Abstract 1691 Differential Response of GCN2 eIF2 Kinase to Specific Amino Acid Limitations in Prostate Cancer

Stress response pathways are critical for cellular adaptation and survival to different environmental cues, such as amino acid (AA) starvation. The eIF2 kinase GCN2 is a major sensor of AA in cells, directing transcriptional and translational changes critical for stress adaptation to AA limitations. GCN2 is activated by interaction with uncharged tRNAs, which accumulate during AA limitation. GCN2 phosphorylates the eukaryotic translation initiation factor 2 (eIF2), resulting in repression of global protein synthesis. In parallel, eIF2 phosphorylation also enhances the translation of key mRNA transcripts, such as that encoding the transcription factor ATF4, which directs the transcription of genes involved AA synthesis, transport, and reclamation. Additionally, GCN2 activation decreases cellular AA consumption by reducing translation, altering metabolism, and modulating the cell cycle. Collectively these events are critical for adaptation and survival to AA starvation. Our laboratory has recently shown that GCN2 is activated in prostate cancer (PCa) and is critical for the maintenance of AA homeostasis. We demonstrated in multiple PCa cell lines in culture and in vivo models that GCN2 inhibition results in AA limitation and reduced proliferation, showing the importance of GCN2 for PCa growth and progression and its potential as a therapeutic target. For our studies on the role of GCN2 and its roles in AA homeostasis in PCa, we utilized the androgen-sensitive and castration-resistant cell lines LNCAP and 22Rv1, respectively. We sought to address which amino acid(s) may be limiting to activate GCN2 in the each of the proliferating cell lines. To determine limiting AAs, LNCaP and 22Rv1 cells were cultured in media lacking either a single or multiple amino acids. Our results show that depletion of each of the amino acids resulted in activation of GCN2, increased eIF2 phosphorylation, and enhanced ATF4 expression, as measured by immunoblotting, and decreased global translation. Although GCN2 activity was increased by depletion of any individual AA, we observed that the degree of GCN2 activation and subsequent translation inhibition vary depending on which AA was depleted. Additionally, we investigated how other nutrient sensing pathways, such as mTORC1 or p53, respond to specific AA starvation, and found that depletion of only a subset of AAs sensed by GCN2 result in activation of these pathways. These results suggest that, unlike other nutrient-sensing pathways, GCN2 can sense and respond to limitation for each AA. We also performed cell cycle analysis by flow cytometry and show that depletion for specific amino acids, such as histidine, results in a GCN2-depedent G1 cell cycle arrest. These findings suggest that GCN2, in response to AA starvation, can regulate cell cycle progression, and this may be critical for stress adaptation. Our results highlight the importance of GCN2 in the adaptation and survival of cells to different types of AA limitation. Unlike other nutrient pathways, GCN2 can sense imbalances of any proteogenic AA and coordinate a stress-mediated adaptive response. Determining the mechanistic features for how GCN2 responds to different types of AA starvation will be important to understand its role in health and disease. NIH GM136331 (Ronald C Wek). Indiana University Melvin and Bren Simon Comprehensive Cancer Center P30CA082709 (Kirk A Staschke).