Targeting DHODH reveals a metabolic vulnerability in AR-positive and AR-negative prostate cancer cells via pyrimidine synthesis and metabolic crosstalk with the TCA and urea cycles

Following recurrence, the cornerstone clinical therapy to treat prostate cancer (PCa) is to inhibit the androgen receptor (AR) signaling. While AR inhibition is initially successful, tumors will eventually develop treatment resistance and evolve into lethal castration-resistant PCa. To discover new anti-metabolic treatments for PCa, a high-throughput anti-metabolic drug screening was performed in PC3 cells, an AR-negative PCa cell line. This screening identified the dihydroorotate dehydrogenase (DHODH) enzyme as a metabolic vulnerability, using both AR-positive and AR-negative models, including the neuroendocrine cell line LASCPC-01 and patient-derived organoids. DHODH is required for de novo pyrimidine synthesis and is the sole mitochondrial enzyme of this pathway. Using extracellular flux assays and targeted metabolomics, DHODH inhibition was shown to impair the pyrimidine synthesis pathway, as expected, along with a significant reprogramming of mitochondrial metabolism, with a massive increase in fumarate (>10-fold). Using ¹³C₆-glucose, it was shown that following DHODH inhibition, PCa cells redirect carbons from glucose toward biosynthetic pathways rather than the TCA cycle. In parallel, using ¹³C₅-glutamine, it was shown that PCa cells use this amino acid to fuel a reverse TCA cycle. Finally, ¹³C₁-aspartate and ¹⁵N₁-glutamine highlighted the connection between pyrimidine synthesis and the urea cycle, redirecting pyrimidine synthesis intermediates toward the urea cycle as a stress response mechanism upon DHODH inhibition. Consequently, combination therapies targeting DHODH and glutamine metabolism were synergistic in impairing PCa cell proliferation. Altogether, these results highlight DHODH as a metabolic vulnerability of AR-positive and AR-negative PCa cells by regulating central carbon and nitrogen metabolism.