The Warburg effect describes a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose even in the presence of oxygen. To better understand how tyrosine kinase signaling, which is commonly increased in tumors, regulates the Warburg effect, we performed phosphoproteomic studies. We found that oncogenic forms of fibroblast growth factor receptor type 1 inhibit the pyruvate kinase M2 (PKM2) isoform by direct phosphorylation of PKM2 tyrosine residue 105 (Y 105 ). This inhibits the formation of active, tetrameric PKM2 by disrupting binding of the PKM2 cofactor fructose-1,6-bisphosphate. Furthermore, we found that phosphorylation of PKM2 Y 105 is common in human cancers. The presence of a PKM2 mutant in which phenylalanine is substituted for Y 105 (Y105F) in cancer cells leads to decreased cell proliferation under hypoxic conditions, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenografts in nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth.

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SUMMARY Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate, and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.

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We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. (Blood. 2006;108:3477-3483)