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EVI1 triggers metabolic reprogramming
the leukemic granulocyte/macrophage progenitor (L-GMP) fraction from Evi1/MF9-recipients (Figure 1E). After the first round of transplantation of BM cells from WT/MF9 or Evi1/MF9, recipient mice receiving Evi1/MF9 cells survived for a significantly shorter time than mice transplanted with WT/MF9 cells (Figure 1F). After a sec- ond round of transplantation with 104 or 103 leukemia cells isolated from primary recipient mice, the survival of sec- ondary recipient mice transplanted with Evi1/MF9 cells was significantly shorter than that of those transplanted with WT/MF9 (Figure 1G). Recipients of Evi1/MF9 cells harbored an increased total number of blast cells in peripheral blood and in the L-GMP fraction in the BM, although there was no difference between these mice and WT/MF9 mice with respect to the total number of GFP+ cells in the BM (Figure 1H-K). A recent report shows that mitochondrial respiration in leukemic stem and progenitor cells is higher than that in differentiated leukemia cells.21,22 Therefore, to examine the metabolic advantages conferred by Evi1/MF9 AML cells, we measured the mitochondrial OCR of the stem cell-enriched c-kit-positive (c-kit+) and differentiated c-kit-negative (c-kit–) fractions from WT/MF9 or Evi1/MF9 mice. The basal and maximum OCR of c-kit+ cells from WT/MF9 mice was higher than that of c-kit– cells (Figure 1L). However, there was no dif- ference in the OCR between c-kit+ and c-kit– cells from Evi1/MF9 mice (Figure 1M). These data indicate that EVI1 activates mitochondrial respiration in differentiated leukemia cells as efficiently as in the stem cell-enriched fraction, thereby providing a metabolic advantage.
Oxidative phosphorylation is activated before enhanced glycolysis during development of EVI1+ MLL-AF9 leukemia
To examine the potential role of EVI1 during develop- ment of MF9 leukemia, we measured mitochondrial func- tion (i.e. OCR) and glycolytic activity (i.e. ECAR) of seri- ally replated colonies of WT/MF9 and Evi1/MF9 leukemia cells at different replating times using a XFp extracellular flux analyzer. Basal OCR and maximal respiratory capaci- ty in normal c-kit+ hematopoietic progenitor cells from Evi1-TG mice were lower than in those from WT mice (Figure 2A). However, after the second round of plating, the OCR of Evi1/MF9 progenitor cells at each replating time was higher than that of WT/MF9 cells (Figure 2B-E). To clarify whether mitochondrial respiration is activated by the metabolic environment in BM, we measured OCR in ex vivo WT/MF9 and Evi1/MF9 leukemia cells isolated from the BM of leukemic mice. Evi1/MF9 mice showed a significantly higher basal and maximal respiratory capaci- ty of OCR than WT/MF9 mice (Figure 2F). Of note, the glycolytic activity of Evi1/MF9 cells increased after the 3rd plating (Figure 2G); in addition, there was no difference between ex vivo ECAR of WT/MF9 leukemia cells and that of Evi1/MF9 cells (Figure 2H). These data suggest that EVI1 activates both OXPHOS and glycolysis; therefore, mitochondrial respiration may play an important role in development of MF9 leukemia and correlate with biologi- cal aggressiveness.
EVI1 expression is associated with metabolic reprograming
To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-flight mass spectrometry-based metabolome profiling of
WT/MF9 and Evi1/MF9 leukemia cells.23 We found signif- icant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. The amounts of F1,6P and lactate in Evi1/MF9 leukemia cells were higher, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in Evi1/MF9 cells (Figure 3A). To identify genes co- expressed with EVI1, we performed DNA microarray analysis of GFP+ whole leukemia cells from WT/MF9 and Evi1/MF9 mice (Figure 3B and Online Supplementary Figure S2) (GSE118096), and compared the results with those for EVI1-over-expressing normal Linlow BM cells (GSE34729).14
Expression profiling of Evi1-transduced normal BM cells14 and MF9 leukemia cells identified upregulation of Idh2 and downregulation of Idh1 in both (Figure 3B and Online Supplementary Figure S2); the expression of Idh1 and Idh2 was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) (Figure 3C). Upregulation of Idh2 may induce increased production of metabolites such as fumarate and malate (metabolites of the TCA cycle). In addition, qRT-PCR revealed increased expression of genes involved in glycolysis including Glut-1 in whole AML cells (Figure 3C and Online Supplementary Figure S3). Collectively, these data indicate that EVI1 induces changes in glucose metabolism and the TCA cycle.
EVI1 activates oxidative phosphorylation via glutaminolysis
To determine how EVI1 regulates mitochondrial metab- olism, we measured mitochondrial mass by flow cytome- try after staining with MitoTracker Deep Red FM. We also measured mitochondrial superoxide production by stain- ing cells with CellROX or MitoSOX. The mitochondrial mass in Evi1/MF9 cells was equivalent to that in WT/MF9 cells (Figure 4A); however, reactive ocygen species (ROS) levels and mitochondrial superoxide production were sig- nificantly higher in Evi1/MF9 cells (Figure 4B and C), sug- gesting that EVI1 expression may facilitate accumulation of ROS. Next, to determine which glycolysis and OXPHOS pathways are important for energy production in Evi1/MF9 cells, we examined the OCR/ECAR ratio in two MF9 murine leukemia lines and various human AML cell lines under conditions of basal respiration. Overall, a higher OCR/ECAR ratio was observed in Evi1/MF9 and EVI1high AML cell lines than in WT/MF9 and EVI1low AML cell lines, suggesting that energy production in EVI1+ leukemia cells is mainly dependent on oxidative phospho- rylation (Figure 4D and E). To measure the dependency, capacity, and flexibility of leukemia cells to oxidize three major mitochondrial fuels (glutamine, long-chain fatty acids and glucose), we used the XF Mito Fuel Flex Test to examine mitochondrial respiration in MF9 cells in the presence or absence of a specific inhibitor of each fuel pathway. Compared with those in WT/MF9 cells, mito- chondria in Evi1/MF9 cells were more dependent on glut- amine than on glucose and fatty acids (Figure 4F). Moreover, when Evi1/MF9 or WT/MF9 cells were cul- tured in the presence of different low concentrations of glucose or glutamine, Evi1/MF9 cells survived significantly longer in the presence of <0.1 g/L glucose (Figure 4G); however, survival decreased significantly when cultured in the presence of <0.2 mM glutamine (Figure 4H), sug- gesting that survival of Evi1/MF9 cells is very dependent on the glutamine concentration.
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