Research ArticleBRAIN TUMORS

KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice

See allHide authors and affiliations

Science Translational Medicine  15 Aug 2018:
Vol. 10, Issue 454, eaar2718
DOI: 10.1126/scitranslmed.aar2718
  • Fig. 1 KHS101 exhibits cytotoxicity in molecularly diverse GBM models.

    (A) PCA in individual cells within the patient-derived GBM and NP1 lines. (B) Radar plots depicting the GBM subtype compartments [classical (C), proneural (P), mesenchymal (M), and neural (N)] of the GBM1 (left), GBM11 (middle), and GBM20 (right) models. (C) Real-time assessment of cellular confluency (normalized to t0 values) in GBM1, GBM11, GBM20, and NP1 models before and after treatment (arrowheads) with DMSO (0.1%) or KHS101 (7.5 μM). A single experiment out of three biological replicates is shown (see file S2 for all data). **P < 0.001 and ****P < 0.0001, two-way analysis of variance (ANOVA). (D) Dose-response curves (normalized to the DMSO control) and the corresponding IC50 (half-maximal inhibitory concentration) values (micromolar; with 95% confidence intervals) are shown for the indicated cell models and KHS101 concentrations after a 5-day treatment period. Data are means ± SD of three biological replicates.

  • Fig. 2 KHS101 selectively induces an autophagic and proapoptotic cell fate across a spectrum of GBM cell models.

    (A) EM and immunocytochemistry [phase-contrast (Phc); anti-LC3B; 4′,6-diamidino-2-phenylindole (DAPI)] images (scale bars, 5 and 25 μm, respectively) of GBM1 and NP1 cells 12 hours after KHS101 (7.5 μM) or DMSO (0.1%) treatments. (B) Immunocytochemistry (phase-contrast; anti-LC3B; DAPI) images (scale bar, 30 μm) of indicated cell models 12 hours after KHS101 (7.5 μM) or DMSO (0.1%) treatments. (C) Quantification of the LC3B-positive cytoplasmic area (percentage) 12 hours after treatment with KHS101 (at the indicated concentrations) or DMSO (D; 0.1%) using the specified cell models. (D) Quantification of CYTO-ID–positive GBM1 cells 12 hours after treatment with DMSO (D; 0.1%) or KHS101 (at the indicated concentrations). (E) Kinetics of caspase 3/7 activation in GBM1 cells treated with KHS101 (7.5 μM) or DMSO (0.1%; data were normalized to to). (F) Relative caspase 3/7 activation (at the 48-hour time point) in response to DMSO (D; 0.1%) or KHS101 (K; 7.5 μM) in the specified cell models. N, negative control (K; 7.5 μM + pan-caspase inhibitor Z-VAD-FMK; 2 μM); P, positive control (staurosporine; 1 μM). Data are means ± SD of three biological replicates, **P < 0.01, Student’s t test (two-tailed).

  • Fig. 3 KHS101 induces acute metabolic stress in GBM cells.

    (A) Kinetics of autophagy induction in CYTO-ID–labeled GBM1 cells upon KHS101 (7.5 μM) or DMSO (0.1%) addition. Individual data points of two biological replicates are shown. (B) Hypergeometric gene enrichment test (left; OXPHOS and TCA cycle gene set enrichment is highlighted), and radar plot (right) indicating marked (more than twofold) alterations in cell cycle/mitosis, metabolic, and stemness pathways in GBM1 cells 24 hours after KHS101 treatment (7.5 μM) compared with the DMSO control (0.1%). FC indicates fold change. (C) qRT-PCR radar charts depicting KHS101-induced (7.5 μM) mRNA expression changes (in relation to the DMSO control; FC range, ≥10 and <30) in GBM1 and GBM20 cell models (left and middle) and the lack of a similar response in NP1 cells, or by TACC3 silencing in GBM1 cells (right). (D) Metabolic phenogram. Basal extracellular flux rates (OCR and ECAR) of the specified cell types are shown in response to vehicle (DMSO; 0.1%) or KHS101 (7.5 μM) treatments. Quadrants indicate the specified metabolic phenotypes. Data are means ± SD of three biological replicates. ****P < 0.0001, Student’s t test (two-tailed, equal variance).

  • Fig. 4 KHS101 impairs relative incorporation of glucose-derived carbon through glycolysis and the TCA cycle in GBM cells.

