Figure 4.
Lack of mitochondrial pyruvate import alters glutamine metabolism and BTZ-driven proteasomal inhibition in MM cells. (A) OCR plot and metabolic parameters of U266 cells treated with the indicated drugs: BTZ (3 nM), UK-5099 (10 μM), or the combination. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (B) LC-MS results of glycolysis and the TCA cycle of monotherapies and combinatorial therapies relative to vehicle control U266 cells (n = 3). U266 cells were treated with BTZ (3 nM), UK-5099 (10 μM), or a combination for 24 hours. (C) Representation of data shown in panel B. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (D) Representation of LC-MS data presented in panel B with a focus on glutamine metabolism and its associated nonessential amino acids. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (E) Chymotrypsin-like proteasome activity was monitored in control (sgCtrl) or MPC1-knockout (sgMPC1 #1 and #2) U266 cells in the presence or absence (vehicle) of BTZ (3 nM) (n = 3). Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005. (F) Similar to panel E, except that U266 cells were treated with either vehicle, BTZ (3 nM), UK-5099 (10 μM), or the combination (n = 3). Significance was determined by two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (G) Similar to panel E, except that glutamine was depleted from the media of U266 cells (basal concentration: 2 mM) (n = 3). Significance was determined using two-way ANOVA followed by a Dunnett test; ∗ P < .0001. (H) U266 cells were treated with BTZ (3 nM) and the glutaminase inhibitor CB-839 (5 μM) for 48 hours, followed by an assessment of cell viability via PI. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .005; ∗∗P ≤ .0001. (I) Schematic representing link between the MPC complex, the metabolic rewiring induced its inhibition, and the proteasomal capacity of MM cells.

Lack of mitochondrial pyruvate import alters glutamine metabolism and BTZ-driven proteasomal inhibition in MM cells. (A) OCR plot and metabolic parameters of U266 cells treated with the indicated drugs: BTZ (3 nM), UK-5099 (10 μM), or the combination. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (B) LC-MS results of glycolysis and the TCA cycle of monotherapies and combinatorial therapies relative to vehicle control U266 cells (n = 3). U266 cells were treated with BTZ (3 nM), UK-5099 (10 μM), or a combination for 24 hours. (C) Representation of data shown in panel B. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (D) Representation of LC-MS data presented in panel B with a focus on glutamine metabolism and its associated nonessential amino acids. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (E) Chymotrypsin-like proteasome activity was monitored in control (sgCtrl) or MPC1-knockout (sgMPC1 #1 and #2) U266 cells in the presence or absence (vehicle) of BTZ (3 nM) (n = 3). Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005. (F) Similar to panel E, except that U266 cells were treated with either vehicle, BTZ (3 nM), UK-5099 (10 μM), or the combination (n = 3). Significance was determined by two-way ANOVA followed by a Dunnett test. ∗P ≤ .05; ∗∗P ≤ .005; ∗∗∗P < .0001. (G) Similar to panel E, except that glutamine was depleted from the media of U266 cells (basal concentration: 2 mM) (n = 3). Significance was determined using two-way ANOVA followed by a Dunnett test; ∗ P < .0001. (H) U266 cells were treated with BTZ (3 nM) and the glutaminase inhibitor CB-839 (5 μM) for 48 hours, followed by an assessment of cell viability via PI. Significance was determined using two-way ANOVA followed by a Dunnett test. ∗P ≤ .005; ∗∗P ≤ .0001. (I) Schematic representing link between the MPC complex, the metabolic rewiring induced its inhibition, and the proteasomal capacity of MM cells.

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