Kazuto Suganuma

Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation.

For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC50: 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a “glycolytic” leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (−) medium showed more growth and survival than glucose (−) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC50: 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (−) FCS (+) medium showed more growth and survival than glucose (+) FCS (−) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid β-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.

Growth inhibition of AML cells with specific chromosome abnormalities by monoclonal antibodies to receptors for vascular endothelial growth factor

By using neutralizing monoclonal antibodies to vascular endothelial growth factor receptor type 1 (VEGFR1) and VEGFR2, we have shown that acute myelogenous leukemia (AML) cells with specific chromosome abnormalities are dependent on VEGF/VEGFR system. AML with t(8;21) is the most dependent subtype on VEGF with both VEGFR1 and VEGFR2. t(15;17)AML cells depend on VEGF with VEGFR1. AML cells with 11q23 abnormalities showed variable dependence on VEGF. The growth of t(11;19)AML cells are most extensively inhibited by anti-VEGFR1 antibody. Then, the growth of Kasumi-1, a t(8;21) cell line was suppressed by either anti-VEGFR1 antibody (p=0.0022) or anti-VEGFR2 antibody (p=0.0029) in a dose-dependent manner. The growth of NB4, a t(15;17) cell line was more potently suppressed by anti-VEGFR1 antibody (p=0.0111) than by anti-VEGFR2 antibody (p=0.0477). These results are quite concordant with the results of clinical samples with t(8;21) or t(15;17). In addition, anti-VEGFR2 monoclonal antibody significantly potentiated the growth inhibitory effect of idarubicin for Kasumi-1. As for downstream signals, we have shown that VEGFR2 transduce growth and survival signals through phosphorylation of Akt and MEK in leukemia cells (Kasumi-1). However, VEGFR1 transduce growth and survival signals through pathways other than MEK and Akt (NB4), although Akt phosphorylation may account for some of the VEGFR1 signals (Kasumi-1). Finally, our data suggested that autocrine pathway of VEGF and VEGFRs observed in AML cells with specific chromosomal translocations have contributed to leukemogenesis as activated signaling of receptor tyrosine kinase.

t(8;21) acute myeloid leukaemia cells are dependent on vascular endothelial growth factor (VEGF)/VEGF receptor type2 pathway and phosphorylation of Akt.

Several anti‐angiogenic drugs have recently been clinically tested for haematological malignancies. To improve the efficacy of molecular target therapy against angiogenic molecules in acute myeloid leukaemia (AML), we examined the dependency of AML cells on the vascular endothelial growth factor (VEGF)/VEGF receptor type2 (VEGFR2) system by using VEGFR2 kinase inhibitor. Nineteen patient AML samples were cultured with or without VEGFR2 kinase inhibitor. All four t(8;21) viable AML cells showed significant reductions when treated with VEGFR2 kinase inhibitor, although VEGFR2 kinase inhibitor did not affect the cell proliferation of five t(15;17) AML samples. Other AML cases showed variable responses. VEGFR2 kinase inhibitor greatly suppressed the growth of Kasumi‐1, a t(8;21) cell line in a dose‐dependent manner through induction of apoptosis, but did not show any significant influence on NB4, a t(15;17) cell line. In addition, VEGFR2 kinase inhibitor potentiated the growth inhibitory effect of cytarabine in Kasumi‐1. Finally, it was shown that the Akt phosphorylation was augmented by VEGF165 in Kasumi‐1, which was abrogated by VEGFR2 kinase inhibitor. NB4 showed undetectable Akt phosphorylation even with VEGF165. These data demonstrated that t(8;21) AML cells are dependent on VEGF through VEGFR2, resulting in the phosphorylation of Akt.

Importance of Glutamine Metabolism in Leukemia Cells by Energy Production Through TCA Cycle and by Redox Homeostasis

Some cancer cells depend on glutamine despite of pronounced glycolysis. We examined the glutamine metabolism in leukemia cells, and found that HL-60 cells most depended on glutamine in the 4 acute myelogenous leukemia (AML) cell lines examined: growth of HL-60 cells was most suppressed by glutamine deprivation and by inhibition of glutaminolysis, which was rescued by tricarboxylic acid (TCA) cycle intermediate, oxaloacetic acid. Glutamine is also involved in antioxidant defense function by increasing glutathione. Glutamine deprivation suppressed the glutathione content and elevated reactive oxygen species most evidently in HL-60 cells. Glutamine metabolism might be a therapeutic target in some leukemia.

Growth of xenotransplanted leukemia cells is influenced by diet nutrients and is attenuated with 2-deoxyglucose

We examined the effects of diet nutrients on xenotransplanted leukemia cells, THP-1 or NB4. THP-1 tumors showed more growth when fed with high fat diet, while NB4 tumors grew more with high carbohydrate diet. Then, administration of 2-deoxyglucose (a glycolysis inhibitor) showed a significant antitumor effect on both tumors: NB4 tumor showed large necrotic areas, while THP-1 tumor did not, but had augmented expression of enzymes for fatty acid oxidation. 2-Deoxyglucose inhibited the growth of NB4 by cell death because main energy producing pathway (glycolysis) was abolished, while 2-deoxyglucose slowed the growth of THP-1 by shifting energy metabolism to fatty acid β-oxidation.

Establishment of a novel human myeloid leukemia cell line, AMU‐AML1, carrying t(12;22)(p13;q11) without chimeric MN1‐TEL and with high expression of MN1

In this study, we established and analyzed a novel human myeloid leukemia cell line, AMU‐AML1, from a patient with acute myeloid leukemia with multilineage dysplasia before the initiation of chemotherapy. AMU‐AML1 cells were positive for CD13, CD33, CD117, and HLA‐DR by flow cytometry analysis and showed a single chromosomal abnormality, 46, XY, t(12;22)(p13;q11.2), by G‐banding and spectral karyotyping. Fluorescent in situ hybridization analysis indicated that the chromosomal breakpoint in band 12p13 was in the sequence from the 5′ untranslated region to intron 1 of TEL and that the chromosomal breakpoint in band 22q11 was in the 3′ untranslated region of MN1. The chimeric transcript and protein of MN1‐TEL could not be detected by reverse‐transcriptase polymerase chain reaction or Western blot analysis. However, the MN1 gene was amplified to three copies detected by array comparative genomic hybridization analysis, and the expression levels of the MN1 transcript and protein were high in AMU‐AML1 cells when compared with other cell lines with t(12;22)(p13;q11‐12). Our data showed that AMU‐AML1 cells contain t(12;22)(p13;q11.2) without chimeric fusion of MN1 and TEL. The AMU‐AML1 cells gained MN1 copies and had high expression levels of MN1. Thus, the AMU‐AML1 cell line is useful for studying the biological consequences of t(12;22)(p13;q11.2) lacking chimeric MN1‐TEL.