Abstract 450: GCN2 protects cancer cells from hypertranslation and metabolic crisis

Abstract

Abstract It is widely assumed that cancer cells rely on high levels of protein synthesis to support growth and proliferation. However, protein synthesis, including ribosome biogenesis, is a highly resource- and energy-demanding process that needs to be coordinated with protein degradation and metabolic programs. GCN2 is an evolutionarily conserved kinase that is primarily activated by uncharged tRNAs when amino acid levels drop and activates the Integrated Stress Response (ISR), which encompasses a global reduction in translation and increased amino acid uptake and generation. GCN2 has been shown to support cancer cell survival under conditions of nutrient scarcity. Here, we used an integrated systems biology approach to delineate the multifaceted role of GCN2 in cancer cells that do not suffer from nutrient shortages. We carried out a broad array of multiomic analyses (RNA-seq, TMT-labelling based proteomics, Ribo-seq, LC-MS metabolomics) of melanoma cells in which GCN2 was inhibited (GCN2iB) or genetically depleted (constitutive and inducible shRNA) in cell culture and in vivo in murine xenografts, supplemented by puromycinylation-based quantification of global protein synthesis. GCN2 inhibition in A375 cells triggered a transcriptome and proteome response that was dominated by the induction of processes linked to protein and ribosome synthesis while suppressing metabolic pathways such as glycolysis and the proteasome. We confirmed these observations in vivo via RNA-seq of tumor samples from mice xenografted with A375 cells in which GCN2 was knocked down by induced shRNA expression and from xenografted mice that were treated orally with GCN2iB. Ribo-seq further demonstrated that, upon GCN2 inhibition, A375 cells expand their translational machinery. Notably, we identified 50 ribosomal genes that were translated at higher rate in GCN2-inhibited cells and whose expression was regulated exclusively at the translational level. GCN2 inhibition/depletion also resulted in an approximately 1.8-fold increase in global protein synthesis and altered the druggable proteome. Moreover, metabolite profiling of GCN2iB-treated cells revealed a loss of metabolic homeostasis including substantial alterations in glycolysis and both amino acid and lipid metabolism. In addition, we observed a significant decrease in ATP levels despite increased translation of mitochondrial ETC complex proteins. Proteomic and translatomic analyses revealed a failure of cells to increase proteasomal subunits and to maintain the production of ECM proteins. Last, but not least, we did not observe a role of GCN2 in preventing or resolving ribosome collisions. Taken together, our findings reveal GCN2 as a master regulator of translation in cancer cells that do not suffer from nutrient scarcity that keeps protein synthesis in check to maintain metabolic homeostasis. Citation Format: Monica Roman-Trufero, Istvan T. Kleijn, Kevin Blighe, Paula Saavedra-Garcia, Jinglin Zhou, Abigail Gaffar, Marilena Christoforou, Mariia Yuneva, Audrey M. Michel, Glenn R. Masson, Vahid Shahrezaei, Holger W. Auner. GCN2 protects cancer cells from hypertranslation and metabolic crisis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 450.