IDH2 stabilizes HIF-1α-induced metabolic reprogramming and promotes chemoresistance in urothelial cancer

Abstract

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine-resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain-of-function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif-1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine-resistant UC cells. Interestingly, IDH2-mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2-mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo-resistant urothelial carcinoma.

Keywords: chemoresistance; hypoxia-inducible factor-1α; isocitrate dehydrogenase 2; metabolomic reprogramming; urothelial carcinoma.

Figure 1. Metabolic differences among WT, CR, and GR UC cells

1. T24 and UMUC3 cisplatin‐resistant (CR) and gemcitabine‐resistant (GR) urothelial cancer (UC) cells were generated by exposing the corresponding wild‐type (WT) cells to up to 3 μM cisplatin (CDDP) or gemcitabine (GEM) over 12 or 18 months, respectively. Brief schema showing the generation of CR and GR cell lines. Metabolites, including CR and GR, were analyzed by using CE‐MS metabolomics.

2. Graphs show changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of GEM, and WST assays were performed 48 h after treatment (n = 3, biological replicates). The data are shown as the mean values ± SDs.

3. Hierarchical clustering of significantly regulated metabolites among T24 and UMUC3 cells, including WT, CR, and GR cells (n = 4, biological replicates).

4. Major metabolites altered in the glycolytic pathway in CR cells and GR cells compared with WT cells. The data are shown as the mean values ± SDs (n = 4) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

5. Bar graph showing the survival of WT, CR, and GR cells in glucose‐ and glutamine‐replete (glu⁺, gln⁺), glutamine limitation (gluΔ, gln⁺), glucose deprivation (glu⁻, gln⁻), glutamine deprivation (glu⁺, gln⁻), or glucose and glutamine deprivation (glu⁻, gln⁻) medium. The table shows the glucose and glutamine concentrations in each medium. Significant differences were observed in GR cells compared with WT and/or CR cells after 48 h. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001. N.D., non‐detectable.

6. WT and GR cells were seeded in 24‐well plates and exposed to 2‐DG and rotenone to measure baseline ECAR and baseline OCR. The data are shown as the mean values ± SDs (n = 3, biological replicates).

7. Baseline OCR/ECAR ratios in WT and GR cells (upper: T24, lower: UMUC3). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. ***P < 0.001.

8. Relative mRNA levels of GLUT1, PKM2, and LDHA, which are enzymes that regulate glycolysis. The data are shown as the mean values ± SEs (n = 3, biological replicates) and were analyzed by Student's t‐test, and the values are plotted relative to the expression levels in WT cells. *P < 0.05, **P < 0.01, ***P < 0.001.

9. Western blot analysis of GLUT1, PKM2, LDHA, and β‐actin compared among WT, CR, and GR cells.

Source data are available online for this figure.

Figure 2. GR cells increases pyrimidine biosynthesis to generate dCTP

1. Schematic of altered mechanisms in GR cells in glycolysis and the pentose phosphate pathway (PPP). Glucose is transferred from aerobic glycolysis to PPP. Glucose flux into the PPP results in increased nucleotide biosynthesis, including purine and pyrimidine biosynthesis.

2. PPP metabolite levels in T24GR and UMUC3GR cells relative to WT cells based on targeted CE‐MS metabolomics The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

3. Levels of endpoint metabolites of the purine synthesis pathway in GR cells relative to those in WT cells as determined by CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. n.s., non‐significant

4. Levels of endpoint metabolites of the pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

5. Relative mRNA expression levels of G6PD, TIGAR, TKT, and CTPS1, which are the genes responsible for activating the PPP and pyrimidine biosynthesis. WT and GR cells are shown. The data are shown as the mean values ± SEs (n = 3, biological replicates). The expression levels were analyzed by Student's t‐test and plotted relative to expression levels in WT cells (left: T24 cells, right: UMUC3 cells). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., non‐significant.

6. Western blot analysis of Hif‐1α, G6PD, TIGAR, TKT, CTPS1, and β‐actin in WT and GR cells under normoxia and hypoxia (left: T24 cells, right: UMUC3 cells).

