An immunocompetent platform for the characterization and discovery of novel therapeutics for hepatocellular carcinoma

项目摘要 肝癌是世界上癌症相关死亡的第四大原因，死亡率正在上升， 这在一定程度上反映了专注于单剂疗法(多药疗法)的历史药物开发活动 靶向的激酶抑制剂和免疫疗法)，对患者的疗效有限。此外，还有一个 这种癌症缺乏有针对性的、个性化的治疗选择。因此，迫切需要确定 新的量身定制的治疗策略。肝细胞癌靶向治疗的研究进展 (肝细胞癌)已经发现小分子抑制剂(例如，lenvatinib)或 生物制品(例如，贝伐单抗)与免疫疗法结合使用，尽管不到30% 患者对这些新的一线疗法有反应。该领域的一个主要差距是缺乏对国际 患者的异质性和未能识别出(I)唯一针对特定于司机的脆弱性的药物，或(Ii) 提高免疫治疗水平。这项建议侧重于一种尚未探索的肝癌治疗设计策略， 它利用体内CRISPR筛查和一种新的患者来源的体外自体肿瘤的使用 有机类T细胞共培养平台为两种药物的药效机制提供新的生物学见解 肿瘤微环境(TME)的关键成分，并允许评估联合治疗 免疫治疗水平的提高。我假设肝细胞癌的药物进展，特别是在 与免疫治疗相结合，依赖于药物对肿瘤T细胞的调节 互动。这一假设将在两个方面得到检验：(1)剖析小说的作用机制 候选药物WNTinib，在使用体内CRISPR筛查的肝细胞癌免疫易治模型中，以及(2) 用于肝癌个体化治疗的新型免疫治疗组合的鉴定。对于目标1， WNTinib的关键靶点(或促进活性或抵抗力的机制)将使用两个 单独的基因组规模CRISPR sgRNA筛选策略：(I)转导基因定义的小鼠肝癌 将有机类化合物原位移植到免疫活性和免疫低下的小鼠中，以及(Ii) 转导OT-I；Cas9和OT-II；Cas9小鼠T细胞并将其移植到表达 卵清蛋白衍生肽SIINFEKL。在WNTinib在场的情况下进行的分析将确定WNTinib的肿瘤 细胞固有的、TME依赖的和T细胞依赖的靶点或机制。对于目标2，我将使用肝细胞癌患者 肿瘤活检以建立器官和肿瘤浸润性淋巴细胞(TIL)培养。这种方法将允许 对肿瘤和T细胞作为单独成分和共同培养的研究。我将把这个平台扩展到 包括单独或与免疫疗法结合使用一组药物进行药物筛选 肝癌临床进展的不同阶段。这项提案的结果将是向前迈出的重要一步 对肝细胞癌用药的多维理解，以及如何联合用药。 提供个性化护理的免疫疗法。

Project Summary Liver cancer is the fourth leading cause of cancer-related death in the world and mortality rates are rising, which, in part, reflects historical drug development campaigns focused on single agent therapies (multi- targeted kinase inhibitors and immunotherapy) that have limited efficacy in patients. Additionally, there are a lack of targeted, personalized therapeutic options for this cancer type. Thus, there is an urgent need to identify novel tailored therapeutic strategies. Recent advances in the therapeutic targeting of hepatocellular carcinoma (HCC) have discovered superiority in efficacy for either small molecule inhibitors (for example, lenvatinib) or biologics (for example, bevacizumab) used in combination with immunotherapy, though less than 30% of patients respond to these new frontline therapies. A major gap in the field is the lack of appreciation for inter- patient heterogeneity and a failure to identify drugs that (I) uniquely target driver-specific vulnerabilities, or (II) improve immunotherapy. This proposal focuses on an unexplored strategy for therapy design in liver cancer, which leverages the usage of in vivo CRISPR screens and a novel patient-derived ex vivo autologous tumor organoid-T cell co-culture platform to provide new biological insight to mechanisms of drug potency on two critical components of the tumor microenvironment (TME), and allow assessment of combination therapies for the improvement of immunotherapy. I hypothesize that drug advancement in HCC, particularly in combination with immunotherapy, is dependent on drug-mediated modulation of tumor-T cell interactions. This hypothesis will be tested in two aims: (1) dissection of the mechanism of action of a novel candidate drug, WNTinib, in immunologically tractable models of HCC using in vivo CRISPR screens, and (2) identification of novel immunotherapy combinations for the personalized treatment of HCC. For Aim 1, WNTinib’s key targets (or mechanism’s contributing to activity or resistance) will be investigated using two separate genome-scale CRISPR sgRNA screening strategies: (I) transducing genetically-defined murine HCC organoids transplanted orthotopically into immunocompetent and immunocompromised mice, and (II) transducing T cells from OT-I;Cas9 and OT-II;Cas9 mice and transplanting them into tumor models expressing the OVA-derived peptide SIINFEKL. Analyses done in the presence of WNTinib will identify WNTinib’s tumor cell intrinsic, TME-dependent, and T cell-dependent targets or mechanisms. For Aim 2, I will use HCC patient tumor biopsies to establish organoids and tumor infiltrating lymphocyte (TIL) cultures. This approach will allow the study of tumor and T cells as separate components and in co-culture together. I will expand this platform to include drug screening, alone or in combination with immunotherapy, using a panel of drugs representing various stages of HCC clinical advancement. Results from this proposal will represent a major step forward for the multidimensional understanding of drug usage in HCC, and how drugs may be used in combination with immunotherapy to provide personalized care.