High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity
Year: 2026
Type: preprint
Abstract: High-resolution measurements of pathway activity make it possible to characterize how signaling responds to changing cellular conditions, to evaluate pathway-targeted inhibitors, and to dissect how sequence variation shapes signaling output. Here, we describe a sequencing-based assay that uses engineered, phospho-state-selective affinity reagents to quantify ERK activation state as a high-resolution, ratiometric readout of RAS/ERK MAPK pathway activity. Profiling ~1,600 BRAF variants at basal and elevated RAS-GTP levels allowed us to assign activity scores to somatic cancer and germline RASopathy variants and to distinguish residues that stabilize autoinhibition from those required for active dimer formation. We identified a large class of dominant negative variants that suppress signaling below endogenous levels through enhanced RAS engagement and an inability to form productive RAF dimers. Measuring the intracellular potency of the RAF inhibitor LXH254 against ~1,000 variants in parallel revealed hinge and activation-loop positions conferring resistance and showed that disrupting the BRAF-MEK interface is broadly sensitizing.
Institution: University of Washington
Language: English
Topic: Melanoma and MAPK Pathways
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: New approaches for investigating the causes and consequences of cellular heterogeneity, Technology to understand genetic variant effects in context, Chemical Genetic Analysis of RAS Signaling, New Molecular Probes For Protein Kinases, The Center for Actionable Variant Analysis; measuring variant function at scale
OpenAlex ID: W7216462567