7. Simple schematic of the glycolytic pathway and the PPP. Glucose flux into the PPP results in increased nucleotide biosynthesis, including purine and pyrimidine biosynthesis. Leflunomide inhibits pyrimidine biosynthesis.

8. The viability of WT and GR cells was assessed by WST assays under treatment with gemcitabine (Gem), leflunomide, or Gem with leflunomide. The data are shown as the mean values ± SDs (n = 3, biological replicates). Comparisons were made with respect to the corresponding controls or the indicated groups, followed by analysis with Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

9. CE‐MS‐based metabolite detection for diphosphate nucleosides in WT and GR cells. The data are shown as the mean values ± SDs (n = 3, biological replicates). The levels in GR cells are presented relative to those in WT control cells and analyzed by Student's t‐test. ***P < 0.001. N.D., non‐detectable.

10. Comparison of baseline dCTP levels in WT and GR cell lines determined by CE‐MS metabolomic analysis. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.

11. dCTP levels as determined by CE‐MS metabolomic analysis under treatment with 1 μM GEM and/or 10 μM deoxycytidine (dC) (left: T24 cells; right: UMUC3 cells). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by one‐way ANOVA with the Bonferroni test. **P ≤ 0.01, ***P ≤ 0.001. n.s., nonsignificant.

12. Effect of deoxycytidine and other nucleosides (deoxyadenosine, deoxyguanosine, and thymidine) at various concentrations with 1 μM GEM in WT cells, as evaluated by WST assays 48 h post‐treatment (upper: T24 cells; lower: UMUC3 cells). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by one‐way ANOVA with the Bonferroni test. *P ≤ 0.05, ***P ≤ 0.001. n.s., non‐significant.

Source data are available online for this figure.

Figure EV1. Stimulation of PPP is responsible for chemoresistance in GR cells

1. Relative mRNA expression levels of DHODH, UMPS, CTPS1, CTPS2, TYMS, IMPDH1, ADSL, ATIC, PFAS, and PRPS1 in WT and GR cells (upper: T24, lower: UMUC3). The data are shown as the mean values ± SEs (n = 3, biological replicates) and were analyzed by Student's t‐test, and plotted relative to expression levels in WT cells. *P ≤ 0.05, ***P ≤ 0.001. n.s., non‐significant.

2. Simple schematic of the glycolytic pathway and the pentose phosphate pathway (PPP). Glucose flux into the PPP results in increased nucleotide biosynthesis, including purine and pyrimidine biosynthesis. Mizoribine inhibits purine biosynthesis.

3. The viability of WT and GR cells was evaluated by WST assays under treatment with gemcitabine (GEM), mizoribine, or GEM with mizoribine. The data are shown as the mean values ± SDs (n = 3, biological replicates). Comparisons were made with respect to the corresponding controls or the indicated groups, followed by Student's t‐test. **P < 0.01, ***P < 0.001. n.s., non‐significant.

4. Bar graph showing the viability of cells exposed to various concentrations of GEM for 48 h and treated with DMSO (GR cells) or 10 μM dC (WT cells) (left: T24 cells; right: UMUC3 cells). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. n.s., non‐significant.

Figure 3. Reductive carboxylation produces 2‐HG to stabilize Hif‐1α

1. Levels of intermediate metabolites of the tricarboxylic acid cycle (TCA) cycle in GR cells relative to those in WT cells based on targeted CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

2. Schematic describing the details of the reductive TCA cycle. Model depicting the pathways for citrate+4 (cit+4) and cit+5 production from [¹³C₅‐] glutamine (glutamine+5). Glutamine+5 is catabolized to α‐ketogutarate+5, which can then contribute to citrate production by two divergent pathways. Normal metabolism in WT cells produces succinate+4, fumarate+4, malate+4, and oxaloacetate+4, which can condense with unlabeled acetyl‐CoA to produce citrate+4. In GR cells, altered mechanisms in the TCA cycle were observed in terms of high glutamate metabolism. The [¹³C₅]‐labeled glutamate‐derived metabolite levels are indicated in black.

