Molecular targeting of DCLK1 signaling in hepatocellular carcinoma

慢性肝病，包括各种原因的肝硬变，是导致 肝细胞癌(HCC)。美国退伍军人管理局医疗保健中肝细胞癌的全国患病率和死亡率 在过去的十年里，系统增加了3倍。尽管接种了乙肝疫苗，但筛查方法有所改进， 和成功的抗病毒治疗，慢性肝病和肝硬变的发生率大幅增加(13%) 从2000年开始。现有的治疗方案(放射、化疗和免疫疗法)不能切除和 转移性肝细胞癌在大多数病例中都没有成功。因此，肝癌仍然是一项重要的医疗保健 挑战。这项建议的基本目标是确定推动转型过程的机制 以及肝脏中的肿瘤生长，并制定策略以改善这些患者的临床结果。在这一探索中， 我们发现双重皮质醇样蛋白1(DCLK1)在慢性病毒性肝炎、肝硬变、 和肝细胞癌，但在正常肝脏中不存在。DCLK1升高与肝细胞癌患者存活率降低有关。最近， 我们发现DCLK1在肝癌细胞中激活了一个非典型的β-连环蛋白(48 KDa)/TCF4信号。然而， 其在慢性肝病和肝细胞癌中的意义以及Dclk1/β-catenin信号轴在肝细胞干细胞分化中的意义是完全的。 未知。利用小鼠肝细胞癌模型，我们证明了DCLK1在肝细胞的某些部位被大量诱导 并可能导致肝细胞癌样肿瘤的生长。我们的初步数据显示DCLK1-表达 肝癌细胞可以极化Kupffer细胞(KCs)为免疫抑制的M2样巨噬细胞。IL-10由 据报道，M2样KCs可促进M1促炎巨噬细胞的选择性凋亡。因此， DCLK1依赖的M2极化有可能促进肝肿瘤的免疫抑制 微环境(TME)。此外，DCLK1激酶活性的抑制导致KEY显著降低。 促炎(肿瘤坏死因子α、IL-1β)和免疫抑制IL-10。这些观察结果表明DCLK1是一种 肝纤维化、免疫调节和肿瘤形成过程的复杂相互联系的中央调节因子 CLDs。我们的中心假设是DCLK1是致癌信号和免疫失调的关键介质。 慢性低密度脂蛋白的组织微环境，共同刺激肝细胞的转化。我们将测试 这一假设具有以下相互关联但独立的具体目标。目标1将确定这些机制 DCLK1通过引入DCLK1的缺失和 DCLK1基因的点突变及其对功能的影响我们将使用DCLK1激酶的抑制剂(DCLK1-IN- 1)和β-连环蛋白/Tcf相互作用(FH535)，以验证DCLK1调节的克隆性、CsC特性和 单核细胞/KCs极化。目的2研究DCLK1激酶抑制对DCLK1/β-连环蛋白的影响 小鼠肝细胞癌TME中调控的信号轴、肝脏转化过程和免疫失调 肝硬变/肝癌模型。最后，目标3将确定DCLK1/β-连环素轴在调节 患者来源的肿瘤异种移植(PDX)模型中的肝癌细胞干性和肿瘤细胞异质性。这个 结果将被DCLK1-IN-1、FH535和联合治疗PDX肿瘤所证实。此外, 我们将对肿瘤进行scRNAseq和磷酸蛋白质组分析，以确定差异磷酸化。 在DCLK1+肿瘤细胞和DCLK1信号网络中由磷酸化介导。这些研究将深化我们的 了解DCLK1对参与肝脏肿瘤发生和发展的复杂信号轴的调节作用 抑制肝组织的先天免疫反应，共同推动肝细胞癌的发生和肿瘤的生长。 此外，该项目将提供潜在的基于抗DCLK1的方法来预防和治疗 肝癌和依赖于DCLK1功能的其他器官的癌症。

Chronic liver diseases (CLDs) including cirrhosis of any etiology are the major risk factors for the development of hepatocellular carcinoma (HCC). The national prevalence and mortality due to HCC in the US VA healthcare systems have increased by 3-fold during the last decade. Despite HBV vaccination, improved screening methods, and successful antiviral treatment for HCV, the incidence of CLD and cirrhosis has substantially (13%) increased since 2000. The existing treatment options (radiation, chemotherapy, and immunotherapy) of unresectable and metastatic HCC have not been successful in the majority of cases. Thus, HCC remains a significant healthcare challenge. The fundamental goal of this proposal is to determine mechanisms driving the transformation process and tumor growth in the liver, and develop strategies to improve clinical outcomes for these patients. In this quest, we found that doublecortin-like kinase 1 (DCLK1) protein is highly expressed in chronic viral hepatitis, cirrhosis, and HCC but not in normal liver. Elevated DCLK1 is associated with decreased survival of HCC patients. Recently, we demonstrated that DCLK1 activates an atypical β-catenin(48 kDa)/TCF4 signaling in hepatoma cells. However, its significance in the CLDs and HCC and DCLK1/β-catenin signaling axis in hepatic cell stemness is completely unknown. Using a mouse model of HCC, we demonstrated that DCLK1 is heavily induced in hepatocytes at sites of injury and likely contributes to the HCC-like tumor growth. Our preliminary data revealed that DCLK1-expressing hepatoma cells can polarize Kupffer cells (KCs) into immunosuppressive M2-like macrophages. IL-10 secreted by M2-like KCs has been reported to promote the selective apoptosis of M1 pro-inflammatory macrophages. Thus, DCLK1-dependent M2 polarization has the potential to promote immunosuppression in the hepatic tumor microenvironment (TME). Furthermore, inhibition of DCLK1 kinase activity resulted in a dramatic reduction in key pro-inflammatory (TNFα, IL-1β) and immunosuppressive IL-10. These observations suggested that DCLK1 is a central regulator of the complex interconnection of fibrogenic, immunoregulatory, and tumorigenic processes in CLDs. Our central hypothesis is that DCLK1 is a key mediator of oncogenic signaling and immune dysregulation in the tissue microenvironment in CLDs, which collectively stimulates the transformation of hepatocytes. We will test this hypothesis with the following interrelated yet independent specific aims. Aim 1 will determine the mechanisms by which DCLK1 mediates immunoregulatory and tumorigenic processes in the liver by introducing deletion and point mutation in DCLK1 and assay for the functional impacts. We will use inhibitors to DCLK1 kinase (DCLK1-IN- 1) and β-catenin/TCF interaction (FH535) to verify DCLK1-regulated clonogenicity, CSC properties, and monocyte/KCs polarization. Aim 2 will determine the effects of DCLK1 kinase inhibition on the DCLK1/β-catenin regulated signaling axis, hepatic transformation process, and immune dysregulation in the HCC TME in a murine model of cirrhosis/liver cancer. Finally, Aim 3 will define the role of the DCLK1/β-catenin axis in the regulation of liver cancer cell stemness and tumor cell heterogeneity in a patient-derived tumor xenografts (PDX) model. The results will be confirmed by the treatment of PDX tumors with DCLK1-IN-1, FH535, and in combination. In addition, we will perform scRNAseq and phosphoproteomic analysis of the tumors to determine differential phosphorylation in DCLK1+ tumor cells and DCLK1 signaling network mediated by phosphorylation. These studies will deepen our understanding of DCLK1 regulation of a complex signaling axis contributing to hepatic tumorigenicity and suppression of innate immune response in the liver tissues, which together drive HCC initiation and tumor growth. In addition, the project will deliver potential anti-DCLK1-based approaches for the prevention and treatment of HCC and cancers of other organs that rely upon DCLK1 functions.