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EVI1 triggers metabolic reprogramming
expression of Idh1/Idh2 is functionally linked to the increased OXPHOS levels observed in Evi1/MF9 cells, we measured OXPHOS in Evi1/MF9 cells transfected with shIdh1 or shIdh2. Evi1/MF9-shIdh2 cells showed marked- ly lower levels of OXPHOS than shScr- and shIdh1-trans- fected cells (Online Supplementary Figure S4D). This sug- gests that upregulation of Idh2 via EVI1 may increase OXPHOS activity, which is important for energy produc- tion in Evi1/MF9 cells.
L-asp inhibits EVI1-mediated oxidative phosphorylation activity
To develop a novel strategy for treatment of Evi1/MF9 cells, we tested the effect of specific inhibitors of glycoly- sis by 2-deoxyglucose (2-DG) and STF31 (a GLUT-1 inhibitor) and glutaminolysis by BPTES (a glutamine syn- thetase inhibitor); L-asparaginase (L-asp) is an enzyme that catalyzes hydrolysis of asparagine and glutamine.
Evi1/MF9 cells were more sensitive to glycolysis and glutaminolysis inhibitors than WT leukemia cells (Figure 5A). Moreover, treatment with L-asp led to marked sup- pression of Evi1/MF9 cell growth (Figure 5B). Analysis of several human AML cells revealed that the IC50 of L-asp against three EVI1high AML cell lines and two primary EVI1+ AML cells was lower than that against EVI1low AML cell lines and two primary EVI1- AML cells (Figure 5C and D). To confirm whether L-asp treatment affects mitochon- drial respiration, we used the XFp extracellular flux ana- lyzer to measure energy metabolism by murine Evi1/MF9 and human EVI1+ AML cells after L-asp treatment. The basal and maximum OCR values for mitochondria in L-asp-treated Evi1/MF9 cells and human EVI1+AML cells were markedly lower than in non-treated cells, suggesting that EVI1+ AML cells are more sensitive to L-asp (Figure 5E). WT/MF9 and Evi1/MF9 do not express surface mark- ers for B- and T-lymphoid cells (Online Supplementary Figure S5A). Addition of L-asp led to a marked reduction in the OCR of normal progenitor cells in Evi1-TG mice (Online Supplementary Figure S5B); however, there was no difference in the IC50 between WT and Evi1-TG mice (Online Supplementary Figure S5C). Some metabolic path- ways may play a role in normal BM progenitor cells but not in leukemia cells. To examine whether suppression of OCR by L-asp depends on glutamine depletion, we stud- ied the effects of high glutamine concentration on the OCR of cultured cells. In WT/MF9, L-asp treatment showed no OCR-suppressing effect, which was not affected by glutamine concentration. Meanwhile, in Evi1/MF9, high concentration of glutamine attenuated the OCR-suppressing effect (Online Supplementary Figure S5D). Taken together, these results indicate that L-asp treatment suppresses growth of EVI1+ AML cells by blocking EVI1- mediated OXPHOS activity.
To examine the therapeutic potential of L-asp in vivo, recipient mice transplanted secondarily with Evi1/MF9 AML cells were treated with L-asp via intraperitoneal injection beginning on day 5 post transplantation; L-asp (1,000 U/kg) was injected five times per week for four weeks (Figure 6A). L-asp treatment led to a significant reduction in the number of GFP+ AML cells in the periph- eral blood (Figure 6B) and extended the survival time of recipient mice (Figure 6C). Although L-asp treatment reduced the total number of leukemia cells in the BM, there was no increase in the number of L-GMP cells in the BM (Figure 6D). Moreover, the basal and maximum OCR
in L-asp-treated BM cells were significantly lower than those in untreated cells (Figure 6E). By contrast, L-asp treatment did not reduce the number of leukemia cells in WT/MF9 mice and did not prolong their survival (Online Supplementary Figure S6A-C). Next, we examined the effect of L-asp treatment in a subcutaneous xenograft model of human AML. Immunodeficient NOG mice were injected with UCSD/AML1 cells and treated with L-asp (Figure 6F). L-asp treatment suppressed the growth of human EVI1high AML tumors effectively (Figure 6G-I). Overall, these findings indicate that inhibition of OXPHOS by L-asp is a potential effective treatment for EVI1high AML.
Low expression of ASNS by MLL-AF9 leukemia cells is associated with high sensitivity to L-asp
High sensitivity to L-asp is due to active glutaminolysis and low expression of asparagine synthetase (Asns). Therefore, we examined expression of the glutamine transporter (Slc1a5) and Asns in MF9 cells. Expression of Asns by Evi1/MF9 cells was significantly lower than that by WT/MF9 cells (Figure 7A). By contrast, expression of Slc1a5 by Evi1/MF9 cells was significantly higher than that by WT/MF9 cells (Figure 7B). WT/MF9 cells trans- fected with shAsns showed a lower IC50 for L-asp (Online Supplementary Figure S7). These findings suggest that sen- sitivity to L-asp correlates with ASNS expression in MF9 leukemia.
Next, we examined expression of IDH2, ASNS, and SLC1A5 in human leukemia cell lines. L-asp-sensitive EVI1high AML cell lines and two primary EVI1+ AML cells showed low expression of ASNS (Online Supplementary Figure S8A and B).
Finally, to determine whether these genes are clinically useful markers for predicting L-asp sensitivity, we exam- ined their expression in leukemia samples from patients in JPLSG AML-05.7 In the MLL-r AML cohort (n=48), there was no significant difference in ASNS expression between EVI1– and EVI1+ AML (Figure 7C). However, expression of ASNS in MF9 AML was significantly lower than that in non-MF9 AML (Figure 7D). Moreover, expression of ASNS in EVI1+MF9 AML (n=11) was significantly lower than that in those with EVI1-MF9 (n=17); however, EVI1 expression did not affect ASNS expression in non-MF9 AML (n=20) (Figure 7E and F). We found no significant dif- ference between EVI1+ and EVI1– MF9 AML patients with respect to expression of IDH2 and SLC1A5 (Figure 7G and H). These findings suggest that lower expression of ASNS in EVI1+MF9 AML is associated with higher sensitivity to L-asp.
Discussion
Previously, it was thought that activation of glycolysis was responsible primarily for metabolic reprogramming at onset of leukemia. In addition, clones that promote the survival of BM cells are produced when genes involved in onset of leukemia directly activate glycolytic metabolism.24-27 However, recent studies report a direct connection between mitochondrial metabolic activity and refractory AML; this is because: (i) production of adeno- sine triphosphate (ATP) by mitochondria is higher in leukemia stem cells than in differentiated blast cells or nor- mal HSC; (ii) chemotherapy-resistance of AML correlates
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