SOX 9驱动的肿瘤发生机制，以及它们在肿瘤发生中的作用， 与AKT信号传导的相互作用仍然知之甚少。根据我们的初步观察，我们的核心假设 HC驱动的ICC肿瘤生长依赖于转录和表观遗传改变， Notch信号传导的效应物，SOX 9和雅普，以及AKT信号传导。 在目的1中，我们的目的是测试共抑制雅普和SOX 9在晚期ICC中的治疗效果，以更好地解决 后期治疗的临床需求。我们将采用遗传性AN-ICC模型以及基于肝毒素的ICC模型 使用创新的诱导型基因调节系统诱导同时的、有条件的和诱导型Sox 9和雅普， 在先进的ICC镇压。在目标2中，我们提出了3个子目标来描述AN的分子机制， 介导的HC驱动的ICC形成。首先，使用ChIP-seq和生物信息学工具，我们将识别出独特的 在HC衍生的ICC形成过程中由Sox 9和雅普调控的重叠下游靶点。第二，我们将阐明 NICD-YAP/TEAD-DNMT 1表观遗传轴在HC驱动的ICC发展中的病理作用， 轴在体内系统中的药理学和遗传学工具，并研究其对ICC肿瘤甲基化组的影响。 为了实现这两个子目标，我们将鉴定Sox 9和雅普的下游效应子，以获得更具选择性和更安全的治疗方法。 在ICC的目标。第三，我们的目标是阐明AKT如何介导HC到ICC的转化， 明白 本研究的成功实施将1）为考虑SOX 9和雅普的共同作用提供必要的证据， 抑制作为精确医学治疗的有吸引力的候选者，理论上这是最有效的方法， 征服致命肿瘤，2）揭示AKT，SOX 9和雅普的关键下游效应子，这将有助于开发 更有效的治疗选择。

In the US, approximately 10,000 patients are newly diagnosed annually with cholangiocarcinoma, and their 5-year survival rate is less than 10%. Heterogeneity in cellular origins and molecular signatures of intrahepatic cholangiocarcinoma (ICC) highlight the demand for biomarkers to stratify tumors and generate targeted therapies. Besides biliary epithelial cells (BECs), hepatocytes (HCs) have been considered as a cellular origin of human ICC. This is evident by rapid HC-driven ICC induction through the co-expression of activated AKT (myrAKT) and Notch intracellular domain (NICD) in mouse HCs using sleeping beauty transposon system. Our preliminary analysis using large ICC patient cohorts demonstrates that the genetic signature of AKT-NICD (AN)-driven murine ICC correlates significantly with ~30% of human ICC, supporting the clinical relevance of this ICC model. Deletion of either Sox9 or Yap delayed ICC formation, and instead induced AN-driven SOX9-/YAP+ or SOX9+/YAP- ICC respectively, which also have been identified in human ICC. These data indicate that Notch independently regulates Sox9 and Yap in HC-driven ICC. Importantly, we found that co-repression of Yap and Sox9 completely prevents Notch- dependent ICC formation. However, the mechanisms of tumorigenesis driven by YAP and SOX9, as well as their interactions with AKT signaling, remain poorly understood. Based on our preliminary observations, our central hypothesis is that HC-driven ICC tumor growth depends on transcriptional and epigenetic alterations driven by two major downstream effectors of Notch signaling, SOX9 and YAP, alongside AKT signaling. In aim 1, we aim to test therapeutic effect of co-repression of YAP and SOX9 in advanced ICCs to better address the clinical need for late-stage therapy. We will employ genetic AN-ICC model as well as liver toxin-based ICC model using innovative inducible gene modulation systems to induce simultaneous, conditional and inducible Sox9 and YAP repression in advanced ICC. In aim 2, we are proposing 3 subaims to delineate the molecular mechanisms underlying AN- mediated HC-driven ICC formation. First, using ChIP-seq and bioinformatic tools, we will identify both the unique and the overlapping downstream targets regulated by Sox9 and Yap during HC-derived ICC formation. Second, we will elucidate the pathologic role of the NICD-YAP/TEAD-DNMT1 epigenetic axis in HC-driven ICC development by modulating this axis with pharmacological and genetic tools in the in vivo system and studying its effects on the methylome of ICC tumors. Pursuing these 2 subaims, we will identify downstream effectors of Sox9 and Yap for more selective and safer therapeutic targeting in ICC. Third, we aim to elucidate how AKT mediates HC-to-ICC transformation, which remains poorly understood. The successful execution of this study will 1) provide an essential evidence for considering SOX9 and YAP co- inhibition as an attractive candidate for precision medicine therapy, which is theorized to be the most effective approach to conquer lethal tumor, and 2) reveal the key downstream effectors for AKT, SOX9 and YAP which will help to develop more potent therapeutic options for ICC.