Abstract 388: Identifying synthetic lethalities in GCN2 KO tumors using an <i>in vivo</i> targeted CRISPRi screen
Authors: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke
Journal: Cancer Research (2024)

Abstract The integrated stress response (ISR) is an intracellular signaling pathway that fosters cell adaptation to diverse stresses. The ISR is mediated by a family of four eIF2 kinases that each sense distinct cellular stresses. Activation of the eIF2 kinases results in reprogrammed gene expression through translational and transcriptional mechanisms that help prevent stress damage. Previously, we reported that prostate cancer (PCa) cells rely on GCN2 eIF2 kinase for maintenance of amino acid (AA) homeostasis to sustain growth (Cordova et al., 2022 eLife 11:e81083). GCN2 inhibition through pharmacological or genetic means results in decreased expression of AA transporters, depletion of intracellular AAs, and reduced growth of PCa cells in culture and mouse xenograft models. Although loss of GCN2 results in a significant reduction in PCa cell growth, minimal cell death is observed. We hypothesize that loss of GCN2 in PCa cells results in vulnerabilities that can be exploited and targeted to induce cell death. To optimize GCN2-targeted therapies for PCa, we utilized a targeted CRISPR-interference (CRISPRi) screen in castration-resistant 22Rv1 cells with functional wild type (WT) GCN2 or those with it deleted (GCN2 KO). The CRISPRi screen was performed in cells grown in culture or grown as tumor xenografts in mice and featured a guide library targeting genes that encode druggable targets, including kinases, phosphatases, and metabolic enzymes. While reduced expression of 152 genes was synthetically lethal with loss of GCN2 in the in vitro screen, more genes (202) were synthetically lethal with loss of GCN2 in vivo. Of note, reduced expression of many metabolic enzymes was synthetically lethal with GCN2 ablation only in the in vivo screen, potentially due to the abundance of nutrients in standard culture media. A group of six metabolism-related genes, identified as hits in the in vivo screen and targets of FDA-approved drugs, were selected for follow-up studies. One of these metabolic enzymes was GART, a trifunctional enzyme that carries out 3 of the 10 enzymatic steps in de novo purine biosynthesis. Based on our findings showing that GCN2 maintains AA levels to support nucleotide pools in PCa cells, we initially focused on this critical enzyme. Inhibition of GART using antifolates, including the FDA-approved Pemetrexed, resulted in minimal cell death of parental and GCN2 KO PCa cells cultured in standard culture media. However, when cultured in reduced nutrient media that better mimicked reduced nutrient availability in vivo, GART inhibition resulted in selective death of GCN2 KO cells. Our CRISPRi screening strategy uncovers new vulnerabilities in GCN2-inhibited PCa cells and reveals a general strategy to identify actionable targets with the potential to combine with GCN2 inhibitors for the treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke. Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen [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 388.