title: Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.
abstract: 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.
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA Bryan_severyn@merck.com.
authors.forename: Bryan
authors.initials: B
authors.lastname: Severyn
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Thi
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Chemical Modeling and Informatics, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Altman
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Lixia
authors.initials: L
authors.lastname: Li
authors.affiliation: Exploratory Biomarkers, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Nagashima
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: George N
authors.initials: GN
authors.lastname: Naumov
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Sathyanarayanan
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Cook
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Erick
authors.initials: E
authors.lastname: Morris
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA.
authors.forename: Bill
authors.initials: B
authors.lastname: Arthur
authors.affiliation: Informatics and Analysis, Merck & Co. Inc., West Point, PA, USA.
authors.forename: Yair
authors.initials: Y
authors.lastname: Benita
authors.affiliation: Informatics and Analysis, Merck & Co. Inc., West Point, PA, USA.
authors.forename: Jim
authors.initials: J
authors.lastname: Watters
authors.affiliation: Informatics and Analysis, Merck & Co. Inc., West Point, PA, USA.
authors.forename: Andrey
authors.initials: A
authors.lastname: Loboda
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Hermes
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: D Gary
authors.initials: DG
authors.lastname: Gilliland
authors.affiliation: Molecular Biomarkers, Merck & Co. Inc., West Point, PA, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Cleary
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Carroll
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Strack
authors.affiliation: Screening and Protein Sciences, Merck & Co. Inc., North Wales, PA, USA.
authors.forename: Matt
authors.initials: M
authors.lastname: Tudor
authors.affiliation: Oncology, Merck & Co. Inc., Boston, MA, USA.
authors.forename: Jannik N
authors.initials: JN
authors.lastname: Andersen
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009154:Mutation; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D054852:Small Molecule Libraries / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: C117307:KRAS protein, human; D054852:Small Molecule Libraries; D016283:Proto-Oncogene Proteins p21(ras)
keywords: MAPK pathway; chemical genomics; colon cancer; gene expression high-throughput screen; xMAP technology