Fig 2: The expression of PFKFB3/ENO2 was regulated by PPFIA4. (A) The correlation between PPFIA4 and PFKFB3 was analyzed in TCGA colorectal adenocarcinoma dataset. (B) The correlation between PPFIA4 and ENO2 was analyzed in TCGA colorectal adenocarcinoma dataset. (C) Expression of PFKFB3 in normal and colon adenocarcinoma tissues in TCGA datasets. (D) Expression of ENO2 in normal and colon adenocarcinoma tissues in TCGA datasets. (E, F) The mRNA (E) and protein (F) expression of PFKFB3/ENO2 in SW403 or SW403-PPFIA4. (G) The protein levels of PFKFB3/ENO2 in HCT116 cells transfected with PPFIA4 shRNA. **p < 0.01, ***p < 0.001.

Fig 3: Resistance is associated with an enhanced glycolytic phenotype. (A) Drug‐naïve (N) or PM‐resistant (Res) cells were treated with the combination of panobinostat and marizomib (25 nm each) for 48 h. After harvesting, cells were labeled with annexin V and propidium iodide, and cell viability was assessed by flow cytometry. The results represent the mean of three independent experiments. Error bars indicate ± SD (****P < 0.0001; naive‐treated vs. resistant cells; unpaired two‐tailed t‐test). (B) Drug‐naïve (upper panel) or PM‐resistant cells (lower panel) were allowed to attach overnight in a 6‐well plate. Cells were harvested and stained with propidium iodide (PI). Cell cycle profile was assessed by flow cytometry. Representative images acquired from three independent experiments for each cell type are shown. (C) Principal component analysis (PCA) plot of RNA‐seq data comparing drug‐naïve control DIPG‐0007 and DIPG‐013 with PM‐resistant cell lines (n = 3). (D) Volcano plot of differential gene expression (FDR, false discovery rate) analysis of RNA‐seq data comparing drug‐naïve vs. PM‐resistant DIPG‐007 (left panel) and DIPG‐013 (right panel) cell lines (n = 3). The number of upregulated genes that are highlighted in red FDR < 0.01, Log2fold > 1.5, and downregulated genes that are highlighted in Blue FDR < 0.01, Log2fold < −1.5 (n = 3). (E) Venn‐diagram showing the number of upregulated genes (upper panel) and downregulated (lower panel) genes (n = 3). (F) Gene set enrichment analysis (GSEA) plots (NES, normalized enrichment score) showing glycolysis and hypoxia gene sets of DIPG‐007 and DIPG‐013 of drug‐naïve vs. PM‐resistant cell lines (n = 3). (G) Hallmark analysis of drug‐naïve vs. PM‐resistant DIPG‐013 cell lines reveals enrichment of hypoxia and glycolytic signatures (among top 10; n = 3). (H) Transcripts per million (TPM) of enolase‐2 in DIPG‐007‐ and DIPG‐013‐naïve vs. ‐resistant cell lines, the results were generated from three biological replicates (****P < 0.0001 naïve vs. resistant, analysis was performed as one‐way ANOVA). Data are displayed as mean with standard deviation. (I) Drug‐naïve (N) or PM‐resistant cells (Res) were allowed to attach overnight in a 6‐well plate. Whole cell lysates were prepared, and western blot analysis was performed with the indicated antibodies (ENO2, enolase‐2). Representative blots acquired from three independent experiments for each cell type are shown. (J) Drug‐naïve (N) and PM‐resistant (Res) U87, SJG2, and DIPG‐013 cells were seeded in 6‐well plates. On the following day, cellular pyruvate dehydrogenase (PDH) activity was assessed as described in the Section 2. The results represent the mean ± SD of three independent experiments (***P < 0.0005; ****P < 0.0001; unpaired two‐tailed t‐test). (K–Q) Quantification of TCA cycle‐related metabolites from U87‐resistant cells and drug‐naïve cells treated with the combination of panobinostat and marizomib for 72 h (ST, short‐term). Untreated control cells (N) received vehicle (DMSO). Citrate (K), cis‐aconitate (L), succinate (M), fumarate (N), malate (O), aspartate (P) and glutamine (Q). The values are represented as mean ± standard deviation of three separate experiments (K–Q). Statistical significance was assessed with Tukey's ANOVA (ns, nonsignificant, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001).

Fig 4: BRD9 enhances glycolysis by targeting ENO2 and ALDOC in colon cancer cells. (A) The ALDOC and ENO2 were identified as BRD9‐regulated genes. The expression of a panel of glucose metabolism‐related genes was assessed by qRT‐PCR in BRD9‐overexpressing SW620 cells. (B) qRT‐PCR assays showing the mRNA levels of ENO2 and ALDOC in BRD9‐deficient and WT cells. (C) Western‐blotting assays detecting the protein levels of ENO2 and ALDOC in the indicated cell samples. (D) ChIP‐qPCR assay of BRD9 binding and H3K27ac in the indicated genes, where KLK2 and BCHE were selected as the positive control. (E, F) Correlation analysis in the TCGA‐COAD cohort revealing the associations between BRD9 and ENO2 or ALDOC, respectively. (G) Kaplan–Meier analysis exhibiting the survival time of patients with high ALDOC and low ALDOC, individually. (H) Kaplan–Meier curve with log‐rank test showing the clinical significance of ENO2 in COAD samples. Data are displayed as mean ± standard deviation. ***p < 0.001, **p < 0.01, *p < 0.05.