Fig 2: IDH1 binds to GA/AU-rich single-stranded RNA. (A) GA- and AU-rich RNA motifs are enriched in IDH1’s RNA targets in all sequences or in the 3′ UTR regions, respectively. (B–E) EMSA analysis of direct binding of purified wild-type to various fluorescence-labeled RNA/DNA probes as indicated. (B) EMSA with the 5 × GAA ssRNA probe containing the overall motif #1. (C) EMSA with ssRNA probes with 2–5 copies of GAA(A) nucleotides. (D) Binding isotherms for IDH1-RNA interactions. Each data point is shown as mean ± SD (n = 3 biological replicates). Error bars represent standard deviations (SD), some of which are too small to be visualized. (E) Summary of RNA-binding activities of IDH1 toward various ssRNA probes used above. Dissociation constants (Kd) are shown. N.D., not detected. GAA and AUUU units are underlined. (F) Relative m6A level of IDH1 RNA targets in ESCs was determined by HPLC-MS/MS following FBioRIP assays. Data are shown as mean ± SD, n = 3. (G) Comparison of RNA-binding activities of wild-type (WT) and oncogenic R132H mutant of IDH1 to the 5× GAA ssRNA probe as shown in the panel (B).

Fig 3: FASN deficiency drives a net cytosol-to-mitochondria citrate flux(A and B) Mass isotopolog analysis of citrate and malate enrichment in FASN-WT and FASN-KO cells incubated with [U-13C]glutamine tracing medium containing 5 μM IDH1 inhibitor (IDH1i; GSK321), 5 μM IDH2i (AGI-6780), and IDH1i plus IDH2i for 4 h. The mean ± SD error bars are displayed. *p values are derived from a one-way ANOVA followed by Dunnett’s multiple comparisons test for the three cell lines under the same treatment (*p < 0.05 compared to FASN-WT, #p < 0.05 compared to FASN-WT/-KO_DMSO group). The experiments were repeated 3 times or more, and n = 3 independent samples.(C) The modeling of metabolic fluxes in FASN-deficient cells. The comparative modeling of metabolic fluxes in FASN-KO2 H460 cells to FASN-WT controls. See Table S1 for definitions of quantitative flux values. The color scale reflects the ratio of each flux in FASN-KO cells relative to FASN-WT controls.(D) Immunoblots of IDH1, CTP, and β-actin in FASN-WT and FASN-KO cells. IDH1, isocitrate dehydrogenase 1; CTP, citrate transport protein.

Fig 4: Interrogation of pY functional role by coupling isotope labeled tracing and CRISPRi-rescue validates regulatory role of IDH1 Y391 and UMPS Y37 on enzyme activity.(A) U-13C5-Glutamine stable isotope tracing scheme to evaluate the role of IDH1 Y391 which is positioned in the NADP binding domain (PDB:1t0l)92. (B) Fraction labeling of citrate and aspartate via oxidative (blue) and reductive (red) TCA cycle over 0, 60, or 120min in A549 sgIDH1 CRISPRi-rescue cell lines (N=4). Knockdown-rescue was achieved by adding 500ng/mL of Dox, replenished daily, to culture media for a total of 96 hours. Total ion counts were normalized to Norvaline internal standard and total protein content, and fraction labeling was calculated by taking the ratio of each labeled species divided by the total pool size. (C) Pool sizes of citrate and aspartate at 120min (N=4). Pool sizes were calculated by summing all species of each metabolite per condition. Data available in Table S8(A – C). (D) Model showing pY391 activating IDH1 to induce reductive carboxylation of aKG to citrate. (E) U-13C5-Glutamine stable isotope tracing scheme for 0, 12, or 24 hours to measure changes in de novo pyrimidine synthesis pathway in A549 sgUMPS CRISPRi-rescue cell lines treated with dox for 96 hours total. (F) Total ion counts of M+4 labeled and total de novo pyrimidine synthesis intermediates carbamoyl aspartate, dihydroorotate, and orotate (N=4). (G) Extracellular orotate and dihydroorotate were measured from media collected at the 24 hours timepoint. Data available in Table S8(D – G). (H) model for pY37-mediated inhibition of UMPS enzyme activity.

Fig 5: IDH1 and IDH2 regulate reductive carboxylation and ccRCC growth.(A) Percent enrichment of 13C relative to [U-13C]glutamine in TCA cycle from orthotopic XP258–derived orthotopic tumorgrafts, cell lines, and subcutaneous xenografts. Tumor-bearing mice were infused with [U-13C]glutamine for 5 hours, and XP258 cells were labeled in culture with [U-13C]glutamine for 4 hours. (B) Western blot showing the abundance of IDH1 and IDH2 in single- and double-knockout (IDH1 + IDH2 KO) cells generated using lentiviral CRISPR-Cas9. Enhanced green fluorescent protein (EGFP) is a control cell line with a guide RNA targeting EGFP. (C) Viable cell content in EGFP, IDH1 KO, IDH2 KO, and IDH1 + IDH2 KO XP258 cells assessed using Promega CellTiter-Glo. RLU, relative light unit. (D) Fractional enrichment of 13C-labeled isotopologues of citrate and malate in cells after 4 hours of culture with [U-13C]glutamine. One-way ANOVA was used to assess the significance of 13C enrichment in citrate and malate (n = 3). (E) Percentage enrichment of citrate M+4 and citrate M+5 relative to [U-13C]glutamine in EGFP, IDH1 KO, IDH2 KO, and IDH1 + IDH2 KO XP258 tumors. Mice were infused with [U-13C]glutamine for 5 hours. Data are from three or more xenografts generated subcutaneously in NOD-SCID mice. One-way ANOVA with a pairwise t test was used to determine the statistical significance. (F) Growth of EGFP, IDH1 KO, and IDH2 KO subcutaneous XP258 xenografts in NOD-SCID mice (n = 10, each cell line). P values were calculated using a mixed-effect two-way ANOVA that assessed the significance of differences in tumor growth over time between EGFP versus IDH1 KO and EGFP versus IDH2 KO tumors. P values: **P < 0.01, ***P< 0.001.