Investigating Immunosuppression in Beta-catenin-mutated Hepatocellular Carcinoma for Improved Precision Medicine Therapeutics

项目总结/摘要 肝细胞癌（HCC）是美国癌症相关死亡率的第6大原因， 由于慢性肝病及其相关后遗症而上升。目前， 由免疫检查点抑制剂（ICI）组成的治疗范例仍然很低，并且存在迫切的 需要新的组合疗法来改善患者死亡率。对目前的国际刑事法院没有反应主要是因为 由于对肿瘤免疫微环境（TIME）以及各种HCC驱动因素如何影响肿瘤免疫的认识不足， 突变导致特定的免疫表型。此外，目前不存在生物标记驱动的 用于患者治疗分层的治疗剂。β-连环蛋白活性（由突变的CTNNB 1癌基因编码）HCC 约占HCC的26-35%，对ICI反应较差，这是由于β-连环蛋白驱动了HCC， 免疫抑制时间和限制效应功能的淋巴细胞重要的抗肿瘤免疫。 我们已经开发了新的β-连环蛋白突变的HCC小鼠模型，其中突变CTNNB 1与 原癌基因MET（β-catenin/hMet）或核因子红细胞2相关因子2（Nrf 2）（β-catenin/hMet）， 连环蛋白/Nrf 2）。这些模型分别代表了所有临床HCC病例的11%和10%。我们的初步 研究表明，β-连环蛋白增强β-连环蛋白突变的HCC的肿瘤发生， 靶向β-连环蛋白促进了驱动抗肿瘤免疫的炎症反应。基于这些 通过观察，我们的总体假设是β-连环蛋白积极抑制适应性免疫反应 靶向β-连环蛋白或其下游免疫调节因子可能会改善对 ICIs。为了研究，我提出了以下具体目标，这将揭示β-连环蛋白的新机制 在HCC TIME中的信号传导，旨在开发精确的医学疗法。具体目标1：我们将 确定β-连环蛋白抑制后激活的免疫细胞，并研究体内是否存在 通过单细胞RNA测序（scRNA-seq）和多重测序， 免疫组化因此，我们将确定β-连环蛋白激活引起的免疫抑制机制， 在β-连环蛋白突变的HCC中。具体目标2：根据我们的初步数据显示干扰素调节因子- 2（IRF 2）抑制，我们假设β-连环蛋白突变的肝癌可能是敏感的， 在IRF 2的重新表达后，由于增强的免疫抑制， 免疫反应我们将使用合成生物学方法选择性地诱导IRF 2在各种不同的表达水平上表达。 肿瘤发生的时间点和监测肿瘤负荷。然后，我们将使用scRNA-seq对淋巴细胞群 以鉴定由IRF 2调节的细胞类型和状态，并测试IFNg（其诱导IRF 2）+ ICI的组合作为免疫调节剂。 治疗方式对培训的贡献：该提案结合了严格的肝脏研究培训 癌症生物学，免疫肿瘤学和先进的生物信息学分析与优秀的临床教育， 帮助我发展成为一名未来的学术医生科学家，站在癌症免疫功能障碍的最前沿。

PROJECT SUMMARY/ABSTRACT Hepatocellular carcinoma (HCC) is the 6th leading cause of cancer-associated mortality in the United States, and is rising due to chronic liver disease and its associated sequalae. Currently, the response rates to current therapeutic paradigms consisting of immune checkpoint inhibitors (ICIs) remain low, and there exist an urgent need for novel combinatorial therapies to improve patient mortality. Lack of response to current ICIs is mainly due to a poor understanding of the tumor immune microenvironment (TIME) and how various HCC driver mutations lead to specific immune phenotypes. Additionally, there currently exist no biomarker-driven therapeutics for patient treatment stratification. ꞵ-catenin-active (encoded by mutated CTNNB1 oncogene) HCCs represent approximately 26-35% of HCCs and respond poorly to ICIs due to ꞵ-catenin driving an immunosuppressive TIME and limiting the effector function of lymphocytes important for anti-tumor immunity. We have developed novel ꞵ-catenin-mutated HCC mouse models where mutant CTNNB1 is co-expressed with either the proto-oncogene MET (ꞵ-catenin/hMet) or nuclear factor erythroid 2–related factor 2 (Nrf2) (ꞵ- catenin/Nrf2). These models represent 11% and 10% of all clinical HCC cases, respectively. Our preliminary studies demonstrate that ꞵ-catenin potentiates tumorigenesis in ꞵ-catenin-mutated HCC, and that directly targeting ꞵ-catenin promotes an inflammatory response driving anti-tumor immunity. Based on these observations, our overarching hypothesis is that ꞵ-catenin actively suppresses the adaptive immune response in the TIME and targeting ꞵ-catenin or its downstream immunomodulatory factors may improve susceptibility to ICIs. To investigate, I propose the following specific aims, which will uncover novel mechanisms of ꞵ-catenin signaling in the HCC TIME, aimed at developing precision medicine therapeutics. Specific Aim 1: We will determine the immune cells activated following ꞵ-catenin inhibition, and investigate whether there is in vivo synergy combining ꞵ-catenin inhibition and ICIs through single-cell RNA-sequencing (scRNA-seq) and multiplex immunohistochemistry. Thus, we will identify mechanisms of immunosuppression caused by ꞵ-catenin activation in ꞵ-catenin-mutated HCC. Specific Aim 2: Based on our preliminary data showing interferon regulatory factor- 2 (IRF2) repression in ꞵ-catenin-mutated HCC, we hypothesize that ꞵ-catenin-mutated HCCs may be sensitized to ICIs, or even show spontaneous tumor regression, upon re-expression of IRF2 as a result of enhanced immune response. We will use synthetic biology approaches to selectively induce IRF2 expression at various timepoints in tumorigenesis and monitor tumor burden. We will then use scRNA-seq on the lymphoid population to identify cell types and states regulated by IRF2, and test combination of IFNg (which induces IRF2) + ICI as a therapeutic modality. Contribution to Training: This proposal combines rigorous research training in liver cancer biology, immuno-oncology, and advanced bioinformatic analyses with an excellent clinical education, to aid my development as a future academic physician-scientist at the forefront of immune dysfunction in cancer.