Many cancer cells have increased rates of aerobic glycolysis, a phenomenon termed the Warburg effect. In addition, in tumors there is a predominance of expression of the M2 isoform of pyruvate kinase (PKM2). M2 expression was previously shown to be necessary for aerobic glycolysis and to provide a growth advantage to tumors. We report that knockdown of pyruvate kinase in tumor cells leads to a decrease in the levels of pyruvate kinase activity and an increase in the pyruvate kinase substrate phosphoenolpyruvate. However, lactate production from glucose, although reduced, was not fully inhibited. Furthermore, we are unique in reporting increased serine and glycine biosynthesis from both glucose and glutamine following pyruvate kinase knockdown. Although pyruvate kinase knockdown results in modest impairment of proliferation in vitro, in vivo growth of established xenograft tumors is unaffected by PKM2 absence. Our findings indicate that PKM2 is dispensable for tumor maintenance and growth in vivo, suggesting that other metabolic pathways bypass its function.M any cancer cells have increased rates of glucose uptake with a concomitant decrease in oxidative phosphorylation, even in the presence of oxygen. This phenomenon of aerobic glycolysis with increased lactate production has been termed the Warburg effect (1). Previous work suggested that expression of pyruvate kinase M2 (PKM2) is a key event in determining this metabolic phenotype, and tumor expression of M2 provides a proliferative advantage in vitro and in vivo (2). In addition, some tyrosine kinases involved in cancer might also be responsible for regulation of the Warburg effect, as it has been shown that they can phosphorylate glycolytic enzymes, including PKM2, and that this phosphorylation may regulate PKM2 activity and promote the Warburg effect and tumor growth (3, 4).Glucose taken up by cells is phosphorylated by hexokinase and subsequently catabolized via glycolysis to phosphoenolpyruvate (PEP). PK catalyzes the dephosphorylation of PEP to pyruvate, generating a molecule of ATP independent of oxygen supply. PK is a tetrameric enzyme encoded by four isozymes (L, R, M1, and M2) that differ in their kinetic properties and tissue expression distribution. The R and the L isoforms are encoded by the same gene but expressed under the control of different tissue promoters, leading to type L expression in tissues with gluconeogenesis, such as the liver, kidney, and small intestine, and type R expression in erythrocytes. The M1 isoform (PKM1) is expressed in muscle and brain, and the M2 isoform, differing only by the differential splicing of one exon, is expressed during embryogenesis, in adipose tissue and pancreatic islets, and is the predominant form found in cancer cells. PKM1 has high affinity for PEP, is not allosterically regulated, and is constitutively active in a tetrameric state. In contrast, PKM2 is allosterically regulated by metabolic intermediates such as fructose-1,6-bisphosphate (FBP) and exists either as a dimer with low affinity for PEP, or...