3. The relative levels of metabolites contributing to citrate production through increased reductive carboxylation [glutamine (gln), glutamate (glu), α‐KG, and citrate] in T24GR cells versus WT cells. Cells were cultured for 24 h in 10 mM glutamine and then cultured for 1 h in medium supplemented with [¹³C5‐] glutamine. ¹³C enrichment in cellular citrate was quantitated by CE‐MS. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

4. ¹³C enrichment in cellular citrate determined by CE‐MS and normalized to the total pool size for the relevant metabolite (left: T24GR, right: UMUC3GR) The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.

5. ¹³C enrichment in cellular malate determined by CE‐MS and normalized to the total pool size for the relevant metabolite (left: T24GR, right: UMUC3GR) The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

6. Relative 2‐hydroxyglutarate (2‐HG) production level based on targeted CE‐MS metabolomics comparing among WT and GR cells (left panel: T24, right panel: UMUC3). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

7. Schematic describing the wild type (WT) IDH2 gain of function which mediated the reductive glutamine metabolism. 2‐HG accumulation driven by WT‐IDH2 suppresses EGLN1/PHD2 expressions to stabilize Hif‐1α expression.

8. Relative mRNA expression levels of IDH2 and EGLN1 genes compared between WT and GR cells (left panel: T24, right panel: UMUC3). The expression levels were analyzed by Student's t‐test and plotted relative to expression levels in WT cells. The data are shown as the mean values ± SEs (n = 3, biological replicates). *P < 0.05, ***P < 0.001.

9. Western blot analysis of IDH2, PHD2, and β‐actin in WT and GR cells (left panel: T24, right panel: UMUC3).

Source data are available online for this figure.

Figure EV2. Whole‐exome sequencing analysis of DNA in UC cell lines

1. Details of the whole‐exome sequencing analysis of DNA in T24WT, T24GR, UMUC3WT, and UMUC3GR cells. The vertical axis shows the genes and their genomic positions. The horizontal axis shows the tumor samples (T24WT, T24GR, UMUC3WT, and UMUC3GR).

2. Details of the alteration rates of genomic positions in each gene. The alteration rates were calculated by the following equation: alteration depth/(alteration depth + reference depth).

Figure 4. Downregulation of HIF1A and IDH2

1. Relative mRNA expression levels of HIF1A, GLUT1, G6PD, TIGAR, TKT, CTPS1, PKM2, and LDHA in cells transfected with siHIF1A#1 and #2 compared with cells transfected with siNTC (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SEs (n = 3, biological replicates) and were analyzed by Student's t‐test, and plotted relative to expression levels in GR cells transfected with siNTC. *P < 0.05, **P < 0.01, ***P < 0.001.

2. Western blot analysis of Hif‐1α, GLUT1, G6PD, TIGAR, TKT, CTPS1, PKM2, LDHA, and β‐actin after transfection with NTC and IDH2‐targeting (siHIF1A#1 and #2) siRNAs (left: T24GR, right: UMUC3GR).

3. Bar graph showing the viability of GR cells exposed to various concentrations of GEM for 48 h after transfection with NTC and HIF1A‐targeting (siHIF1A#1 and #2) siRNA (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01, ***P < 0.001.

4. Relative mRNA expression levels of IDH2, GLUT1, G6PD, TIGAR, TKT, CTPS1, PKM2, and LDHA in cells transfected with siIDH2#1 and #2 compared with cells transfected with siNTC (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SEs (n = 3, biological replicates) and were analyzed by Student's t‐test, and plotted relative to expression levels in GR cells transfected with siNTC. *P < 0.05, **P < 0.01, ***P < 0.001.

5. Western blot analysis of IDH2, PHD2, HIF‐1α, G6PD, GLUT1, TIGAR, TKT, CTPS1, PKM2, LDHA, and β‐actin after transfection with NTC and IDH2‐targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR).

6. Relative 2‐HG production level based on targeted CE‐MS metabolomics in GR cells after transfection with NTC and IDH2‐targeting (siIDH2#1 and #2) siRNA (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05. n.s. = non‐significant.

