Fig 2: PFKP Y64 phosphorylation by EGFR enhances AKT-mediated ß-catenin S552 phosphorylation and nuclear translocation of ß-catenin and expression of its downstream genes. (A) Nuclear fractions of U87/EGFRvIII cells with or without PFKP depletion and with or without reconstituted expression of WT Flag-rPFKP or Flag-rPFKP Y64F mutant were prepared Immunoblotting analyses were performed with the indicated antibodies. (B) U87/EGFRvIII cells with or without PFKP depletion and with or without reconstituted expression of WT Flag-rPFKP or Flag-rPFKP Y64F mutant in the presence or absence of CA ß-catenin expression were transfected with TOP-FLASH or FOP-FLASH. The relative levels of luciferase activity were normalized to the levels of luciferase activity of Renilla control plasmid. Data represent the means ± SD of three independent experiments. *P < 0.001, based on the one-way ANOVA. (C,D) The mRNA expression levels (C) and the protein expression levels (D) of CCND1 and MYC in U87/EGFRvIII cells with or without PFKP depletion and with or without reconstituted expression of WT Flag-rPFKP or Flag-rPFKP Y64F mutant in the presence or absence of CA ß-catenin expression were determined by real-time PCR and immunoblotting analyses with the indicated primers and antibodies, respectively. Data represent the means ± SD of three independent experiments. *P < 0.001, based on the one-way ANOVA.

Fig 3: PFKP expression is required for EGFR activation-induced nuclear translocation and transactivation of ß-catenin. (A) Serum-starved U87/EGFR cells with the indicated shRNAs (left panel) were treated with or without EGF (100 ng/ml) for 9 h. The cells were harvested for the isolation of nuclear fractions (right panel). Immunoblotting analyses were performed with the indicated antibodies. (B) U87/EGFRvIII cells were stably expressed with control shRNA or PFKP shRNA. Immunofluorescent staining was performed with an anti-ß-catenin antibody. (C) U87/EGFR cells with or without PFKP depletion were transfected with TOP-FLASH or FOP-FLASH, which was followed by EGF treatment for 6 h. Luciferase activity was measured. The relative levels of luciferase activity were normalized to the levels of untreated cells and to the levels of luciferase activity in the Renilla control plasmid. Data represent the means ± SD of three independent experiments. *P < 0.001, based on the Student's t-test. (D,E) Serum-starved U87/EGFR cells with or without depleted PFKP were treated with or without EGF for the indicated periods of time. The mRNA expression levels (D) and the protein expression levels (E) of CCND1 and MYC in U87/EGFR cells were determined by real-time PCR and immunoblotting analyses with the indicated primers and antibodies, respectively. Data represent the means ± SD of three independent experiments. *P < 0.001, based on the Student's t-test.

Fig 4: PFKP Y64 phosphorylation-induced ß-catenin transactivation promotes brain tumor growth. (A,B) A total of 5 × 105 PFKP-depleted U87/EGFRvIII cells with reconstituted expression of the indicated proteins was intracranially injected into athymic nude mice. After 2 weeks, the mice were euthanized and examined for tumor growth. Hematoxylin-and-eosin–stained coronal brain sections show representative tumor xenografts (A). Tumor volumes were measured (B). Data represent the means ± SD of seven mice (bottom panel). *P < 0.001, #P < 0.001, based on the one-way ANOVA; n.s., not significant. Scale bar, 2 mm. (C) EGFR-phosphorylated PFKP Y64 promotes AKT activation-mediated nuclear translocation of ß-catenin, ß-catenin transactivation, TCF/LEF-induced transcription of CCND1 and MYC, leading to tumor development.

Fig 5: THZ1 disturbs the cellular metabolism of B-ALL cells by downregulating c-MYC-mediated metabolic enzymes. (A) Relative mRNA expression of c-MYC was measured by qRT-PCR in THZ1- and DMSO-treated NALM6 and REH cells. (B) Protein expression of c-MYC was determined by Western blot in THZ1- and DMSO-treated NALM6 and REH cells. (C) Relative mRNA expression of GLUT1 was measured by RNA-SEQ in THZ1- and DMSO-treated NALM6 cells. (D,E) Heatmap of glucose, amino acid, and nucleotide metabolism-related genes was analyzed by RNA-SEQ in THZ1- and DMSO-treated NALM6 cells. (F) Protein expression levels of glucose and nucleotide metabolism-related genes, such as HK1, LDHA, and PFKP, were detected by Western blot in THZ1- and DMSO-treated NALM6 and REH cells. B-ALL cells were treated with THZ1 for 24 h. Values were shown as mean ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001.