Fig. 3: Mitochondrial complex I inhibition induces distinct metabolic reprogramming followed by reductive metabolism of glutamine in NOTCH1-mutated cell lines, uncovering a potential metabolic mechanism of acquired resistance under OxPhos blockade.

a ATP levels for NOTCH1-mutated T-ALL (n = 4) treated 96 h in media with: no nutrients, pyruvate (Pyr), glutamine (Gln), glucose (Glc), Gln+Pyr, Glc+Pyr, Glc+Gln, Glc+Pyr+Gln, (mean ± SD, n = 4 independent experiments, 3 replicates); one-way ANOVA: *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001. b Basal OCR of 4 NOTCH1-mutated T-ALL cell lines treated 24 h as in (a), (mean ± SD, n = 3 independent experiments, 4 replicates); one-way ANOVA: *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001. c Metabolic phenotype (basal OCR/basal ECAR ratio) as in (a); (mean ± SD, n = 3 independent experiments, 4 replicates). d Extracellular acidification rate (ECAR) in NOTCH1-mutated T-ALL cell line PF-382 after IACS-010759 treatment (0–123 nM, 4 h); Glycolysis Stress Test (mean ± SD, n = 3 independent experiments, 4 replicates). e Basal ECAR of NOTCH1-wt (n = 3) and -mutated (n = 6) cell lines treated 24 h in media with Gln only (blue) and after acute injection of Glc (black); (Glycolysis Stress Test), (mean ± SD, n = 3 independent experiments, 4 replicates); two-way ANOVA: ***p = 0.0002. f Basal OCR in NOTCH1-wt (n = 3) and -mutated (n = 6) cell lines treated as in (e); (mean ± SD, n = 3 independent experiments, 4 replicates); two-way ANOVA: *p = 0.0250. g Basal ECAR of NOTCH1-mutated (n = 5) and -wt (n = 3) cell lines treated with IACS-010759 (0–123 nM, 4 h); (mean ± SD, n = 3 independent experiments, 4 replicates); two-tailed t test: ****p < 0.0001. h Glycolytic capacity in NOTCH1-mutated (n = 5) and -wt (n = 3) cell lines treated with IACS-010759 (0–123 nM, 4 h), Glycolysis Stress Test; (mean ± SD, n = 3 independent experiments, 4 replicates); one-way ANOVA: ****p < 0.0001. i ATP production (%) for NOTCH1-mutant (n = 8) and -wt (n = 3) cell lines treated with IACS-010759 (0–123 nM, 4 h), ATP Rate Assay; (mean ± SD, n = 3 independent experiments, 4 replicates); one-way ANOVA: ****p < 0.0001. j Levels of citrate and α-ketoglutarate and citrate/α-ketoglutarate ratio over time in NOTCH1-mutated cell line PF382; (mean ± SD, n = 1, 4 replicates); two-way ANOVA: ****p < 0.0001. k Stable isotope-resolved metabolomics ultra-high-pressure liquid chromatography MS analysis (SIRM) of NOTCH1-mutant PF-382 cell line cultured with 13C5,15N2-glutamine, treated with DMSO or IACS-010759 (10 nM, 12 h); (mean ± SD, n = 1, 4 replicates). two-tailed t-test; *p < 0.05; **p < 0.005; ***p < 0.001; and ****p < 0.0001.