Introduction Tyrosine kinase inhibitors (TKI) have dramatically changed the prognosis of patients with leukaemia. Unfortunately, they fail to achieve durable remissions for many patients with advanced BCR-Abl leukaemia or acute myelogenous leukaemia. Myeloid leukemic cells depend on mitochondrial oxidative phosphorylation (OxPhos) and/or glycolysis to produce ATP and macromolecules for survival and intense proliferation. Particularly, leukemic cells driven by oncogenes, like BCR-Abl or FLT3 ITD , present high glycolysis rate. Here we investigated metabolic modifications induced by several TKI in leukemic cells and we propose new strategies combining TKI and inhibitors of metabolism for patients with BCR-Abl and FLT3 ITD leukemias Material and methods Several myeloid leukemic cell lines as well as patient samples were examined. Effects of drugs on viability were determined by flow cytometry with Annexin-V/Sytox staining. Glucose and mitochondrial metabolism changes were determined using XFe24 Seahorse and by mass spectrometer using glucose- C 13 or glutamine-C 13 . Results and discussions Under normoxic and hypoxic conditions, the constitutive activation of oncogenic tyrosine kinases supports glucose metabolism of leukemic cells. Consequently, inhibition of BCR-Abl or FLT3 ITD by TKI leads to severe metabolic changes including drastic reduction in glucose uptake and glycolysis, as well as decrease in mitochondrial metabolism. However, a sub-population of leukemic cells maintains viability even after several days of in vitro treatment with TKI and is characterised by higher mitochondrial membrane potential, confirming the primordial role of mitochondrial metabolism in the survival of TKI-treated cells. Interestingly, the association of TKI and L-Asparaginase (l-ase), an enzyme that depletes extracellular asparagine and glutamine, decreases OxPhos resulting in the eradication of TKI-tolerant cells. Moreover, the association of TKI and l-ase kills BCR-Abl + leukemic cells from patients with acquired resistance to TKI. By a co-culture of mesenchymal (MS-5) and leukemic cells, we have also demonstrated that the presence of MS-5 managed to rescue TKI-treated cells, nevertheless the addition of l-ase eradicates the persistent cells that survived under TKI treatment alone, without affecting MS-5 cells viability. Conclusion These results might enable the development of new therapeutic strategies based on anti-metabolic cooperativity combining TKI and L-Ase in patients who develop myeloid leukaemia or in patients with acquired resistance to TKI.