Abstract A122: Dual targeting of <i>de novo</i> and salvage nucleotide synthesis pathways as a therapeutic strategy in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) displays remarkable metabolic flexibility, utilizing both de novo and salvage nucleotide synthesis pathways to support proliferation under nutrient-limited conditions. Although inhibitors of dihydroorotate dehydrogenase (DHODH), a critical enzyme in de novo pyrimidine biosynthesis, have demonstrated strong preclinical efficacy, they have failed to translate into clinical success across various cancers. In this study, we characterize the activity of a novel DHODH inhibitor, across multiple PDAC cell lines and identify differential sensitivity patterns driven by compensatory resistance mechanisms. Methods: A panel of human and mouse PDAC cell lines (HPAF-II, Mia-Paca-2, PANC-1, L3.6pl, AsPC-1, KPC) was treated with the DHODH inhibitor across a concentration range of 0.003 µM to 100 µM to assess differential sensitivity. Next, to characterize the mechanism of cell death, we performed western blot analysis to evaluate markers of apoptosis (cleaved caspase-3, PARP cleavage), ferroptosis (GPX4, 4-HNE), and senescence (Cyclin B1, p21). Resistant PDAC cell lines were further analyzed to investigate compensatory survival mechanisms, including salvage pathway activation (radiolabeled transport assay), autophagy (western blot for autophagy markers- LC3B, p62), and macropinocytosis (Dextran uptake). Syngeneic pancreatic orthotopic xenograft mouse models were utilized for studying in-vivo efficacy (non-invasive imaging) and tumor immune microenvironment (flow cytometry analysis). Results: Cell viability assays revealed marked differences in sensitivity to DHODH inhibition across PDAC cell lines, with L3.6pl being the most sensitive and HPAF-II the most resistant, exhibiting nearly a 100-fold difference in IC50 values. Mechanistic investigations revealed that DHODH inhibition in sensitive cell lines induces apoptosis, ferroptosis, reduced proliferation, triggered G2/M cell cycle arrest, culminating in cell death. In contrast, resistant cell lines preserve viability through upregulation of the salvage pathway, autophagy, and macropinocytosis. Our findings show that salvage pathway activation mediates DHODH resistance by facilitating the uptake and recycling of extracellular nucleosides into pyrimidine nucleotides, bypassing the need for de novo synthesis. In-vivo studies revealed that combined inhibition of DHODH and equilibrative nucleoside transporter 1 (ENT1) significantly remodels the tumor immune microenvironment, leading to increased infiltration of CD4+ and CD8+ T cells in PDAC tumors in syngeneic mouse models. Conclusion: These findings demonstrate that resistance to DHODH inhibition in PDAC is mediated by metabolic reprograming through the salvage pathway and nutrient scavenging mechanisms. Combined targeting of de novo and salvage nucleotide biosynthesis represents a promising strategy to overcome resistance and enhance antitumor immunity in PDAC, supporting further translational investigation. Citation Format: Shivam Vij, Arnav Joshi, Tejinder kaur, Rajgopal Govindarajan. Dual targeting of de novo and salvage nucleotide synthesis pathways as a therapeutic strategy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A122.