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

Area of Science:

• Oncology
• Molecular Biology
• Pharmacology

Background:

• The RAS-MAPK pathway is crucial for cellular functions and is frequently dysregulated in colorectal cancers, driving neoplasm development.
• Mutations in this pathway lead to aberrant signaling, making it a promising target for therapeutic interventions.

Purpose of the Study:

• To develop and validate a gene expression-based high-throughput screening (GE-HTS) assay for identifying modulators of the RAS-MAPK pathway.
• To discover novel compounds targeting the RAS-MAPK pathway for potential colorectal cancer therapies.

Main Methods:

• Developed a 26-gene signature for RAS-MAPK pathway activity using the Affymetrix QuantiGene Plex 2.0 system.
• Performed primary and confirmatory GE-HTSs using KRAS mutant colon cancer cells and a chemical library.
• Validated hits through dose-response assays, additional cell line testing, and in vitro colony formation assays.

Main Results:

• The GE-HTS assay achieved a 1.4% hit rate and successfully enriched for compounds targeting MEK and EGFR.
• Demonstrated high sensitivity (0.84) and selectivity (1.00) for the assay.
• Confirmed active compounds across multiple assays and cell lines.

Conclusions:

• The developed GE-HTS assay is a robust tool for identifying novel compounds and mechanisms that modulate the RAS-MAPK pathway.
• This assay can facilitate larger, unbiased chemical screens for developing new colorectal cancer therapeutics.