Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic plasticity that underlies its aggressive growth and therapeutic resistance. However, genetic determinants that modulate PDAC sensitivity to glycolytic inhibition remain incompletely defined. Here, we performed a pooled genome-wide CRISPR-Cas9 dropout screen in PDAC cells under 2-deoxy-D-glucose (2-DG) selection and identified FGD5 as a key regulator of glycolytic dependency. Loss of FGD5 sensitized PDAC cells to 2-DG, reduced clonogenic growth and stem-like properties, and was associated with a shift from glycolysis toward oxidative phosphorylation. Clinically, FGD5 expression was elevated in PDAC tissues and correlated with unfavorable patient outcomes. Mechanistically, FGD5 interacted with and stabilized PGK1 by limiting STUB1-mediated ubiquitination and proteasomal degradation. Pharmacological perturbation with CB-5083, a compound prioritized through structure-guided screening, modulated FGD5-PGK1-associated proteostasis readouts, increased PGK1 ubiquitination, reduced PGK1 abundance, and cooperated with 2-DG to suppress PDAC growth in vitro and in vivo. Moreover, lactate-associated H3K18 histone lactylation was linked to increased FGD5 transcription, consistent with a feed-forward regulatory connection between glycolysis, epigenetic modification, and metabolic adaptation. Collectively, our findings position FGD5 as a metabolic vulnerability in PDAC and provide a mechanistic framework supporting combinatorial strategies that couple glycolysis inhibition with perturbation of FGD5-PGK1-associated proteostasis. A genome-wide CRISPR-Cas9 screen identified FGD5 as a metabolic vulnerability that increases PDAC sensitivity to the glycolysis inhibitor 2-deoxy-D-glucose (2-DG). In PDAC cells, FGD5 interacts with PGK1 and supports PGK1 stability by limiting STUB1-dependent ubiquitination and proteasomal degradation, thereby sustaining aerobic glycolysis and lactate production. Glycolysis-derived lactate is associated with increased H3K18 histone lactylation (H3K18la) at the FGD5 promoter, accompanied by elevated FGD5 transcription, consistent with a metabolic-epigenetic regulatory link. Pharmacological perturbation with CB-5083 attenuates FGD5-PGK1-associated proteostasis readouts, whereas 2-DG inhibits glycolytic flux and lactate-associated H3K18la. Together, CB-5083 and 2-DG cooperate to suppress PDAC growth by coupling perturbation of the FGD5-PGK1-associated proteostasis axis with glycolytic inhibition.
CRISPR Gene Editing