Abstract Cancer stem cells (CSCs) pose a critical challenge in Pancreatic cancer (PC) management because of their unique ability to survive treatment and drive tumor relapses. CSCs exhibit metabolic flexibility to adapt to stress by switching between glycolysis and oxidative phosphorylation. Pyruvate dehydrogenase kinase 3 (PDK3) governs this transition by phosphorylating and inactivating the pyruvate dehydrogenase complex (PDHA), thereby promoting a glycolytic phenotype. Understanding the metabolic reprogramming that sustains cancer stem cells may reveal strategies to disrupt their survival and propagation. In this study, RNA sequencing of spheroid-based PCSC model followed by unbiased in-silico analysis identified 14 upregulated metabolic regulatory genes, with PDK3 emerging as the top candidate. PDK3 expression was elevated in CSC subtype population, pancreatic tumor tissues from patients and Kras; PdxCre (KC) mouse models, but minimal in normal pancreatic tissues. Dual-IHC staining demonstrated co-expression of PDK3 with the stemness marker CD44 in human pancreatic tumor tissues, highlighting its association with CSC phenotypes. Stable lentiviral-mediated shRNA knockdown (KD) or pharmacologic inhibition with artesunate (a PDK3 targeting compound identified in this study) significantly reduced stemness markers (CD44, SOX2, and CD133), glycolytic genes (GLUT1 and LDHA), and phosphorylated PDHA levels. Functionally, PDK3 depletion decreased the side-population fraction, impaired sphere-formation, and colony-forming capacities, suggesting suppressed sustenance and self-renewal properties. Metabolomic profiling by LC-MS revealed lactic acid as one of the most enriched metabolites in scramble PC cells compared to PDK3 KD cells. Consistently, metabolomic analysis revealed a significant reduction in lactic acid release and glucose uptake upon PDK3 KD. Seahorse analysis further demonstrated that PDK3 depletion lowered ECAR and elevated OCR, indicating a metabolic shift from glycolysis toward oxidative phosphorylation. Interestingly, elevated lysine lactylation and SOX2 expression were observed in control and lactate-treated PC cells, whereas both were markedly reduced upon glycolysis inhibition and PDK3 KD, indicating a strong dependence on PDK3-mediated metabolic activity. Mechanistically, immunoprecipitation and immunofluorescence revealed that PDK3 promotes lactate-dependent lysine lactylation of SOX2, enhancing its stability and stemness activity. Orthotopic implantation of PDK3 KD human PC cells resulted in significantly reduced tumor growth in vivo, underscoring PDK3’s pivotal role in driving CSC-mediated tumor progression. In conclusion, our findings establish PDK3 as a central metabolic regulator of PCSC maintenance and tumor progression, offering new opportunities to develop therapies that disrupt lactate-fueled stemness in pancreatic cancer. Citation Format: Neelanjana Gayen, Nivedeta Krishna Kumar, Ayoola O. Ogunleye, Palanisamy Nallasamy, Venkatesh Varadharaj, Saravanakumar Marimuthu, Surinder K. Batra, Moorthy P. Ponnusamy. Metabolic control of pancreatic cancer stemness through PDK3 dependent SOX2 lactylation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2189.
Gayen et al. (Fri,) studied this question.