Targeting PGK1 enhances enzalutamide efficacy in resistant prostate cancer

Enzalutamide is a standard AR-targeting therapy for advanced prostate cancer (PCa), yet intrinsic and acquired resistance remain major barriers to durable response. To identify regulators of enzalutamide sensitivity, we performed a kinome-wide CRISPR-Cas9 knockout screen in the intrinsically resistant 22Rv1 cell line. Phosphoglycerate kinase 1 (PGK1), a key glycolytic enzyme, emerged as a top negatively selected gene, indicating that its loss increases enzalutamide susceptibility. PGK1 inhibition or genetic deletion reduced viability, colony formation, migration, and invasion in 22Rv1 cells, validating its role in maintaining the aggressive phenotype of resistant PCa. Bulk RNA-seq analysis of PGK1 knockdown cells revealed increased AR signaling pathway activity, suggesting compensatory AR reactivation following metabolic disruption. Combination MTT assays showed synergistic efficacy between PGK1 inhibition and enzalutamide, supporting a metabolic-hormonal interaction that enhances AR-directed therapy response. Mechanistically, Seahorse analysis demonstrated that PGK1 inhibition decreases glycolytic rate in resistant PCa cells, indicating impaired metabolic compensation as a potential mechanism of sensitization. Future work includes xenograft studies using 22Rv1 and LNCaP PDX models, ATP flux assays, mitochondrial stress test, lipid metabolism, and pathway-level analysis. These findings identify PGK1 as a metabolic vulnerability and highlight its therapeutic potential to overcome enzalutamide resistance in advanced prostate cancer.