Genomic Analysis of Tumor Context Vulnerabilities in Human Metastatic Melanoma

描述(申请人提供)：尽管经过几十年的研究，转移性恶性黑色素瘤仍然是一种不治之症。只有少数被诊断为转移性黑色素瘤的患者对化疗有临床反应，而治愈的患者更少；播散性疾病患者的5年存活率从5%到18%不等。该项目的重点和潜在目标是通过功能基因组筛选和体内验证来确认黑色素瘤中的特定基因变化，这些变化提供了可以在晚期黑色素瘤中利用的遗传脆弱性的背景。具体地说，我们建议进行高通量功能性RNA干扰(RNAi)筛查，以系统地识别调节黑色素瘤细胞敏感性的基因(特定目标1)。随后，我们将通过以下方式验证这些基因的作用(特定目标2)：在体外确认siRNA介导的基因敲除，使用组织微阵列(TMAS)表征人类黑色素瘤的蛋白质表达，执行高含量的机制分析以阐明这些基因在体外调节黑色素瘤细胞敏感性的机制，并评估在体外结合选择剂抑制这些基因的有效性。最后，我们将评估我们验证的基因靶点是否可以添加到体内针对黑色素瘤异种移植模型的分子信息联合疗法(特定目标3)。我们假设，这种基于功能的基因组方法将确定与肿瘤进展相关的遗传扰动的背景，这些背景对于使黑色素瘤细胞对化疗更具抗药性至关重要。我们推测，这些有效的基因可以促进对这些肿瘤相关脆弱性的联合靶向，并可能与临床开发直接相关。相关性：被诊断为晚期黑色素瘤的患者的长期预后令人沮丧，五年存活率从5-18%不等。这项拟议的研究旨在确定转移性黑色素瘤细胞获得的遗传脆弱性，这些脆弱性可能被利用来提高对这种疾病的临床干预效果。

DESCRIPTION (provided by applicant): Despite decades of research, metastatic malignant melanoma remains an incurable disease. Only a minority of patients diagnosed with metastatic melanoma show a clinical response to chemotherapy, while still fewer are cured; the 5-year survival rate for patients with disseminated disease ranges from 5-18%. The focus and underlying goal of this project is to identify specific genetic changes in melanoma, confirmed through both functional genomic screening and in vivo validation, which provide a context of genetic vulnerability that can be exploited in advanced stage melanoma. Specifically, we are proposing to undertake a high-throughput functional RNA interference (RNAi) screen to systematically identify genes that mediate melanoma cell sensitivity (Specific Aim 1). We will subsequently validate the role of these genes (Specific Aim 2) by confirming siRNA-mediated gene knockdown in vitro, characterizing protein expression in human melanomas using tissue microarrays (TMAs), performing high-content mechanistic assays to elucidate the mechanism by which these genes mediate melanoma cell sensitivity in vitro, and evaluating the effectiveness of inhibiting these genes in vitro in combination with the selecting agent. Lastly, we will evaluate whether our validated genetic targets can add to molecularly-informed combination therapies in vivo against xenograft models of melanoma (Specific Aim 3). We hypothesize that this functional-based genomic approach will identify the context of genetic perturbations associated with tumor progression that are critical to rendering melanoma cells more resistant to chemotherapy. We speculate that these validated genes could facilitate the combined targeting of these tumor-context vulnerabilities and could be of direct relevance to clinical exploitation. Relevance: The long-term prognosis for patients diagnosed with advanced stage melanoma is dismal, with a five-year survival rate ranging from 5-18%. The proposed research aims to identify genetic vulnerabilities acquired by metastatic melanoma cells that may be exploited to improve the effectiveness of clinical intervention in this disease.