RAF inhibitors activate the integrated stress response by direct activation of GCN2

Rebecca Gilley (), Andrew M. Kidger, Graham Neill, Eve Morrison, Paul Severson, Dominic P. Byrne, Niall S. Kenneth, Gideon Bollag, Chao Zhang, Taiana Maia de Oliveira, Patrick A. Eyers, Richard Bayliss, Glenn R. Masson and Simon J. Cook ()
Additional contact information
Rebecca Gilley: Babraham Research Campus
Andrew M. Kidger: Babraham Research Campus
Graham Neill: University of Dundee
Eve Morrison: Babraham Research Campus
Paul Severson: 1 Bolivar Drive
Dominic P. Byrne: University of Liverpool
Niall S. Kenneth: University of Liverpool
Gideon Bollag: 1 Bolivar Drive
Chao Zhang: 1 Bolivar Drive
Taiana Maia de Oliveira: AstraZeneca
Patrick A. Eyers: University of Liverpool
Richard Bayliss: University of Leeds
Glenn R. Masson: University of Dundee
Simon J. Cook: Babraham Research Campus

Nature Communications, 2025, vol. 16, issue 1, 1-19

Abstract: Abstract Paradoxical activation of wild type RAF by chemical RAF inhibitors (RAFi) is a well-understood ‘on-target’ biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that requires the translation initiation factor eIF2α. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2α-dependent Integrated Stress Response (ISR). Rather, RAFi exposure activated GCN2, an alternate eIF2α kinase, leading to eIF2α-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2α antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic ‘bell-shaped’ concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by a GCN2 kinase dead mutation. A M802A GCN2 gatekeeper mutant was activated at lower RAFi concentrations, demonstrating that RAFi bind directly to the GCN2 kinase domain; this is supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation is a previously unappreciated off-target effect that may modulate tumour cell responses.

Date: 2025
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DOI: 10.1038/s41467-025-65376-w

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