Fig. 4: MTHFD2 inhibitors induce DNA damage and apoptosis via thymine-less death.
a, HL-60 cell viability on MTHFD2 inhibitors, MTX or PMTX ± thymidine 50 μM at 96 h. Data from one representative experiment of two performed in duplicate are displayed as means. b, Thymidine rescue of DNA replication on TH7299 in THP-1 cells. Scale bar, 5 μm. Scatter dot plots show the mean and distribution of RF speeds per treatment (n (fibers per condition) = 105 (except TH7299 + thymidine, n = 100)). ***P < 0.001; one-way ANOVA (F = 99.79, d.f. = 3). NS, not significant. c, Cell-cycle analysis of DNA damage (γH2AX) on MTHFD2 inhibitors (TH7299 3 μM, TH9028/TH9619 50 nM) in HL-60 cells. Per sample, at least 15,000 events were gated (Extended Data Fig. 5). Graphs show the percentage of γH2AX-positive cells as means ± s.d. from one of two independent experiments performed in triplicate. *P = 0.0179, ***P < 0.001; one-way ANOVA (FS(24h) = 4.937, FS(48h) = 303.4, FG2/M(24h) = 2.968, FG2/M(48h) = 310, d.f. = 3). d, THP-1 cell protein levels of checkpoint and cell death markers on TH7299. β-Actin was used as a loading control. A representative experiment is shown (n = 2). e, Subcellular fractionation of THP-1 cells for replication markers on TH7299 (24 h, 10 μM). Chr., chromatin-bound fraction; Sol., soluble fraction (cytoplasmic and nuclear). Histone H3 and β-actin were used as loading controls for the chromatin-bound and soluble fractions, respectively. A representative experiment is shown (n = 2). f, DNA fiber assay on TH7299 (3 μM), TH9619/MTX (50 nM) and VE-821 (1 μM) in HL-60 and THP-1 cells compared with nontumorigenic LCL-534 and LCL-889 cells. Scale bar, 5 μm. Scatter dot plots show the mean and distribution of RF speeds for each treatment. From left to right: n (fibers per condition) = 198, 227, 244, 182, 255, 201, 278, 461, 240, 219, 229, 223, 152, 250, 165, 176, 344, 183, 447 and 326. A representative of two independent experiments is shown. ***P < 0.001; one-way ANOVA with Holm–Šídák correction for multiple comparisons (FHL-60 = 233.9, FTHP-1 = 187, FLCL-534 = 165.3, FLCL-889 = 435.1, d.f. = 4).
