Extended Data Fig. 5: MTHFD2i induce replication stress selectively in cancer cells, rescuable by thymidine.
(a) THP-1 cell viability upon MTHFD2i or methotrexate (MTX) ± thymidine 50 μM (96 h). Data from one representative experiment are displayed as means, n = 2. (b) SW620 cell viability upon TH7299, TH9619, MTX or 5-fluorouracil (5-FU) ± thymidine 50 μM (96 h). Data from one representative experiment are displayed as means, n = 2. (c) TH9619 reduces serine-derived carbon-flux into purine synthesis. Graphs show mass isotopologue distribution (MID) of intracellular ATP, ADP, and AMP upon [U-13C]-serine tracer in response to 24 h treatment with 0.3, 1, or 20 μM TH9619 or 50 nM MTX in HL-60 cells; mean ± SEM of three independent experiments each measured in triplicate. wells. Reduced labelling from [U-13C]-serine of ATP, ADP, and AMP as well as increased relative percentage of unlabeled M + 0 isotopologues of ATP, ADP, and AMP is indicative of a reduced flux of 13C-carbon units from serine to purine synthesis upon TH9619 treatment. MTX serves as a positive control for purine synthesis inhibition. (d) DNA fiber assay upon TH9028 50 nM, MTX 50 nM and VE-821 1 μM in AML and non-malignant cells. Scale bar, 5 μm. Dotplots show the distribution and calculated mean of replication fork speed per treatment. From left to right, n (fibers/condition) = 165; 345; 192; 227; 220; 284; 237; 279; 142; 192; 218; 171; 192; 170; 114; 248. Shown is a representative out of two independent experiments. * P < 0.05, *** P < 0.001; one-way ANOVA with Dunnett’s multiple comparison test (FHL-60 = 492.6, FTHP-1 = 771.6, FLCL-534 = 624, FLCL-889 = 770.6, DF = 3). (e) Replication origin firings upon TH7299 (10 μM, 24 h) in THP-1 cells. Data are displayed as means ± SD, n = 20. *** P < 0.001; one-way Mann-Whitney rank test (F = 39, DF = 1). (f) Cell cycle analysis of DNA damage (γH2AX) induction upon MTHFD2i treatment in HL-60 cells. Per sample, at least 15,000 events were gated. (g) Thymidine rescue of cell cycle progression upon TH7299 treatment in THP-1 cells. At least 10,000 events were analyzed/condition. Bar graphs represent the percentage of cells in each phase, displayed as means, n = 2.
