Abstract A053: Targeting pyrimidine metabolic vulnerabilities via DHODH inhibition in ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is a lethal malignancy with poor survival. While initial treatments can be effective, recurrence and resistance remain major problems, suggesting the need for new therapies. One promising target is nucleotide metabolism, as EOC cells require elevated pyrimidine pools to support rapid proliferation and DNA repair. Pyrimidines are synthesized via de novo synthesis and salvage pathways. Dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pathway, has shown therapeutic potential in some cancers but remains underexplored in EOC. Here, we evaluate the efficacy of a novel DHODH inhibitor, HOSU-53, and investigated the molecular mechanisms for refractory to treatment in EOC. We tested a panel of human EOC cell lines and found that half were hyper-susceptible to HOSU-53 in vitro. By comparing uridine-free and uridine-supplemented conditions, we showed that the vulnerability to DHODH inhibitors was driven by the deficiency in extracellular uridine salvage. Isotope-labeled uridine tracing confirmed efficient uptake in resistant cells, but not in sensitive ones. Based on this, we classified the lines as salvage-proficient or -deficient. In vivo studies using xenograft models derived from salvage-deficient cells demonstrated strong tumor suppression upon treatment significant tumor suppression upon treatment, establishing DHODH inhibition as a promising therapeutic approach in a subset of EOC. Since uridine uptake is mediated by SLC28A and SLC29A transporter families, we investigated their function, expression, and localization in several salvage-proficient EOC cell lines. We observed substantial intertumoral heterogeneity in transporter expression and subcellular distribution, suggesting that different EOC cells may rely on distinct transporter subtypes or localization mechanisms to support uridine salvage. This heterogeneity may account for the inconsistent responses in prior attempts to combine DHODH and SLC29A inhibitors. To facilitate clinical stratification, we developed a functional assay to assess uridine-uptake using 5-ethynyl uridine (5-EU). After cellular uptake and incorporation into nascent RNA, 5-EU is fluorescently tagged via click chemistry, enabling the visualization of uridine salvage activity at single-cell level. Applied to multiple primary EOC samples and cell lines, the assay revealed minimal 5EU signal in salvage-deficient cells, and strong labeling in salvage-proficient ones. This 5EU assay offers scalable platform to identify tumors with impaired uridine salvage that may be responsive to DHODH inhibition. In summary, our study highlights DHODH inhibition as a promising strategy for EOC, particularly in tumors with impaired salvage. We identified metabolic heterogeneity in uridine uptake as a determinant of therapeutic response and established the 5-EU assay as a practical tool for stratifying patients based on metabolic phenotype. Together, these findings provide a strong rationale for the development of targeted and combination therapies to improve clinical outcomes in ovarian cancer. Citation Format: Linzhou Wang, Na Li, Yajing Yang, Jessica Miao, Aidan Li, Elsa Wani, Chad Bennett, Sandip Vibhute, Ruoning Wang, Selvendiran Karuppaiyah, Jiangjiang Zhu, Qi-En Wang. Targeting pyrimidine metabolic vulnerabilities via DHODH inhibition in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A053.