Fig. 3

Resistant cells maintain anaplerotic capacity during cyst(e)inase treatment making mitochondrial metabolism a synergistic target. a TCA cycle intermediates and related metabolite levels 6 and 24 h after 250 nM cyst(e)inase treatment in MIA-PaCa2 and Panc1 cells (n = 3 cultures). Color bar shows Log₂ scale. b Predicted induction of pyruvate carboxylase (PC) activity by MIA-PaCa2 cells to replenish pools of oxaloacetate (OAA) and subsequently aspartate (Asp) via [U-¹³C]-glucose. Glc glucose, Pyr pyruvate, Lac lactate, Ac-CoA acetyl-CoA, Cit citrate, α-KG α-ketoglutarate, Glu glutamate, Gln glutamine, Succ succinate Fum fumarate, Mal malate, GLS glutaminase, SDH succinate dehydrogenase, ETC electron transport chain, ATP syn ATP synthase. c Mass isotopologue analysis of aspartate in MIA-PaCa2 (top) and Panc1 (bottom) cells cultured in [U-¹³C]-glucose and treated with cyst(e)inase for 6 h (n = 3 cultures). d Cell survival of BxPC3 cells treated with combination of cyst(e)inase (Cys) and CB-839 for 48 h. Values are relative to untreated control, which is not shown (n = 3 cultures for each condition). e Isobologram of the effect of the combination of cyst(e)inase and CB-839 (data from 3–4 independent experiments). Dashed line represents 5% error to distinguish true synergy from experimental variability. f Relative cell survival 48 h after treatment with either 200 nM Cys alone or in combination (single or double) with 0.5 mM GSH ethyl ester (GSH) and 5 mM methyl aspartate (Asp) (n = 3 independent experiments). g Isobologram of the effect of the combination of cyst(e)inase and tigecycline (data from 3–4 independent experiments). All data represent mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; compared to untreated controls c or to Cys treatment f; two-sided Student’s t test c or two-way ANOVA with Bonferroni’s method for multiple-comparison test f. All experiments were repeated (in exact or similar form) three times or more