PKM isoform splicing shifts are indicative of the increased anabolic need of leukemic cells. (A) Simplified overview of glycolysis and regulation by PKM isoforms. PKM is the rate-limiting factor of glycolytic outflow, with PKM1 favoring channeling carbon from glucose to mitochondria for energy production. PKM2 generally has lower catalytic activity, restricting exit from glycolysis and guaranteeing the availability of anabolic intermediates. The inhibitors used in this study and their intervention points are schematically indicated. 3-Phosphoglycerate is a precursor of Ser and therefore indirectly also of glycine and glutathione synthesis. For clarity, the pentose-phosphate cycle that is necessary for production of nucleotide bases was omitted. (B) Seahorse analysis of metabolic changes induced by Ptbp1 knockdown. Murine control and knockdown cells were subjected to measurements of OCRs and ECARs. Relevant differences are shaded blue. (C) Determination of 50% inhibitory concentrations for inhibitors of glycolysis (2-deoxy-glucose), Ser biosynthesis (CBR5884), and energy production (rotenone). The experiment was performed with murine MLL-ENL transformed cells and graphs depict relative proliferation as determined by standard MTT tests. (D) Effect of metabolic drugs on colony formation. The number of CFUs was determined after a 48-hour transient treatment of leukemic cells (MLL-ENL transduced) and normal HSPCs (Kit/CD117⁺ fraction of bone marrow including GMPs) with inhibitors or a solvent control in the indicated concentrations. Top: the mean and standard deviation of the relative number of colonies recorded in triplicate experiments. Bottom: a representative example of methylcellulose cultures after staining of colonies with INT.