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Mutations in KRAS, particularly at codon 12, are frequent in adenocarcinomas of the colon, lungs and pancreas, driving carcinogenesis by altering cell signalling and reprogramming metabolism. However, the specific mechanisms by which different KRAS G12 alleles initiate distinctive patterns of metabolic reprogramming are unclear. Using isogenic panels of colorectal cell lines harbouring the G12A, G12C, G12D and G12V heterozygous mutations and employing transcriptomics, metabolomics, and extensive biochemical validation, we characterise distinctive features of each allele. We demonstrate that cells harbouring the common G12D and G12V oncogenic mutations significantly alter glutamine metabolism and nitrogen recycling through FOXO1-mediated regulation compared to parental lines. Moreover, with a combination of small molecule inhibitors targeting glutamine and glutamate metabolism, we also identify a common vulnerability that eliminates mutant cells selectively. These results highlight a previously unreported mutant-specific effect of KRAS alleles on metabolism and signalling that could be potentially harnessed for cancer therapy.

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.

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