Fig 2: ME1 and IDH2 synergistically inhibit cancer growth and metastasis.A. Tumor volume progression in mice subcutaneously xenografted with A549 cells treated as indicated (n = 6). siME1s (5nmol/animal) were injected intratumorally every other day and AGI-6780 (60 mg/kg) was administrated orally everyday.B. Tumor weights in mice subcutaneously xenografted with A549 cells treated as indicated (n = 6).C. Photograph and size comparison of excised tumors (n = 6).D. Representative ME1, Ki-67 immunostaining and TUNEL in xenografted tumors (n = 6). Scale bar, 100 μm.E. Representative immunohistochemical staining of IDH2 in xenograft tumors with in vivo ME1 knockdown (n = 6). Scale bar, 100 μm.F. Bioluminescence images of mice treated as indicated were obtained on day 60. AGI-6780 (60 mg/kg) was administrated orally everyday.G. Left, representative images of metastatic nodules on the surface of lungs in nude mice. Right, metastatic nodules were counted and recorded with or without a microscope (n = 6).H. Representative images of H&E-stained lung sections (n = 6). Scale bar, 200 μm.Data are represented as mean ± SD. Statistical significance was determined by Student's t-test. **p < 0.01, ***p < 0.001.

Fig 3: The AMPK-FoxO1 axis underlies adaptive IDH2 upregulation.A. Immunoblot analysis of FoxO1, p-AMPK and AMPK expression in MCF-7 and A549 cells with control transfection or ME1 knockdown. β-Tubulin was used as the loading control.B. Immunoblot analysis of p-AMPK, AMPK, FoxO1 and IDH2 in MCF-7 cells treated with AICAR (1 mM), compound C (5 μM) or both for 24 h. β-Tubulin was used as the loading control.C. Immunoblot analysis of p-AMPK, AMPK, FoxO1 and IDH2 in MCF-7 cells treated with AICAR (1 mM), AS1842856 (0.2 μM) or both for 24 h. β-Tubulin was used as the loading control.D. Immunoblot analysis of FoxO1 and IDH2 in MCF-7 cells with control transfection or ME1 knockdown and treated with or without AS1842856 (0.2 μM) for 24 h. β-Tubulin was used as the loading control.E. Dual-luciferase reporter assay in MCF-7 cells with control transfection or ME1 knockdown (n = 3).F. Dual-luciferase reporter assay in MCF-7 cells treated with AICAR (1 mM), compound C (5 μM) or both for 24 h (n = 3).G. Dual-luciferase reporter assay in MCF-7 cells treated with AICAR (1 mM), AS1842856 (0.2 μM) or both for 24 h (n = 3).H. The pathway through which IDH2 is activated by ME1 knockdown in a manner dependent on AMPK-FoxO1 can contribute to the replenishment of the cytosolic NADPH pool to reduce redox stress and support lipogenesis.Data are represented as mean ± SD. The data represent at least two independent experiments. Statistical significance was determined by Student's t-test. ***p < 0.001. See also Supplementary Fig. 5.

Fig 4: ME1 and IDH2 synergistically induce cancer cell apoptosis.A. Effect of IDH2 inhibition by 2.5 μM AGI-6780 for 24 h, ME1 knockdown and the combined treatment on intracellular ROS levels in MCF-7 and A549 cells (n = 3).B. Effect of IDH2 inhibition by 2.5 μM AGI-6780 for 24 h, ME1 knockdown and the combined treatment on labeled fatty acid abundance levels in MCF-7 and A549 cells cultured in medium containing 10 mM [U-13C] glucose for 72 h (n = 4).C. Effect of IDH2 inhibition by 2.5 μM AGI-6780 for 24 h, ME1 knockdown and the combined treatment on apoptosis rate in MCF-7 and A549 cells (n = 3).D. Intracellular ROS levels in MCF-7 and A549 cells after ME1 knockdown and dual targeting of ME1 and IDH2 in culture with or without 10 mM NAC treatment for 24 h (n = 3).E. Relative viability of MCF-7 and A549 cells after ME1 knockdown and dual targeting of ME1 and IDH2 in culture with or without 10 mM NAC treatment for 24 h (n = 5).F. Effect of combinatorial targeting ME1 and CTP on relative cell viability of MCF-7 and A549 cells (n = 6).Data are represented as mean ± SD. The experiments in B were conducted once, and all the other experiments were repeated at least twice. Statistical significance was determined by Student's t-test. **p < 0.01, ***p < 0.001. See also Supplementary Fig. 6.

Fig 5: Enzymatic activities of IDH (a), COX (b), and ATPase (c). The data are presented as means ± SD. ANOVA analysis: two-way (n = 11 per group): S, stage effect; one-way (n = 11 per group): * difference between stage III and stage IV.