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.

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.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and frequently metastasizes to LNs. Identifying metastasis-promoting factors is of immense clinical interest, as the prognosis for patients with even a single unilateral LN metastasis is extremely poor. Here, we report that p90 ribosomal S6 kinase 2 (RSK2) promotes human HNSCC cell invasion and metastasis. We determined that RSK2 was overexpressed and activated in highly

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529, which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2, unlike Y529 phosphorylation, which facilitates ERK binding. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, as well as the subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.

3142 The Warburg effect describes a pro-oncogenic metabolic switch in which cancer cells, including leukemia cells, take up more glucose than normal tissue, yet use less glucose for oxidative phosphorylation and favor glycolysis even in the presence of oxygen (aerobic glycolysis). However, the molecular mechanisms underlying the Warburg effect remain unclear. Growth factor (GF) receptors are believed to play a key role in programming cancer cell metabolism. These GF receptors are expressed in many hematopoietic malignancies as constitutively activated tyrosine kinase mutants. Thus, we examinined whether tyrosine kinase signaling — commonly upregulated in hematopoietic malignancies — regulates the Warburg effect to contribute to leukemogenesis and disease progression. We performed phospho-proteomics studies and found that pyruvate kinase M2 isoform (PKM2), which is a rate-limiting enzyme of glycolysis, is tyrosine phosphorylated in leukemia cells expressing FGFR1 fusion tyrosine kinases, which are associated with 8p11 leukemia/lymphoma syndrome. We also found that 8p11 leukemogenic FGFR1 directly phosphorylates and inhibits PKM2. Recent seminal studies from Dr. Lew Cantley's group demonstrated that the enzymatic activity of PKM2 is inhibited by phosphotyrosine binding; PKM2 expression is important for aerobic glycolysis and provides a growth advantage to tumors. However, it remains unclear which dedicated tyrosine kinase pathways are physiologically responsible for this regulation and whether PKM2 itself is tyrosine phosphorylated to achieve inhibition of PKM2 in cancer cells. Here we report that FGFR1 inhibits PKM2 by direct phosphorylation at Y105. This consequently inhibits the formation of tetrameric, active PKM2 by disrupting cofactor fructose-1,6-bisphosphate (FBP) binding in a putative “inter-molecule manner”, where one molecule in an active PKM2 tetramer, when phosphorylated, may function as an inhibitory binding partner to the other sister molecules. In addition, phosphorylation of PKM2 at Y105 is common in many human leukemia cell lines expressing oncogenic tyrosine kinases such as BCR-ABL, FLT3-ITD, and JAK2V617F. Furthermore, expression of the PKM2 Y105F mutant in cancer cells following RNAi-mediated knockdown of endogenous PKM2 leads to decreased cell proliferation under hypoxia, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to program cancer cell metabolism and promote tumor growth. This may represent a common, acute molecular mechanism to regulate the Warburg effect, in addition to the chronic changes that are believed to be regulated by hypoxia inducible factor 1 and Myc. Disclosures: No relevant conflicts of interest to declare.