Fasting sensitizes hepatocellular carcinoma to sorafenib by curtailing metabolic flexibility Jelena Krstic 1, Isabel Reinisch 1, Katharina Schindlmaier 1, Christoph Nössing 1,2, Maria R. Depaoli 3, Jeta Ramadani-Muja 3, Meritxell Huch 4, Roland Malli 3,5 and Andreas Prokesch 1,5 1 Gottfried Schatz Research Center for Cell Signaling, Metabolism & Aging, Division of Cell Biology, Histology and Embryology, Medical University of Graz, 8010 Graz, Austria 2 Cancer Research UK Beatson Institute, Garscube Estate, Glasgow, UK. 3 Gottfried Schatz Research Center for Cell Signaling, Metabolism & Aging, Division of Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Austria 4 Max Planck Institute of Molecular Cell Biology and Genetics, 01307, Dresden, Germany 5 BioTechMed-Graz, 8010 Graz, Austria Metabolic flexibility is an important mediator of therapy resistance in cancer. However, while cancer cells rewire their metabolic pathways to adapt to nutritional microenvironment, they also reveal metabolic vulnerabilities which can be therapeutically exploited. Hepatocellular carcinoma (HCC) is one of the deadliest cancers with very few molecular therapeutic opportunities. Sorafenib, the most widely used clinical drug for advanced HCC provides limited extension in median overall survival of three months due to primary or acquired resistance. We explored whether nutrient deprivation can be used as an adjuvant therapy to sorafenib in the treatment of HCC. We show that fasting can sensitize resistant HCC to sorafenib in HCC-derived cells, xenografts, and in patient-derived organoids. We found that sorafenib acts as potent inhibitor of mitochondrial respiration, causing resistant HCC cells to switch to glycolysis for survival. Synergistically, reduced nutrients prevent this Warburg shift and lead to sensitisation to sorafenib in vitro and in vivo. Functional experiments show that glucose is the limiting nutrient crucial for curtailing this metabolic flexibility. We further show that p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting, as p53 knock out cells and xenografts remain resistant under starvation. p53 is also necessary for the sorafenib-enhancing effect of intermittent fasting in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for advanced-stage, and possibly even early-stage, HCC.