Fig 2: Effects of SHIN1 and PRTX on one-carbon flux through the mitochondrial and cytoplasmic compartments. A, Schematic of one-carbon flux through the mitochondrial and cytosol compartments using [2,3,3-2H]L-serine. The 2H atoms in [2,3,3-2H]L-serine (blue circle) in the mitochondria are metabolized to generate [2H]formate. [2H]Formate in the cytosol is converted to [2H]10-formyl THF and [2H]5,10-methylene THF, resulting in [2H]dTMP and [2H]dTTP (as M+1). Reversal of SHMT1 activity in the cytosol results in 2H atoms in [2,3,3-2H]serine (red) being metabolized to [2H]5,10-methylene THF, which is utilized by thymidylate synthase to synthesize [2H]dTMP and [2H]dTTP (as M+2). B and C, Total serine (B) and glycine (C) and the serine-isotopomer distributions in SK-ES-1 cells treated with vehicle (DMSO), 10 nmol/L PRTX, or 10 µmol/L SHIN1. The increase in the M+1 and M+2 serine isotopomers in the presence of the inhibitors is a direct reflection of accumulated [2,3,3-2H]L-serine accompanying the loss of SHMT2 activity, combined with low levels of one-carbon flux through SHMT2 and MTHFD2 in mitochondria and SHMT1 in the cytosol. D, Total dTTP pools are shown with the isotopomer distributions (M+0, M+1, M+2) for untreated and inhibitor-treated SK-ES-1 cells compared with DMSO-treated cells. E–G, GAR (E), AICAR (F), and inosine monophosphate (IMP; G) pools were measured in vehicle and inhibitor-treated cells. An unpaired t test was performed for total serine, total glycine, total dTTP, and IMP in comparison with the DMSO-treated sample. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 3: Progressive loss of the ability of relevant metabolites to rescue the proliferation of Ewing sarcoma cells after one-carbon metabolism inhibition. A–C, Rescue by exogenous adenine, thymidine, formate, and hypoxanthine addition of the growth inhibition induced by 72 hours of treatment of SK-ES-1 and TC-71 cells with one-carbon inhibitors. D and E, Rescue by metabolite supplementation of the growth inhibition induced by 7-day treatment of SK-ES-1 and TC-71 cells with SHIN1 (D) and PRTX (E). F and G, Rescue effect of delayed (at day 3) metabolite addition on the growth inhibition induced by 7-day treatment of SK-ES-1 and TC-71 cells with SHIN1 (F) and PRTX (G). A–G, *, P < 0.001 vs. DMSO. H and I, Exogenous glycine deprivation impairs the ability of hypoxanthine, adenine, thymidine, and formate (concentrations as in A–C) to rescue the proliferation impairment consequent to SHMT1/2 inhibition with SHIN1. Glycine-free RPMI 1640 was supplemented with 1.33 mmol/L (10×), 133 µmol/L (1×), or 13.3 µmol/L (10%) glycine. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Depletion of SHMT2, but not SHMT1, impairs proliferation of Ewing sarcoma cells. A, shRNA-mediated depletion of SHMT1 (constitutive, shSHMT1) or SHMT2 (inducible, ishSHMT2) in SK-ES-1 and TC-71 cells. B, Incucyte live analysis of cell proliferation upon depletion of SHMT1 and SHMT2 with two shRNAs each. LKO: empty vector. C, Representative colony-forming assays of SK-ES-1 and TC-71 cells upon depletion of SHMT1 or SHMT2. D, Quantitative results from the experiment in C. *, P < 0.01.

Fig 5: Progressive loss of the ability of relevant metabolites to rescue the proliferation of Ewing sarcoma cells after one-carbon metabolism inhibition. A–C, Rescue by exogenous adenine, thymidine, formate, and hypoxanthine addition of the growth inhibition induced by 72 hours of treatment of SK-ES-1 and TC-71 cells with one-carbon inhibitors. D and E, Rescue by metabolite supplementation of the growth inhibition induced by 7-day treatment of SK-ES-1 and TC-71 cells with SHIN1 (D) and PRTX (E). F and G, Rescue effect of delayed (at day 3) metabolite addition on the growth inhibition induced by 7-day treatment of SK-ES-1 and TC-71 cells with SHIN1 (F) and PRTX (G). A–G, *, P < 0.001 vs. DMSO. H and I, Exogenous glycine deprivation impairs the ability of hypoxanthine, adenine, thymidine, and formate (concentrations as in A–C) to rescue the proliferation impairment consequent to SHMT1/2 inhibition with SHIN1. Glycine-free RPMI 1640 was supplemented with 1.33 mmol/L (10×), 133 µmol/L (1×), or 13.3 µmol/L (10%) glycine. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.