Defining Melanoma Therapeutic Avenues by Integrative Functional Genomics

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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.