Fig. 5: MTHFD2 inhibitors exacerbate uracil misincorporation into DNA.

a, Proposed mechanism for the antitumor effect of MTHFD2 inhibitors via thymine-less-induced RS. MTHFD2 supports de novo thymidylate synthesis by providing CH₂-THF. On MTHFD2 inhibition, accumulation of dUMP promotes uracil misincorporation, causing DNA damage. Failing to repair these lesions, cells undergo RF collapse and cell death. A combination of MTHFD2 and dUTPase inhibitors further increases uracil misincorporation and apoptosis. Thymidine supplementation bypasses this, rescuing DNA replication and cell viability. CDA, cytidine deaminase; DSBs, double-strand breaks; dUTPasei, dUTPase inhibitor; TK, thymidine kinase. b, Comet assay in THP-1 cells. Scale bar, 2 μm. Dot plots represent comet tail moment per cell and bars display the mean (n = 200 cells per condition). ^****P < 0.0001; one-way Kruskal–Wallis test with Dunn’s multiple comparison correction (F = 904.2, d.f. = 9). c, Genomic uracil incorporation on TH7299 in THP-1 cells (10 μM, 48 h). Bars represent dU lesions as fold-change over DMSO displayed as means (n = 2). One of two independent experiments is shown. ^**P < 0.01, ^***P < 0.001; one-way ANOVA (F = 156.4, d.f. = 2). d, Genomic uracil incorporation in HL-60 cells (TH9619 100 nM ± dUTPase inhibitor 10 µM, MTX 100 nM, 5-FU 10 μM ± thymidine 10 μM, 48 h). Bars represent dU lesions per million deoxynucleotides (MdN). Data are displayed as pooled means ± s.d. from two independent experiments performed in triplicate. ^**P = 0.0063, ^****P < 0.0001; two-way ANOVA with Holm–Šídák correction for multiple comparisons (F_treatment = 48.64, F_thymidine = 87.47, d.f._treatment = 5, d.f._thymidine = 1). e, Comet assay in THP-1 cells on TH7299 ± dUTPase inhibitor (10 μM, 24 h). Dot plots represent tail moment per cell and bars display the mean (n = 200 cells per condition). ^***P < 0.001, ^****P < 0.0001; one-way Kruskal–Wallis test with Dunn’s multiple comparison correction (F = 361.4, d.f. = 9). f, THP-1 cell viability on TH9028–dUTPase inhibitor combination ± thymidine 10 μM at 72 h. One of three independent experiments is shown. Data are displayed as means (n = 2). g, Apoptosis analysis in THP-1 cells. Approximately 15,000 events per condition were analyzed. Bars represent annexin-V-positive populations as means (n = 2). ^*P_24h = 0.0151, ^*P_72h = 0.0213, ^**P = 0.0029, ^***P = 0.0002; one-way ANOVA (F = 43.89, d.f. = 5). One of two independent experiments is shown.

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