7. Relative dCTP level based on targeted CE‐MS metabolomics in GR cells after transfection with NTC and IDH2‐targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05. n.s., non‐significant.

8. Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 h after transfection with NTC and IDH2‐targeting (siIDH2#1 and #2) siRNA (left: T24GR, right: UMUC3GR) The data are shown as the mean values ± SDs (n = 3, biological replicates).

Source data are available online for this figure.

Figure EV3. IDH2 down regulation suppresses metabolic rewiring

1. The entire heat map showing intracellular levels of metabolites in T24GR cells transfected with NTC and IDH2. For each cell line, four independently derived cell lines are shown, and the levels were normalized to those in WT cells.

2. PPP metabolite levels in T24GR cells transfected with siIDH2 compared with GR cells transfected with NTC based on targeted CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

3. The intermediate metabolite levels of the TCA cycle in T24GR cells transfected with NTC and siIDH2 based on targeted CE‐MS/MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

4. Levels of endpoint metabolites of the purine and pyrimidine synthesis pathway in GR cells relative to those in WT cells as determined by CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05. n.s., non‐significant.

5. The levels of +5 metabolites and +4 metabolites, including early metabolites and late metabolites, in the TCA cycle compared between T24GR cells transfected with NTC and those transfected with siIDH2. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

6. 13C enrichment in cellular citrate and malate between T24GR cells transfected with NTC and those transfected with siIDH2 as determined by CE‐MS; the levels were normalized to the total pool size of the relevant metabolite. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

Figure 5. IDH2 overexpression recapitulate metabolic rewiring

1. Relative mRNA expression levels of IDH2, EGLN1, Hif‐1α, G6PD, TIGAR, TKT, and CTPS1 between J82 wild‐type cells (J82WT) and J82 cells with IDH2 overexpression (J82‐IDH2ox cells). The data are shown as the mean values ± SEs (n = 3, biological replicates). The expression levels were analyzed by Student's t‐test and are plotted relative to the expression levels in WT cells. *P < 0.05, **P < 0.01, ***P < 0.001.

2. Western blot analysis of IDH2, PHD2, Hif‐1α, G6PD, TIGAR, TKT, CTPS1, and β‐actin in J82WT and J82‐IDH2ox cells.

3. Hierarchical clustering of significantly regulated metabolites in J82WT and J82‐IDH2ox cells (n = 3, biological replicates each).

4. Levels of intermediate metabolites of the tricarboxylic acid (TCA) cycle based on targeted CE‐MS/MS metabolomics in GR cells relative to WT cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, ***P < 0.001.

5. Relative 2‐HG production level based on targeted CE‐MS metabolomics in J82‐IDH2ox cells compared with WT cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. ***P < 0.001.

6. ¹³C enrichment in cellular α‐KG, as determined by CE‐MS and normalized to the total pool size of the corresponding metabolite. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.

7. ¹³C enrichment in cellular malate determined by CE‐MS and normalized to the total pool size of the corresponding metabolite. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01, ***P < 0.001.

8. Major metabolites in the glycolytic pathway altered in J82‐IDH2ox cells compared with WT cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

9. PPP metabolite levels in J82‐IDH2ox cells compared with WT cells based on targeted CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

10. Relative dATP, dTTP, and dCTP level based on targeted CE‐MS metabolomics in J82‐IDH2ox cells compared with WT cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P ≤ 0.01, ***P ≤ 0.001. N.D., non‐detectable.

11. Graph showing the viability of J82WT and J82‐IDH2ox cells exposed to various concentrations of GEM for 48 h. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. ***P < 0.001.

Source data are available online for this figure.

Figure 6. Increased intracellular NADPH levels leads to cross resistance to CDDP

1. Changes in cytotoxicity between WT and GR T24 and UMUC3 cells exposed to increasing concentrations of CDDP for 48 h. The data are shown as the mean values ± SDs (n = 3, biological replicates).

