Gu TL, Goss VL, Reeves C, Popova L, Nardone J, Macneill J et al.. Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia. Blood 108: 4202-4204

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Blood (Impact Factor: 10.45). 01/2007; 108(13):4202-4. DOI: 10.1182/blood-2006-06-026666
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The 8p11 myeloproliferative syndrome (EMS) is associated with translocations that disrupt the FGFR1 gene. To date, 8 fusion partners of FGFR1 have been identified. However, no primary leukemia cell lines were identified that contain any of these fusions. Here, we screened more than 40 acute myeloid leukemia cell lines for constitutive phosphorylation of STAT5 and applied an immunoaffinity profiling strategy to identify tyrosine-phosphorylated proteins in the KG-1 cell line. Mass spectrometry analysis of KG-1 cells revealed aberrant tyrosine phosphorylation of FGFR1. Subsequent analysis led to the identification of a fusion of the FGFR1OP2 gene to the FGFR1 gene. Small interfering RNA (siRNA) against FGFR1 specifically inhibited the growth and induced apoptosis of KG-1 cells. Thus, the KG-1 cell line provides an in vitro model for the study of FGFR1 fusions associated with leukemia and for the analysis of small molecule inhibitors against FGFR1 fusions.

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    • "Interestingly, immunoblotting results show that FGFR1 as a receptor tyrosine kinase is co-localized with PDHK1 and its substrate PDHA1 in mitochondria (Figure 1D right). Moreover, TKI258 treatment significantly decreased phosphorylation levels of PDHA1 at S293 in human myeloid leukemia KG1a cells harboring a FOP2-FGFR1 fusion protein (Gu et al., 2006) (Figure 1E; left) and lung cancer NCI-H1299 cells overexpressing FGFR1 (Marek et al., 2009) (Figure 1E; right). In consonance with this, targeting PDHK1 by a PDHK inhibitor, dichloroacetate (DCA), or shRNA results in decreased S293 phosphorylation levels of PDHA1 in FGFR1-expressing cancer cells (Figure 1F). "
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    ABSTRACT: 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 the 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.
    Molecular cell 12/2011; 44(6):864-77. DOI:10.1016/j.molcel.2011.10.015 · 14.02 Impact Factor
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    • "STAT3-decoy ODN also induced cell death of MCF-7 cells, in which a low but detectable STAT3 activation has been previously observed [42]. However, the STAT3-decoy ODN had no effect on the acute myeloid leukemia cell line KG1, in which STAT5, rather than STAT3, is activated [43] (Figure 4A). The mutated STAT3-decoy ODN had no effect in any of the three cell lines (Figure 4A). "
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    ABSTRACT: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.
    BMC Cell Biology 04/2011; 12(1):14. DOI:10.1186/1471-2121-12-14 · 2.34 Impact Factor
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    • "Compared to IMAC, PhosphoScan should better dissect phosphorylation profiles, specifically identifying tyrosine-phosphorylated peptides. This approach has already been successfully used in the phospho-profiling of primary [23] and metastatic [24] lung cancer, in acute leukemia samples [25], in Bcr/Abl positive cell lines [26], AML cell lines [27] and Hodgkin lymphoma cell lines [28]. Since tyrosine phosphorylation regulates the activity of many proteins, the PhosphoScan approach can provide a picture of the most active pathways in the analyzed samples. "
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    ABSTRACT: Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.
    03/2011; 34(2):141-53. DOI:10.1007/s13402-011-0019-7
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