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

Area of Science:

• Molecular Biology
• Cellular Signaling
• Cancer Therapeutics

Background:

• Paradoxical activation of wild-type RAF by RAF inhibitors (RAFi) is a known on-target effect.
• The Integrated Stress Response (ISR) pathway, involving eIF2α phosphorylation, regulates cellular stress.
• The Unfolded Protein Response (UPR) is a key component of the ISR.

Purpose of the Study:

• To investigate the mechanism by which RAF inhibitors activate the ISR.
• To determine if RAFi-induced ISR activation is dependent on known ISR activators like PERK.
• To explore the role of GCN2 kinase in RAFi-mediated ISR activation and its potential clinical relevance.

Main Methods:

• Treatment of cells with various RAF inhibitors (RAFi).
• Analysis of ATF4 and CHOP expression, key ISR markers.
• Inhibition of PERK, GCN2 (using inhibitors, RNAi, or knock-out), and ISRIB.
• In vitro and cellular activation assays for GCN2.
• Site-directed mutagenesis of GCN2 (kinase-dead and gatekeeper mutants).
• Mechanistic structural modeling of RAFi-GCN2 interaction.

Main Results:

• RAFi induce ERK1/2-independent UPR activation, including ATF4 and CHOP expression, dependent on eIF2α.
• RAFi-induced ATF4/CHOP expression is not reversed by PERK inhibition but requires GCN2 activation.
• RAFi directly activate GCN2 kinase in vitro and in cells, exhibiting a bell-shaped concentration-response curve.
• GCN2 inhibition or antagonism reverses RAFi-induced ATF4/CHOP expression, confirming GCN2's essential role.
• RAFi bind directly to the GCN2 kinase domain, as evidenced by gatekeeper mutant studies and structural modeling.

Conclusions:

• RAF inhibitors activate the ISR through direct, paradoxical activation of the GCN2 kinase.
• This GCN2-mediated ISR activation is an off-target effect of RAFi, independent of ERK1/2 signaling.
• The findings suggest that GCN2 activation by RAFi may impact tumor cell survival and clinical outcomes.