1. Supplementary Figure 1
Oligomycin,
1µM
FCCP,
Rotonone ,1µM Antimycin A, 1µM
Figure S1. Oxygen consumption rate (OCR) and basal extracellular acidification rate (ECAR) of BaF3 and BaF3/ITD cells monitored with a Seahorse XF24 analyzer as described in Materials and Methods.

2. Supplementary Figure 2 a b c ** BaF3/ITD BaF3
Akt
β-Actin
Akt siRNA
Scr siRNA
Control
BaF3
Figure S2. (a) Glycolytic activity in BaF3 and BaF3/ITD cells after treatment with sorafenib (18 hrs), as measured by glucose uptake, lactate and ATP production levels. . (b-c) Glycolytic activity in BaF3/ITD cells after treatment with LY (20μM) or knockdown of Akt by siRNA. Non-targeting Scramble siRNA (Scr) was used as a negative control. All data were shown as means ± SD of three experiments; **, P<0.01.

3. Supplementary Figure 3
Figure S3. MAPK/ERK kinase inhibitor caused minimum inhibition of glucose uptake, lactate and ATP production in BaF3/ITD cells (5 and 10μM for 24 hrs). All data were shown as means ± SD of three experiments; * P<0.05. .

4. Supplementary Figure 4 a b
Figure S4. (a) Comparison of mitochondrial transmembrane potential in BaF3 and BaF3/ITD cells after treatment of 20μM 3-BrOP at various time points, as measured by a potential-sensitive probe Rhodamine-123 (numbers indicate subpopulation of cells that lost transmembrane potential). (b) Comparison of cell death in BaF3 and BaF3/ITD cells induced by treatment of 3-BrOP for 18 hrs. Cell death was measured by annexin-V/PI assay (numbers indicate subpopulation of cell death).

5. Supplementary Figure 5
Figure S5. Comparison of cell death in BaF3 and BaF3/ITD cells induced by 1 μM sorafenib, 7.5 μM 3-BrOP (Br), or their combination sequentially as indicated (numbers indicate subpopulation of cell death).

6. Supplementary Figure 6 a b
Control
3-Brop b
Figure S6. (a) Glucose uptake and ATP levels in HL60 and MV4-11 cells before and after treatment of 20μM 3-BrOP for 3 hrs. (b) Comparison of cell death in human leukemia cells with FLT3 wild type (FLT3/WT, HL60 and ML-1) and FLT3/ITD mutation (MOLM-13 and MV4-11) induced by treatment of 3-BrOP for 18 hrs. Cell death was measured by annexin-V/PI assay (numbers indicate cell death percentage).

7. Supplementary Figure 7
Figure S7. Comparison of cell death in FLT3 wild type (HL60, ML-1) and FLT3/ITD mutated (MOLM13, MV4-11) leukemia cells induced by 1 μM sorafenib, 7.5 μM 3-BrOP (Br), or their combination sequentially as indicated (numbers indicate subpopulation of cell death).

8. Supplementary Figure 8 a b 2-DG, 24h
4% % % %
4% % % %
Control mM mM mM
BaF3
ITD
Figure S8. (a) Cell proliferation inhibited by 2-DG in BaF3 and BaF3/ITD cells was measured by 72 hr MTT assay. (b) Comparison of cell death in BaF3 and BaF3/ITD cells induced by treatment of 2-DG for 24 hrs. Cell death was measured by annexin-V/PI assay (numbers indicate subpopulation of cell death).

9. Supplementary Figure 9
Ctrl DG 2.5 mM DG 5 mM DG 7.5 mM 2-DG 10 mM
MV4-11 (FLT3/ITD)
Sorafenib 10 nM
Sorafenib + 2-DG
HL-60 (FLT3/WT)
Sorafenib 10 nM
Sorafenib + 2-DG
ML-1 (FLT3/WT)
Sorafenib 10 nM
Sorafenib + 2-DG
Figure S9. Comparison of cell death in FLT3 wild type (FLT3/WT: HL60, ML-1) and FLT3/ITD mutated (MV4-11) leukemia cells induced by 10 nM sorafenib, 2.5, 5, 7.5 and 10 mM 2-DG, or their combination for 24 hrs (numbers in red indicate subpopulation of cell death of MV4-11 cells; numbers in black indicate subpopulation of viable cells of HL60 and ML-1).

10. Supplementary Figure 10 a b
Figure S10. Glycolytic inhibitor 2-DG enhances the therapeutic effect of sorafenib in primary leukemia cells from AML patients with FLT3/ITD. (a) Quantitative comparison of percentage of viable cells (annexin-V/PI assay) after treatment with various concentrations of 2-DG for 24 hrs in AML patients with wild type (FLT3/WT) (n=5) and FL3/ITD (n=3). Data were presented as means ± SEM. *, P < 0.05; **, P < (b) Primary leukemia cells isolated from FLT3/ITD or FLT3/wild type (FLT3/WT) AML patients were treated with 1 μM sorafenib for 42 hrs, 2.5 mM 2-DG for 24 hrs, or their combination sequentially (sorafenib for 18 hrs followed by 2-DG for 24 hrs). The percentage of cell death was determined by annexin-V/PI assay. Values represent mean percentage of cell death population ± SEM. (FLT3/ITD, n=3; FLT3/WT, n=5). NS, non-significant.