Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway

• DOI: 10.1042/bcj20240015
• Publication Date: 2024-02-21T14:51:41Z
• Authors: Laura Weatherdon; Kate Stuart; Megan Cassidy; Alberto Moreno de...; Hanneke Okkenhaug; Markus Muellener; Grahame Mckenzie; Simon J. Cook; Rebecca Gilley
• Keywords: 0301 basic medicine; 03 medical and health sciences; Signaling

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) BRAF MEK1/2 (melanoma colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new proteins), inhibitor modalities regulators this pathway, which may be drug targets, continues increasingly involves cell-based screens with small molecules or genetic such as RNAi, CRISPR protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V BRAFV600E harbour stably integrated EGR1:EmGFP reporter gene can detected by flow cytometry, high-content microscopy immunoblotting. BRAFV600E-driven EmGFP inhibited ERK1/2 (MEKi ERKi). BRAFi inhibit but enhance response KRASG12V, recapitulating paradoxical activation wild type RAF proteins. In addition molecules, iDab6, encoding RAS-specific antibody fragment KRASG12V- not expression. Finally, substitution bacterial nitroreductase allowed drive death presence pro-drug, allow selection promote survival. These should prove useful identify KRAS- BRAF-dependent (drug target discovery) well screening triaging 'hits' from discovery screens.