2. Representative image showing the effect of CDDP in GR xenograft UC mouse models.

3. Graph showing the cytotoxic effect of CDDP and GEM in WT and GR cells orthotopically implanted mouse models (n = 5 mice per group). UMUC3GR cells (2 × 10⁶ cells) were implanted subcutaneously into the flanks of nude mice. After 1 weeks of implantation, the groups were treated with GEM (i.p. injection of 50 mg/kg weekly) or CDDP (intraperitoneal injection of 15 mg/kg on weekly). The mean growth of tumor volume (mm³) ± SD for each group is shown. Data were analyzed by Student's t‐test. **P < 0.01. n.s., non‐significant.

4. Immunohistochemistry (IHC) staining of Ki‐67 in UMUC3WT and GR cells exposed to CDDP/GEM. Ki‐67 staining quantitation of UMUC3WT and UMUC3GR cells exposed to CDDP/GEM are shown as bar graph The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by one‐way ANOVA with the Bonferroni test. **P < 0.05, ***P < 0.001. n.s., non‐significant.

5. Schematic of the mechanism by which GR UC cells acquire an antioxidant defense. Bypass of the oxidative PPP via aerobic glycolysis is suspected to be responsible for increased NADPH production. The increased NADPH leads to an increase in the GSH level. The increase in the GSH level decreases ROS generation and leads to the acquisition of cross‐resistance to CDDP.

6. NADPH/NADP ratios in WT and GR cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

7. Intracellular GSH levels in WT and GR cells after 24 h of exposure to the DMSO, CDDP (1 μM and 10 μM). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01. n.s., non‐significant.

8. Intracellular ROS generation in WT and GR cells after 24 h of exposure to CDDP (1 and 10 μM). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. ***P < 0.001. n.s., non‐significant.

9. NADPH/NADP ratios of GR cells transfected with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.

10. Intracellular GSH levels in GR cells transfected with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

11. ROS generation levels in GR cells transfected with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01. n.s., non‐significant.

12. Graph shows the viability of WT and GR cells exposed to various concentrations of GEM for 48 h after transfection with NTC, IDH2 siRNA#1, and siRNA#2 (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates).

13. Treatment in vivo using orthotopically implanted mouse models (n = 5 mice per group). We implanted UMUC3GR cells (2 × 10⁶ cells) transfected with #siNTC for 48 h, UMUC3GR cells (2 × 10⁶ cells) transfected with siIDH2#1 and siIDH2#2 for 48 h, and then injected subcutaneously into the flank of each BALB/c‐nu/nu mice. One week after implantation, cells were treated with CDDP (i.p. injection of 15 mg/kg weekly). The mean tumor volume (mm³) ± SD for each group is shown. Data were analyzed by Student's t‐test. *P < 0.05.

Figure EV4. Suppression of oxidative PPP lowers NADPH level

1. Simple schematic of the glycolytic pathway and the pentose phosphate pathway (PPP). The G6PD inhibitor (G6PD‐i) inhibits the oxidative bypass pathway coupling glycolysis to the PPP.

2. Bar graph showing the viability of GR cells exposed to various concentrations of CDDP and GEM for 48 h and treated with DMSO or G6PD‐i (10 μM) (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

3. Total NADPH levels in T24GR and UMUC3GR cells after 48 h of exposure to DMSO or G6PD‐i (10 μM). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

4. NADPH/NADP ratio in T24GR and UMUC3GR cells after 48 h of exposure to DMSO or G6PD‐i (10 μM). The data are shown as the mean values ± SDs (n = 3, biological replicates). Data were analyzed by Student's t‐test and are plotted relative to the levels in DMSO group. *P < 0.05, **P < 0.01.

5. Intracellular GSH levels in GR cells after 48 h of exposure to DMSO or G6PD‐i (10 μM) (left: T24GR, right: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01, ***P < 0.001.

Figure 7. AGI6780 restores chemosensitivity in chemo‐resistant UC cells

1. Graph showing the viability of GR cells exposed to various concentrations of GEM for 48 h in combination with DMSO or 10 μM AGI6780 (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates).

2. Relative 2‐HG production level based on targeted CE‐MS metabolomics in GR cells exposed to DMSO or 10 μM AGI6780 for 24 h (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01.

