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EVI1 triggers metabolic reprogramming
targets such as CD52 and GPR56 for the treatment of patients with EVI1high leukemia; however, these potential targets did not prove effective in practice.10,11
Dysregulation of metabolic pathways occurs through aberrant expression of transcription factors such as MYC.12,13 Transcriptional changes resulting from aberrant- ly activated MYC lead to increased glucose uptake and glycolytic activity; it also stimulates glutaminolysis and lipid biosynthesis during leukemogenesis. It is, however, still not known whether MLL positively regulates meta- bolic pathways. Therefore, we focused on metabolic reprogramming of MF9 AML with high EVI1 expression (EVI1+MF9 AML). A recent study shows that EVI1 alters cellular metabolism and increases activity of CKMT1, which affects mitochondrial respiration and ATP produc- tion.14 However, further studies are needed to resolve the metabolic relationship between MLL and EVI1.15,16
Here, we developed transgenic (TG) mice with high expression of Evi1 in hematopoietic progenitor cells and established a mouse model of EVI1+MF9 AML. We then examined metabolic changes in these cells during leukemia development using an XFp extracellular flux ana- lyzer. We found that the leukemia cells showed accelerat- ed oxidative phosphorylation (OXPHOS) prior to activa- tion of glycolysis, along with greater dependency on glut- amine as an energy source. In addition, treatment with L- asparaginase (L-asp) effectively inhibited proliferation of Evi1+MF9 AML cells by suppressing OXPHOS. Therefore, although patients with EVI1+MF9 AML usually have a poor prognosis, treatment with specific inhibitors of ener- gy metabolic reprogramming induced by EVI1 (e.g. L-asp) may improve their outcomes.
Methods
TG mice
The +24 mCNE-mhsp68p vector was kindly provided by Dr. Motomi Osato (Kumamoto University, Kumamoto, Japan).17 +24mCNE is a stem cell-specific enhancer of Runx1, which is located +24 Kb downstream from the transcription start site in mice. Therefore, it is also called eR1(+24m) and is referred to here- after as eR1. The mEvi1 transgenic construct was engineered by introducing mouse Evi1 cDNA into the EcoR1 site of the eR1- mhsp68p vector. A 7.67 kb Sap1/BsaA1 fragment of eR1-mhsp68p mEvi1 was then microinjected into fertilized mouse eggs (C57BL/6), and TG offspring were screened by polymerase chain reaction (PCR) using the following primers: 5′-ggc cac cat ggc gta tta gg-3′ and 5′-tct tcc agc gga tag aat gg-3′. TG mice were back- crossed onto a C57BL/6 background and maintained as heterozy- gotes. All procedures were approved by the University of Miyazaki Animal Care and Use Committee.
Retroviral bone marrow transduction assays
The pMIG-FLAG-MLL-AF9 plasmid was a kind gift from Daisuke Nakada (Addgene plasmid # 71443).18 Plat-E cells were transiently transfected with MSCV vectors harboring the pCL-Eco plasmid using polyethylenimine (PEI). At four days post injection of wild-type (WT) and Evi1-TG mice with 5-fluorouracil (5-FU) (150 mg/kg; Kyowa Kirin), bone marrow (BM) cells were harvest- ed and incubated for 24 hours (h) in X-Vivo 15 (Lonza, Allendale, NJ, USA) supplemented with 50 ng/mL SCF, 50 ng/mL TPO, 10 ng/ mL IL-3, and 10 ng/ mL IL-6 (all from Peprotech, Rocky Hill, NJ, USA). After incubation, cells in retronectin (Clontech, Mountain View, CA, USA)-coated plates were spin-infected
[490 g for 45 minutes (min) at 20°C] with retroviral supernatant supplemented with polybrene (8 mg/mL). BM cells (500,000) were then transplanted into lethally irradiated C57BL/6 mice. For sec- ondary transplantations, GFP+ cells from primary recipient mice were transplanted into sublethally irradiated recipients.
Measurement of oxygen consumption rate
Oxygen consumption rate (OCR) was measured using an XFp extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). Leukemia cells were suspended in XF Assay Medium sup- plemented with 10 mM glucose, 1 mM pyruvate, and 2 mM glut- amine. Cells (200,000 per well) were seeded in a culture plate pre- coated with Cell-Tak (Fisher Scientific). The plate was centrifuged and left to equilibrate for 60 min in a CO2-free incubator before being transferred to the XFp extracellular flux analyzer. The Mito Stress Test was performed as follows: 1) basal respiration was measured in XF Base Medium (1 mM sodium pyruvate, 10 mM glucose, 2 mM glutamine); 2) oligomycin (1 mM) was injected to measure respiration linked to ATP production; 3) the uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 mM) was added to measure maximal respiration; and 4) rotenone and antimycin A (0.5 mM each) were applied together to measure non-mitochondrial respiration. Measurement of fuel dependency and fuel flexibility was performed according to the manufacturer’s Mito fuel flex test kit protocol (Agilent Technologies).
Statistical analysis
Statistical analysis was performed using a paired Student’s t-test, the Mann-Whitney U test, or the log-rank test as appropri- ate. Calculations were performed using Prism software (GraphPad). Group data are expressed as the mean±standard devi- ation. No mice were excluded from data analysis.
Results
EVI1 induces aggressive MLL-AF9 leukemia to activate mitochondrial respiration
Initially, we created a series of TG mice with high Evi1 expression (Evi1-TG) in lineagelowSca-1+c-kit+ (LSK) and granulocyte/macrophage progenitor (GMP) cells (Figure 1A) under the control of the eR1 enhancer, which shows hematopoietic-specific enhancer activity in both zebrafish and mouse models (Online Supplementary Figure S1A).17 There were no differences in blood cell counts or the per- centage of normal hematopoietic progenitors including hematopoietic stem cells (HSC), multipotent progenitors (MPP), and GMP between WT and Evi1-TG mice (Online Supplementary Figure S1B and C). Evi1-TG mice do not develop leukemia and hematopoietic disorders until at least 16 weeks.
To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used a murine model in which AML is driven by the MF9 oncogene.19,20 Murine hematopoietic progenitor cells from WT and Evi1- TG were transduced with retrovirus encoding both MF9 and GFP, and serial colony forming assays were per- formed. BM cells showing MF9 expression were trans- planted into irradiated syngeneic mice (Figure 1B). The colony assay revealed that the colony numbers and per- centages of GFP+ AML cells from Evi1-TG were higher than those from WT in vitro (Figure 1C and D). After estab- lishing primary MF9-induced AML in WT or Evi1-TG (WT/MF9 or Evi1/MF9) mice, we noted significantly high- er expression of Evi1 in GFP+ whole leukemia cells and in
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