Fig 2: MTHFD2 promotes PD-L1 transcription through cMYC O-GlcNAcylation.a The GSEA analysis of the transcriptome (as indicated in Fig. 2a) in SW1990 cells transfected with indicated siRNAs, were performed with the GeneSet “Genes regulated in primary epithelial cell overexpressing cMYC” in MSigDB C6 (oncogenic signatures) and with the GeneSet “Genes having at least one occurrence of the transcription factor binding site MYC/MAX_B in the regions spanning up to 4 kb around their transcription starting sites” in MSigDB C3 (motif) transcription factor targets database. b SW1990 cells with siMTHFD2 transfected with indicated expressing vectors, PD-L1 mRNA levels were analyzed by real-time PCR. c SW1990 cells with siMTHFD2 treated with 0.3 mM UDP or 0.6 mM UTP for 24 h, immunoblotting analyses were performed using the indicated antibodies. d Analysis of the apparent half-life time of wild type and mutant cMYC in cycloheximide treated cells transfected with indicated vectors. e, f The cell lysates of SW1990 cells expressing WT or T58A Flag-cMYC with indicated plasmid (e) or siRNA (f) were subjected to immunoprecipitation with an anti-Flag antibody, and the precipitates (loading volume adjusted to be equal precipitated cMYC) and whole lysates (equal total protein amount loading) were analyzed by immunoblotting using indicated antibodies. g, h SW1990 cells expressed with WT or T58A Flag-cMYC transfected with indicated siRNAs, were treated with 0.3 mM UDP or 0.6 mM UTP for 24 h. PD-L1 mRNA levels were analyzed by real-time PCR (g); cell lysates were subjected to immunoprecipitation with an anti-Flag antibody, and the precipitates (loading volume adjusted to be equal precipitated cMYC) and whole lysates (equal total protein loading) were analyzed by immunoblotting using indicated antibodies (h). In (b) and (g), the values are presented as mean ± s.e.m (n = 3 independent experiments); p values (Student’s t test, two-sided) with control or the indicated groups are presented (also see Supplementary Fig. 6).

Fig 3: The EWS‐FLI1 oncoprotein regulates the expression of genes required for the mitochondrial one‐carbon cycle. (A) A schematic illustrating the incorporation of serine and glycine into the one‐carbon cycle and production of reducing equivalents for cells. (B) The levels of formate in TC32 and SKNMC cells 48 h post‐transfection of either siNeg or siFLI1; treatment with CBR5884 40 μM for 24 h; or treatment with NCT503 10 μM for 24 h (mean ± SEM of three independent experiments). (C) Exon level expression and fold‐change (Log2 TPM) from RNA‐seq of the expression of MTHFD2 and MTHFD1L in control (siNeg) and EWS‐FLI1‐silenced (siFLI1) TC32 cells. (D) Violin plots (including median and quartiles) of the expression of MTHFD2 and MTHFD1L in EWS‐FLI1 positive EWS primary tumors (n = 58), EWS‐FLI1 positive EWS cell lines (n = 33) and normal tissues (n = 42). (E) qPCR analysis of the expression of the indicated genes (relative to siNeg‐transfected cells, mean ± s.e.m., n = 3) in EWS‐FLI1‐silenced TC32 cells (48 h). (F) Immunoblots of the indicated proteins in lysates prepared from TC32 cells, siRNA‐transfected as indicated. (G) Fold‐enrichment of EWS‐FLI1‐co‐precipitating DNA determined by qPCR of genomic DNA spanning putative EWS‐FLI1 binding sites. Data shown were calculated as a percent of input DNA and the fold‐enrichment of the anti‐FLI1 antibody compared to IgG; triplicate samples of each treatment in each experiment. (B, E, and G) Two‐way ANOVA (adjusted P‐value) ***P < 0.001. (D) Unpaired t‐test, adjusted for multiple comparisons; **q < 0.01, ***q < 0.001

Fig 4: MTHFD2 deacetylation inhibits NADPH production and breast cancer growth. (A) Characterization of stable re-expression of MTHFD2 in ZR-75-30 cells. WT MTHFD2 or the KR mutant was reintroduced into MTHFD2-knockdown cells. The knockdown efficiency and re-expression of Flag-tagged MTHFD2 were determined by western blotting. (B and C) K50 acetylation of MTHFD2 modulates the NADP+/NADPH ratio and ROS level. MTHFD2-knockdown ZR-75-30 cells re-expressing WT MTHFD2 (shMTHFD2 + MTHFD2WT) or its KR mutant (shMTHFD2 + MTHFD2KR) were cultured under folate deprivation followed by NAM incubation for 6 h. The NADP+/NADPH ratios (B) and ROS levels (C) were determined by a spectrofluorometer. (D) Re-expression of WT MTHFD2, but not the KR mutant, protects cells from oxidative injury. MTHFD2-knockdown ZR-75-30 cells re-expressing WT MTHFD2 (shMTHFD2 + MTHFD2WT) or the KR mutant (shMTHFD2 + MTHFD2KR) were treated with indicated concentrations of H2O2 for 12 h. Cell viability was assayed. (E) The KR mutant significantly reduces tumor growth by xenograft assay. ZR-75-30 breast cancer cells were transplanted to nude mice. After tumor harvest, tumor weight and size were measured. (F) Working model. SIRT4 deacetylates MTHFD2 at K50 residue in response to folate deprivation, which promotes proteasomal degradation of MTHFD2 via CUL3-triggered ubiquitylation, leading to an inhibition of NADPH production and cancer cell proliferation. Therefore, stressful signal of inadequate folate is sensed by the SIRT4–MTHFD2 axis to cause disorder of cellular redox by reducing MTHFD2-catalyzed NADPH production and hamper proliferation of breast cancer cells. Data shown represent mean ± SD obtained from triplicate independent experiments. n.s. indicates no significance, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: MTHFD2 is acetylated at K50. (A) Pan-acetylation of MTHFD2 is increased by NAM treatment. HEK293T cells with Flag-tagged MTHFD2 overexpression were treated with NAM for 6 h. Lysine acetylation of immunopurified MTHFD2 was detected with anti-acetyl lysine (pan-ac) antibody. Relative ratios of MTHFD2 acetylation were normalized by MTHFD2 protein level. (B) The pan-acetylation level of WT MTHFD2 is much higher than that of the KQ or KR mutant. Flag-tagged WT MTHFD2 and KQ and KR mutants were ectopically expressed into HEK293T cells and purified by Flag beads. Pan-acetylation of immunopurified MTHFD2 was analyzed by western blotting. (C) Pan-acetylation of WT MTHFD2 rather than that of the KQ or KR mutant is increased by NAM treatment. Flag-tagged WT MTHFD2 and KQ and KR mutants were ectopically expressed in HEK293T cells treated with or without NAM and purified by Flag beads. Pan-acetylation of immunopurified MTHFD2 was analyzed by western blotting. (D) K50 site-specific acetylation antibody [acMTHFD2 (K50)] recognizes WT MTHFD2, but not its KQ and KR mutants. Flag-tagged WT MTHFD2 and its KQ and KR mutants were immunopurified with Flag beads and blotted with acMTHFD2 (K50) antibody. (E) Endogenous MTHFD2 is acetylated at K50 in multiple cell lines. HEK293T, MCF-7, and ZR-75-30 cells were treated with NAM for 6 h. K50 acetylation of immunopurified endogenous MTHFD2 was detected by western blotting.