3. Relative dCTP level based on targeted CE‐MS metabolomics in GR cells exposed to DMSO or 10 μM AGI6780 for 24 h (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01, ***P < 0.001.

4. Graph showing the viability of GR cells exposed to various concentrations of CDDP for 48 h in combination with DMSO or 10 μM AGI6780 (upper: T24GR, lower: UMUC3GR). The data are shown as the mean values ± SDs (n = 3, biological replicates).

5. NADPH/NADP ratio in GR cells exposed to DMSO or 10 μM AGI6780 for 24 h. The data are shown as the mean values ± SDs (n = 3, biological replicates). The data were analyzed by Student's t‐test (upper: T24GR, lower: UMUC3GR). *P < 0.05, **P < 0.01.

6. Intracellular ROS generation in GR cells exposed to DMSO or 10 μM AGI6780 for 24 h. The data are shown as the mean values ± SDs (n = 3, biological replicates). The data were analyzed by Student's t‐test (upper: T24GR, lower: UMUC3GR). *P < 0.05, **P < 0.01.

7. In vivo treatment of orthotopically implanted mouse models (n = 5 mice per group). UMUC3GR cells (2 × 10⁶ cells) were implanted subcutaneously into the flanks of nude mice. One week after implantation, the groups were treated with GEM alone (i.p. injection of 50 mg/kg weekly), AGI6780 alone (i.p. injection of 50 mg/kg daily), or a combination of GEM and AGI6780 (i.p. injection of 50 mg/kg GEM weekly and i.p. injection of 50 mg/kg AGI6780 daily). The mean tumor volume (mm³) ± SD for each group is shown. Data were analyzed by Student's t‐test. **P < 0.01.

8. Representative images of tumors upon necropsy.

9. Body weights of mice with the indicated treatments. The data are shown as the mean values ± SDs (n = 5 mice per group), and were analyzed by Student's t‐test. n.s. = non‐significant.

10. IHC staining for IDH2, Ki‐67, CAIX, TIGAR, TKT, and CTPS1 in groups with GEM alone, AGI6780 alone, and the combination of GEM and AGI6780.

11. In vivo treatment of orthotopically implanted mice (n = 5 mice per group). UMUC3GR cells (2 × 10⁶ cells) were implanted subcutaneously into the flanks of nude mice. One week after implantation, the groups were treated with CDDP alone (weekly i.p. injection of 15 mg/kg), AGI6780 alone (daily i.p. injection of 50 mg/kg), or a combination of GEM and AGI6780 (weekly i.p. injection of 15 mg/kg CDDP and daily i.p. injection of 50 mg/kg AGI6780). The tumor volume (mm³, mean ± SD) in for each group is shown. Data were analyzed by Student's t‐test. **P < 0.01.

12. Representative images of tumors upon necropsy.

13. Body weights of mice treated as indicated. The data are shown as the mean values ± SDs (n = 5 mice per group) and were analyzed by Student's t‐test. n.s. = non‐significant.

Source data are available online for this figure.

Figure 8. High IDH2 expressed patients follow poor prognosis

1. Representative immunostaining of IDH2, CAIX (surrogate marker of Hif‐1α), TIGAR, TKT, and CTPS1 in surgical specimens from upper tract urothelial carcinoma (UTUC) patients. The upper panel represents the immunostaining of metabolic enzymes of early‐stage UTUC tumors, whereas the lower panel represents the images of invasive UTUC tumors. The power field scale bar = 100 μm.

2. Comparison of histoscores (H‐scores) for each metabolic enzyme divided by early‐ or advanced‐stage tumors. The H‐score was calculated by applying the following formula: mean percentage × intensity (range, 0–300). Violin plots were divided by pTa‐2: 116 UC patients and pT3‐4: 98 UC patients. Data were analyzed by Student's t‐test.

3. Heat map describing the IHC score in UTUC patients treated with GEM and CDDP (GC) adjuvant chemotherapy (n = 74). The heat map of metabolic enzymes in patients was classified by chemo‐sensitivity with information for age, sex, pT stage, grade, and death.

