TY - JOUR
T1 - RAF inhibitors activate the integrated stress response by direct activation of GCN2
AU - Gilley, Rebecca
AU - Kidger, Andrew M.
AU - Neill, Graham
AU - Morrison, Eve
AU - Severson, Paul
AU - Byrne, Dominic P.
AU - Kenneth, Niall S.
AU - Bollag, Gideon
AU - Zhang, Chao
AU - Maia de Oliveira, Taiana
AU - Eyers, Patrick A.
AU - Bayliss, Richard
AU - Masson, Glenn R.
AU - Cook, Simon J.
N1 - Publisher Copyright:
© The Author(s) 2025.
PY - 2025/11/17
Y1 - 2025/11/17
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/105022102506
U2 - 10.1038/s41467-025-65376-w
DO - 10.1038/s41467-025-65376-w
M3 - Article
C2 - 41249187
AN - SCOPUS:105022102506
SN - 2041-1723
VL - 16
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 10033
ER -