Defining Melanoma Therapeutic Avenues by Integrative Functional Genomics

尽管基于基因组标准的肿瘤分类和患者分层 提供巨大的临床潜力，识别肿瘤遗传的关键效应因素 改变-并发展强有力的治疗途径来拦截它们-代表着一种 翻译肿瘤学的巨大障碍。大规模研究的最新进展 “扰动”方法(例如，病毒RNAi和小分子筛选)很有效 承诺缓解这种瓶颈；然而，它们在癌症中的系统应用 生物学将显著受益于强大的体外癌症模型 基因组的多样性在患者身上表现出来。恶性黑色素瘤提供了一条丰富的途径 这一点：与其他实体肿瘤不同，这种致命的恶性肿瘤的细胞很容易 在体外培养，从而提供了一个多样化和易处理的基因组和 功能研究。 因此，我们建议应用池RNAi和小分子微阵列 对一组具有基因特征和患者来源的患者进行顺序筛查 黑色素瘤细胞系。我们将组装一个针对所有表达基因的慢病毒文库 位于黑色素瘤中被放大的基因组部分，并执行 对20个黑色素瘤株进行RNAi联合筛查 常见的黑色素瘤基因组改变。‘Hits’(例如，选择性耗尽的shRNA)将 与基因组模式相关以确定黑色素瘤的(Onco)基因靶点 放大。我们将使用一系列细胞来验证最有希望的靶基因 体外存活分析(阵列RNAi格式)和体内肿瘤形成分析 (使用shRNA“迷你池”)。最后，我们将确定与顶部结合的候选配体 通过进行小分子微阵列筛选来定位(Onco)候选蛋白质 表位标记的蛋白质构建。如果成功，这个项目应该详细说明一个频谱 靶蛋白和潜在的先导化合物与常见的基因变化有关 黑色素瘤。此外，这些努力应该形成一种“平台化”的综合方法。 适用于所有存在可治疗的体外模型的癌症。

Although tumor classification and patient stratification based on genomic criteria offers tremendous clinical potential, discerning critical effectors of tumor genetic alterations—and developing robust therapeutic avenues to intercept them—represents a formidable obstacle to translational oncology. Recent advances in large-scale `perturbagen' approaches (e.g., viral RNAi and small molecule screening) hold great promise to alleviate such bottlenecks; however, their systematic application in cancer biology would benefit markedly from robust in vitro cancer models that fully encompass the genomic diversity manifest in patients. Malignant melanoma offers a rich avenue in this regard: unlike other solid tumors, cells from this lethal malignancy are readily cultured in vitro, thereby providing a diverse and tractable system for genomic and functional studies. Accordingly, we propose to apply pooled RNAi and small molecule microarray screening sequentially to a panel of genetically characterized and patient-derived melanoma cell lines. We will assemble a lentiviral library targeting all expressed genes located within the portion of the genome that is amplified in melanoma, and perform pooled RNAi screening across a panel of 20 melanoma lines representative of the most prevalent melanoma genomic alterations. `Hits' (e.g., selectively depleted shRNAs) will be correlated with genomic patterns to identify (onco)gene targets of melanoma amplifications. We will validate the most promising target genes using a series of cell survival assays in vitro (in arrayed RNAi format) and tumor formation assays in vivo (using shRNA `mini-pools'). Finally, we will identify candidate ligands that bind the top target (onco)protein candidates by performing small-molecule microarray screens using epitope-tagged protein constructs. If successful, this project should elaborate a spectrum of target proteins and potential lead compounds linked to common genetic changes in melanoma. Moreover, these efforts should inform a “platformizable” integrated approach applicable to all cancers for which tractable in vitro models exist.