4. Kaplan–Meier curves showing the cancer‐specific survival (CSS) of 74 UTUC patients treated with GC adjuvant chemotherapy. Metabolic enzymes were classified according to the results of receiver operating curve (ROC) analysis. Data were compared with the log‐rank test.

5. Representative immunostaining of IDH2, CAIX, TIGAR, TKT, and CTPS1 in surgical specimens from muscle invasive urothelial carcinoma (MIBC) patients with neoadjuvant GC chemotherapy. Case 1 was a transurethral resection of bladder tumor (TURBT) specimen that induced a pathological complete response (pT0) in a radical cystectomy (RC) specimen. Case 2 was indicated as residual pT3 after neoadjuvant GC therapy. The power field scale bars = 200 and 50 μm.

6. Heat map describing the IHC score in MIBC patients treated with GC chemotherapy (n = 32). The heat map of metabolic enzymes in patients classified by IDH2 expression with information for age, sex, pT stage, grade, and death.

7. Details of high and low expression of CAIX, TIGAR, TKT, and CTPS1 classified with IDH2 expression.

8. Kaplan–Meier method showing the CSS of 32 MIBC patients treated with GC chemotherapy. The cutoffs for metabolic enzyme levels were determined according to the results of ROC analysis. Data were compared with the log‐rank test.

Figure 9. Mechanism of metabolic reprogramming

Graphical summary of the metabolic basis of GEM resistance in UC cells. GR cells demonstrate 2‐HG production by reductive glutamine metabolism stimulated by IDH2 gain of function. The increase of 2‐HG production suppresses PHD2 function and stabilizes Hif‐1α expression, which regulates the key metabolic enzymes TIGAR, TKT, and CTPS1, which are involved with aerobic glycolysis and PPP bypass. With the stimulation of aerobic glycolysis metabolism, it promotes the generation of dCTP for acquiring therapeutic competition against GEM. At the same time, oxidative PPP stimulation increases NADPH/NADP production and enhances antioxidant defense, which facilitates cross‐resistance to CDDP. Thus, inhibition of IDH2 suppress the entire metabolic reprogramming and therefore restore both GEM and CDDP sensitivity in UC cells.

Figure EV5. AGI6780 suppresses the entire metabolic reprogramming

1. The viability of J82WT and J82‐IDH2ox cells was assessed by WST assays under treatment with GEM, mizorubine, GEM with mizorubine, leflunomide, or GEM with leflunomide. The data are shown as the mean values ± SDs (n = 3, biological replicates). Comparisons were made with respect to the corresponding controls or the indicated groups, followed by analysis with Student's t‐test. **P ≤ 0.01, ***P ≤ 0.001. n.s., non‐significant.

2. Complete heatmap showing the intracellular levels of metabolites in J82‐IDH2ox cells treated with AGI6780. For each cell line, data for three individual biological replicates are shown, and the levels were normalized to those in J82‐IDH2ox cells.

3. Major metabolites in the glycolytic pathway altered in J82‐IDH2ox cells treated with AGI6780 compared with untreated J82‐IDH2ox cells. The data are shown as the mean values ± SDs (n = 3, biological replicates). The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05.

4. Levels of intermediate metabolites of the tricarboxylic acid (TCA) cycle based on targeted CE‐MS metabolomics in J82‐IDH2ox cells treated with AGI6780 relative to untreated J82‐IDH2ox cells based on targeted CE‐MS metabolomics. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

5. Relative 2‐HG production level based on targeted CE‐MS metabolomics in J82‐IDH2ox cells treated with AGI6780 compared with untreated J82‐IDH2ox cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.

6. PPP metabolite levels based on targeted CE‐MS metabolomics in J82‐IDH2ox cells treated with AGI6780 relative to untreated J82‐IDH2ox cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. *P < 0.05, ***P < 0.001.

7. Relative dCTP level based on targeted CE‐MS metabolomics in J82‐IDH2ox cells treated with AGI6780 compared with untreated J82‐IDH2ox cells. The data are shown as the mean values ± SDs (n = 3, biological replicates) and were analyzed by Student's t‐test. **P < 0.01.