Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context

• Published in: Journal of biomolecular screening Vol. 21; no. 9; p. 989
• Main Authors: Severyn, Bryan, Nguyen, Thi, Altman, Michael D, Li, Lixia, Nagashima, Kumiko, Naumov, George N, Sathyanarayanan, Sriram, Cook, Erica, Morris, Erick, Ferrer, Marc, Arthur, Bill, Benita, Yair, Watters, Jim, Loboda, Andrey, Hermes, Jeff, Gilliland, D Gary, Cleary, Michelle A, Carroll, Pamela M, Strack, Peter, Tudor, Matt, Andersen, Jannik N
• Format: Journal Article
• Language: English
• Published: United States 01-10-2016
• Subjects: Apoptosis - drug effects; Cell Line, Tumor; Cell Proliferation - drug effects; Dose-Response Relationship, Drug; Gene Expression Profiling - methods; Gene Expression Regulation - drug effects; High-Throughput Screening Assays - methods; Humans; MAP Kinase Signaling System - drug effects; Mutation; Neoplasms - drug therapy; Neoplasms - genetics; Proto-Oncogene Proteins p21(ras) - genetics; Small Molecule Libraries - isolation & purification; Small Molecule Libraries - pharmacology; MAPK pathway; gene expression high-throughput screen; xMAP technology; colon cancer; chemical genomics
• ISSN: 1552-454X
• DOI: 10.1177/1087057116658646

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway.