Fig. 1: TH9619 and TH9975 target MTHFD1(DC) but not mitochondrial MTHFD2.
From: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells
a, 1C metabolism flux between mitochondria and cytosol/nucleus. b–e, Dose–response curves of SW620 cells treated for 96 h with TH9619 (b), TH9975 (c), DS18561882 (d) or SHIN1 (e) in the presence of 50 μM thymidine, 1 mM sodium formate or vehicle (cultured in RPMI-FBS), means ± s.d. (n = 3). f, [U-13C]serine-derived formate release rate of SW620 WT, MTHFD2−/− and SHMT1−/− cells treated for 24 h with the indicated concentrations of TH9619 and TH9975 or 50 nM MTX (cultured in RPMI-FBS), means ± s.d. (n = 3 for control and TH9619, n = 2 for TH9975, n = 1 for MTX); one-way ANOVA (analysis of variance) with Tukey’s multiple comparisons test. g, Quantification of mitochondrial membrane potential in SW620 WT cells treated with 1 µM of TH9619 or TH9975 or 0.5 µM MTX for 48 h. FCCP was used as positive control. Data are displayed as means ± s.d. (n = 4, n = 5 for FCCP); one-way ANOVA with Dunnett’s test for multiple comparisons. h,i, CETSA analysis of MTHFD2 stabilization in SW620 WT cells treated for 3 h with 10 µM TH9619 or vehicle. MTHFD2 stabilization was assessed by western blot by normalizing remaining MTHFD2 signal to HSP60 in the mitochondrial fractions (h) or lamin A/C in the nuclear fractions (i). j, Representative immunoblots of one out of two independent experiments. k, DARTS analysis of MTHFD1 stabilization in SW620 lysates that were incubated with 10 µM TH9619 or vehicle for 30 min, followed by protein digestion at increasing pronase concentration and assessment of MTHFD1 degradation by western blot. MTHFD1 signal was normalized to SOD1. Shown is a representative out of three independent experiments. l, Mean pIC50 (−log of half-maximum inhibitory concentration) values for indicated MTHFD1/2 inhibitors assessed for their inhibitory activity against MTHFD1(DC) WT or MTHFD1(DC) mutant (Q100A), mean (from left to right n = 4, 9, 2, 19, 5, 15, 5, 6, 4, 8); unpaired, two-tailed t-test. m, CRISPR-Select cassette for MTHFD1-Q100A was delivered to SW620 cells. The graph shows the ratio of MTHFD1-Q100A versus WT on day 2 and day 25 of 10 μM TH9619 treatment normalized to day 2 value, means ± s.d. (n = 4); paired, two-tailed t-test.
