Figure 6.
MondoA provides leukemia stress resistance by limiting Oxphos and fatty acid synthesis via decreased PDH activity. (A) Long-term proliferation measured by direct cell counting. Control Cas9-only Nalm6 B-ALL cell line, MondoA overexpression, and MKO, grown under normoxia (20% O2) (n = 6 for each time point, P < .001 at 96 hours and 120 hours). (B) Mitochondrial respiration determined by cellular OCR in control, MKO clones, and Nalm6 cells with MondoA overexpression using a Seahorse extracellular flux analyzer (Agilent). (C) Glycolysis determined by ECAR in control, MKO clones, and Nalm6 cells with MondoA overexpression using a Seahorse extracellular flux analyzer (Agilent). (D) Basal respiration as measured OCR before oligomycin injection (P < .05 and P < .001, 2-way ANOVA with Bonferroni posttest). (E) ATP production is measured as the difference between basal respiration and OCR levels after oligomycin injection. (F) Maximal respiration as measured by OCR levels after FCCP injection (P < .05 and P < .01, 2-way ANOVA with Bonferroni posttest). (G) Glycolysis as measured as the difference between basal ECAR and ECAR levels after glucose injection. (H) Gas chromatography–mass spectrometry quantification of glucose uptake from, and lactate secretion into, culture medium of the indicated cell lines (P ≤ .0001, 2-way ANOVA with the Tukey multiple comparisons test). (I) PDH activity assessed by NADH production. PDH activity in control, MKO, and MOE Nalm6 cells assessed by the colorimetric PDH activity assay kit (n = 3 for each time point, P < .001, 2-way ANOVA with the Bonferroni posttest). (J) Western blot showing MondoA, total PDK1, pPDH (Ser293), total PDH, TXNIP, α-tubulin, and GAPDH as loading controls in Cas9-only control Nalm6 cells, MKO, and MOE cells. (K) PDK1 mRNA in MKO clones compared with CTRL and MOE clones measured by qRT-PCR (P < .001, 2-way ANOVA with the Bonferroni posttest). (L) Heat map showing PDK1 gene coexpression with MondoA in B-ALL. RNA-Seq data of 194 primary ALL patient samples (TARGET) from Xena browser. (M) Pharmacological inhibition of Nalm6 cell proliferation with PDK-inhibitor dichloroacetic acid (DCA; 5 mM) as measured by live cell analysis with image-based cytometry. (N) Pharmacological inhibition of Nalm6 cell proliferation with MondoA-inhibitor SBI477 (10 µM) as measured by live cell analysis with image-based cytometry. (O) Line graph and (P) western blot showing PDK1 protein relative density in Nalm6 control cells and MOE cells after 16 hours of SBI477 treatment at various concentrations (0, 5, 10 µM). DMSO, dimethyl sulfoxide. *P < .05; **P < .01; ***P < .001; ****P < .0001.

MondoA provides leukemia stress resistance by limiting Oxphos and fatty acid synthesis via decreased PDH activity. (A) Long-term proliferation measured by direct cell counting. Control Cas9-only Nalm6 B-ALL cell line, MondoA overexpression, and MKO, grown under normoxia (20% O2) (n = 6 for each time point, P < .001 at 96 hours and 120 hours). (B) Mitochondrial respiration determined by cellular OCR in control, MKO clones, and Nalm6 cells with MondoA overexpression using a Seahorse extracellular flux analyzer (Agilent). (C) Glycolysis determined by ECAR in control, MKO clones, and Nalm6 cells with MondoA overexpression using a Seahorse extracellular flux analyzer (Agilent). (D) Basal respiration as measured OCR before oligomycin injection (P < .05 and P < .001, 2-way ANOVA with Bonferroni posttest). (E) ATP production is measured as the difference between basal respiration and OCR levels after oligomycin injection. (F) Maximal respiration as measured by OCR levels after FCCP injection (P < .05 and P < .01, 2-way ANOVA with Bonferroni posttest). (G) Glycolysis as measured as the difference between basal ECAR and ECAR levels after glucose injection. (H) Gas chromatography–mass spectrometry quantification of glucose uptake from, and lactate secretion into, culture medium of the indicated cell lines (P ≤ .0001, 2-way ANOVA with the Tukey multiple comparisons test). (I) PDH activity assessed by NADH production. PDH activity in control, MKO, and MOE Nalm6 cells assessed by the colorimetric PDH activity assay kit (n = 3 for each time point, P < .001, 2-way ANOVA with the Bonferroni posttest). (J) Western blot showing MondoA, total PDK1, pPDH (Ser293), total PDH, TXNIP, α-tubulin, and GAPDH as loading controls in Cas9-only control Nalm6 cells, MKO, and MOE cells. (K) PDK1 mRNA in MKO clones compared with CTRL and MOE clones measured by qRT-PCR (P < .001, 2-way ANOVA with the Bonferroni posttest). (L) Heat map showing PDK1 gene coexpression with MondoA in B-ALL. RNA-Seq data of 194 primary ALL patient samples (TARGET) from Xena browser. (M) Pharmacological inhibition of Nalm6 cell proliferation with PDK-inhibitor dichloroacetic acid (DCA; 5 mM) as measured by live cell analysis with image-based cytometry. (N) Pharmacological inhibition of Nalm6 cell proliferation with MondoA-inhibitor SBI477 (10 µM) as measured by live cell analysis with image-based cytometry. (O) Line graph and (P) western blot showing PDK1 protein relative density in Nalm6 control cells and MOE cells after 16 hours of SBI477 treatment at various concentrations (0, 5, 10 µM). DMSO, dimethyl sulfoxide. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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