High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity
- 1University of Washington
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.
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BibTeX
@misc{adebanjo2026highresolution,
title = {{High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity}},
author = {A. F. Adebanjo and J. J. Simon and F. Banales Mejia and D. Fowler and D. J. Maly},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.30.755370},
url = {https://www.biorxiv.org/content/10.64898/2026.09.30.755370v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - High-resolution measurement of ERK activation state reveals determinants of BRAF regulation and drug sensitivity
AU - A. F. Adebanjo
AU - J. J. Simon
AU - F. Banales Mejia
AU - D. Fowler
AU - D. J. Maly
PY - 2026
DA - 2026/09/30
PB - bioRxiv
DO - 10.64898/2026.09.30.755370
UR - https://www.biorxiv.org/content/10.64898/2026.09.30.755370v1
AB - 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.
ER -