Fig 2: PGK1 phosphorylates VCP at Ser 746. (A) Lactate production in KBM5-T315I cells after treatment with NG52 (0, 6.25, 12.5, 25, 50 μmol/L) for 24 h. (B) Cell viability changes in KBM5-T315I cells after treatment with PKM2-IN-1 (0, 0.375, 0.75, 1.5, 3 μmol/L) for 24 h, as detected by CCK-8 assay. (C) Flowchart illustrating the kinase-deficient mutant of PGK1 (T378P). (D, E) Phosphorylation reactions of VCP protein, PGK1-WT, and PGK1-T378P, VCP-WT, VCP-S746A, VCP-S748A in the presence or absence of ATP were conducted in a defined system. VCP phosphorylation was detected using a pan-phospho-serine/threonine antibody. (F) KBM5-T315I cells were treated with 50 μmol/L NG52 for 24 h. Cells were then immunoprecipitated with an anti-VCP antibody, followed by Western blot analysis. Phosphorylation was detected using a pan-phospho-serine/threonine antibody. (G–I) PGK1-OE and PGK1-KD cells were immunoprecipitated with an anti-VCP antibody, followed by Western blot analysis. Phosphorylation was detected using a pan-phospho-serine/threonine antibody; PGK1-KD cells were transfected with PGK1-WT cDNA or PGK1-T378P cDNA. WT and T378P cells were immunoprecipitated with an anti-VCP antibody, followed by Western blot analysis. Phosphorylation was detected using a pan-phospho-serine/threonine antibody. Error bars: SEM, n = 3. Statistical significance of differences in mean values: ∗∗P < 0.01.

Fig 3: Chemical screening and identification of CPU-216 directly bound to PGK1. (A) A summary of the search for small molecular compounds that target PGK1. (B) Inhibition of ATP production by candidate molecules at 10 μmol/L for 12 h in HEK-293T. (C) ATP production activity was assessed by adding various synthetic derivatives of flavonoids individually 6 μmol/L for 12 h in HEK-293T. (D) The ATP production of CPU-216 (0, 2, 4, 6 μmol/L) in HEK-293T at 12 h. (E) The inhibitory effect of CPU-216 (0, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 nmol/L) on PGK1 enzyme activity. (F) The kinase profiling selectivity investigation of CPU-216 at a concentration of 0.5 μmol/L was carried out by ICE Bioscience using the ICEKP Kinome Panel, based on 80 kinase activity assays. (G) Direct binding of PGK1 to CPU-216. (H) CETSA melt curve of PGK1 in KBM5-T315I cells, with and without the presence of CPU-216 (6 μmol/L), after heat treatment. (I) Immunoblot analysis of PGK1 in KBM5-T315I cells treated with 0, 2, 4, and 6 μmol/L CPU-216 and pronase (1:50). (J) The interaction between CPU-216 and PGK1 was detected by Microscale Thermophoresis (MST). (K) Molecular docking analysis of CPU-216 and PGK1. (L) CML cells were treated with 0–80 μmol/L CPU-216 for 12 h, and cell viability was assessed using the CCK-8 assay. (M) KBM5-T315I-PGK1-WT, PGK1-OE, PGK1-KD, and PGK1-T378P cells were treated with 0–80 μmol/L CPU-216 for 12 h, and cell viability was assessed using the CCK-8 assay. (N) KBM5-T315I-VCP-KD cells were transfected with VCP-WT cDNA or VCP-E689A cDNA. WT and E689A cells were treated with 0–80 μmol/L CPU-216 for 12 h, and cell viability were assessed using the CCK-8 assay. (O) The effects of CPU-216 combination with imatinib or ponatinib, on the growth and viability of KBM5-T315I cells. Synergy score (δ-score) >10: synergy; <–10: antagonism. Error bars: SEM, n = 3. Statistical significance of differences in mean values: ∗P < 0.05, ∗∗P < 0.01.

Fig 4: In vivo studies demonstrated the anti-T315I-mutant CML activity of CPU-216. (A, B) The effects of imatinib, NG52, and CPU-216 on tumor growth in KBM5-T315I cell-bearing xenograft models were investigated, with measurements of tumor volume (Error bars: SEM, n = 5. Statistical significance of differences in mean values: ∗P < 0.05, ∗∗P < 0.01.). (C, D) The expression of LC3B-II and SQSTM1 in xenograft tumor tissue samples from KBM5-T315I cell-bearing mice was analyzed by immunofluorescence. Green or red fluorescence indicates the expression of LC3 and SQSTM1, respectively, while nuclei were stained with DAPI. Samples were evaluated using a confocal microscope (FV1000; Olympus) with FV10-ASW2.1 acquisition software (Olympus) at room temperature (original magnification 1000 ×; immersion objective 60 × with immersion oil type). (E) Mice were sacrificed after treatment with CPU-216, and their tissues were resected and fixed in 4% formalin for hematoxylin and eosin (H&E) analysis. Images were acquired at 20 × magnification. (F, G) Cell proliferation in KBM5-T315I cells was assessed by Ki-67 staining after treatment with imatinib, NG52, and CPU-216 (Error bars: SEM, n = 3. Statistical significance of differences in mean values: ∗P < 0.05, ∗∗P < 0.01). (H) The expression levels of autophagy-related proteins under the treatment of NG52 and CPU-216. (I) In NG52 or CPU-216-treated tumor tissues, VCP phosphorylation was detected. (J, M) Effects of imatinib, CPU-216, and imatinib plus CPU-216 on the spleen weight in KBM5-T315I cells-bearing NOD/SCID mice were assessed. Representative photographs of the spleens were also displayed. (K) HuCD45 expression was examined in cells from the blood of each group of KBM5-T315I cells-bearing mice by flow cytometry. (L) Kaplan–Meier survival plots for KBM5-T315I cell-bearing NOD/SCID mice were presented (Error bars: SEM, n ≥3, compared with the control group. Statistical significance of differences in mean values: ∗P < 0.05, ∗∗P < 0.01).

Fig 5: Inhibition of PGK1 induces autophagy in T315I-mutant CML cells by increasing the deubiquitination of Beclin 1. (A, B) Expression levels of LC3B-II and Beclin 1 were analyzed by Western blot in PGK1-OE, PGK1-KD, PGK1-T378P cells, β-actin was used as a loading control. (C) Co-IP experiments demonstrated an interaction between VCP and Beclin 1. (D) Western blot analysis of the level of ubiquitination in KBM5-T315I cells treated with NG52 (50 μmol/L) and 10 μmol/L MG132 for 24 h. (E) Transmission electron microscopy (TEM) was used to observe lysosomal morphology following treatment with 50 μmol/L of NG52. (F, G) The expression levels of autophagy-related proteins under different concentrations of NG52 treatment. (H) Flow cytometric analysis of fluorescence intensity values of autophagy and GEOmean analysis. (I, J) Transcriptional expression of autophagy-related genes under different concentrations of NG52 treatment. (K) The effect of NG52 on LC3B accumulation in KBM5-T315I cells at different time points. (L) The expression levels of BCR-ABL were analyzed by Western blot in KBM5-T315I cells after NG52 (0, 12.5, 25, 50 μmol/L) treatment for 24 h in the presence or absence of 5 mmol/L 3-MA, β-actin was used as a loading control. (M) Expression levels of BCR-ABL were analyzed by Western blot in PGK1-OE, PGK1-KD, and PGK1-MT cells, β-actin was used as a loading control. Error bars: SEM, n ≥3. Statistical significance of differences in mean values: ∗P < 0.05, ∗∗P < 0.01.