Mechanisms of liver metastasis and associated resistance to immunotherapy

项目摘要 黑色素瘤的发病率在过去三十年中翻了一番，预计在2010年将超过100，000例。 2020年的美国。2019年，美国有近7,000人死于黑色素瘤。黑色素瘤很容易 通过淋巴和血行途径传播。最常见的转移部位包括淋巴 淋巴结、肺、肝和脑虽然新的疗法，如免疫疗法，提高了生存率， 对于患有转移性疾病的患者，转移模式可影响对此类疗法的反应。特别是， 肝转移患者对免疫检查点的应答率和生存率较低， 与转移到其他器官的患者相比，抑制剂。肝转移的分子基础 发展以及对药物反应和耐药性的影响知之甚少。这部分是由于有限的 类似于患者中观察到的转移模式的临床前模型，以及基因组的稀疏性， 人肝转移性病变的表征。该提案的目标是确定黑色素瘤的驱动因素 肝转移（MLM）和免疫治疗相关耐药机制。为此，我们 表征并使用了一种新的同基因小鼠模型，该模型忠实地再现了基因组、转移和 在患者中观察到的反应模式。我们进行了大规模的体内CRISPR-Cas9筛选， 促进MLM发展的扰动，但不是原发性肿瘤生长或转移到其他器官 网站.在这个建议的第一个目标，我们阐明了细胞内在的信号机制，促进MLM。接下来， 使用集成的高重成像，免疫分析和单细胞测序，我们确定了免疫 MLM浸润和并发肺转移以及免疫室如何影响对 临床上使用的免疫疗法。最后，我们收集了大量肝转移患者的活检样本， 和来自其他器官部位的并发转移以及外周血单核细胞（PBMC）， 基因组和免疫分析。总之，这些研究将提供一个基因组决定因素的景观， MLM的信号传导机制和免疫环境及其对免疫治疗反应的影响 并有可能确定合理药物开发的新途径。

PROJECT SUMMARY The incidence of melanoma has doubled in the last three decades and is projected to exceed 100,000 cases in the US in 2020. Almost 7,000 people died of melanoma in the US in 2019. Melanoma has a strong propensity to spread both via lymphatic and hematogenous routes. The most common sites of metastasis include lymph nodes, lung, liver and brain. Although novel therapies, such as immunotherapies have improved survival of patients with metastatic disease, metastatic patterns can influence responses to such therapies. In particular, patients with metastases to the liver have a lower response and survival rates in response to immune checkpoint inhibitors compared to patients with metastases to other organs. The molecular underpinnings of liver metastasis development and the impact on drug response and resistance are poorly understood. This is in part due to limited pre-clinical models that resembles metastatic patterns seen in patients, and a sparsity of genomic characterization of human liver metastatic lesions. The goal of this proposal is to determine drivers of melanoma liver metastasis (MLM) and mechanisms of associated resistance to immunotherapies. For this purpose, we characterized and used a novel syngeneic mouse model that faithfully recapitulates genomic, metastatic and response patterns seen in patients. We performed a large-scale in vivo CRISPR-Cas9 screen and identified perturbations that promote development of MLM, but not primary tumor growth or metastasis to other organ sites. In the first aim of this proposal, we elucidate the cell intrinsic signaling mechanisms promoting MLM. Next, using integrated high-plex imaging, immune-profiling and single-cell sequencing, we determine the immune infiltrates of MLM and concurrent lung metastases and how the immune compartment impacts the response to clinically used immunotherapies. Finally, we assembled a large cohort of patient biopsies from liver metastases and concurrent metastases from other organ sites as well as peripheral blood mononuclear cells (PBMCs) for genomic and immune profiling. Together, these studies will provide a landscape of genomic determinants, signaling mechanisms and immune environments of MLM and their impact on responses to immunotherapies and have the potential to identify novel avenues for rationale drug development.