    (A) Gas chromatography–mass spectrometry stable isotope analysis of methoximation and silylation-derivatized metabolites extracted from NP1 and GBM1 cells after a 4-hour treatment with KHS101 (7.5 μM) or DMSO (0.1%) in medium containing U-13C glucose. Graph shows the fractional enrichment (percentage) in the isotopologs of glucose. (B to L) Fractional enrichments of fructose 6-phosphate (F6P) (B), dihydroxyacetone phosphate (DHAP) (C), glyceraldehyde 3-phosphate (GAP) (D), glycerol 3-phosphate (G3P) (E), phosphoenolpyruvate (PEP) (F), lactate (G), citrate (H), succinate (I), fumarate (J), malate (K), and aspartate (L). The x axis indicates the mass isotopomers (which are designated as M0, M1, M2…Mn, where n is the number of labeled atoms in the molecule) in the specified metabolites (corrected for 13C natural abundance; lactate M2 is not shown, as enrichment above natural abundance was not detected). Data are means ± SEM of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, one-way ANOVA (Tukey post hoc test).

  • Fig. 5 KHS101 interacts with mitochondrial HSPD1.

    (A) Two-dimensional SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting of GBM1 cell lysates (20 to 40% ammonium sulfate–precipitated fraction) detecting KHS101-BP–labeled protein in the presence or absence of unlabeled KHS101 (as specified) after photocrosslinking (30 min) and biotin-tag labeling (click chemistry reaction using biotin-azide). Asterisk: 60 kDa. Right inlay shows the relative reduction of candidate compound-protein complex signal (%; spots 1–4) in the presence of unlabeled KHS101. Median of three technical repeats (back dots) is shown. Spot 1 corresponded to HSPD1 (identified by proteomics analysis after protein spot excision). (B) Specific in vitro binding of recombinant human HSPD1 with biotinylated KHS101 (KHS101-bio) was detected by silver staining of SDS-PAGE gels in the presence/absence of unlabeled KHS101, precipitated with streptavidin-conjugated agarose beads. Asterisk, 60 kDa. (C) HSPD1 mitochondrial (M)–to–cytoplasmic (C) ratio in GBM1 and NP1 cells as assessed by immunoblot quantification. Black dots represent biological replicates (median ± SD is shown). **P < 0.01, Student’s t test (two-tailed, equal variance). (D) Relative mitochondrial HSPD1 protein expression (percentage; normalized to control values as assessed by immunoblot analysis) 6 hours after DMSO (D; 0.1%) or KHS101 (K; 7.5 μM) treatment in GBM1 cells. (E) HSPD1 mRNA expression (fold changes) in GBM1 cells treated with DMSO (D; 0.1%) or KHS101 (K; 7.5 μM). SD of three biological repeats (black dots) is shown.

  • Fig. 6 KHS101 induces HSPD1-dependent aggregation of metabolic enzymes.

    (A) HSPD1/HSPE1 substrate refolding activity in the presence of KHS101 (IC50, 14.4 μM). Data are means ± SD of three replicates. (B) HSPD1 complex substrate refolding activity in the presence of HB072 (inactive KHS101 analog) and MC (mitochondrial HSPD1-binding compound). Data are means ± SD of three replicates. (C) Left: Silver staining of aggregated (pellet) and soluble (supernatant) mitochondrial fractions (solubilized with 0.5% NP-40) from NP1 and GBM1 cells treated with DMSO (D; 0.1%) or KHS101 (K; 7.5 μM) for 1 hour. Right: KHS101-induced protein enrichment as assessed for the aggregated/pellet (P) and soluble/supernatant (S) fractions in GBM1 versus NP1 cells. Data are GBM1/NP1 ratios of three biological replicates ± SD. **P < 0.01, Student’s t test (two-tailed, equal variance). Aggregated proteins were identified by mass spectrometry (table S2). (D) KHS101-GBM protein aggregation represented in a predicted (STRING) interaction network of proteins (homo sapiens; network edges; confidence; line thickness indicating strength of data support). Only glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shared between NP1 and GBM1 cells. Green, red, blue, and yellow colors represent enzymatic functions in protein folding, glycolysis, OXPHOS, and glycine metabolism, respectively. (E) Quantitative proteome analysis identifying differentially regulated proteins and the specified enrichment sets in GBM1 cells treated with KHS101 (7.5 μM) for 1 hour. Data (log fold change) are calculated from change in average protein levels between 1 hour and t0. The −log10 of the Benjamini and Hochberg false discovery rate–adjusted P values was obtained from group-wise comparison (red line depicts P = 0.05).

  • Fig. 7 KHS101 significantly attenuates GBM growth in vivo.

    (A) Confocal microscopic images (scale bar, 40 μm) and quantification of NAD(P)H AF in the vehicle (n = 4) or KHS101 (n = 4) treatment groups (using three different tissue sections per specimen). Nuclei were stained with propidium iodide (PI). (B) Immunocytochemistry-based quantification of HK2- and MKI67-positive tumor area in the vehicle (V; n = 5) or KHS101 (K; n = 6) treatment groups (using ≥3 different tumor sections per specimen). (C) Bean plot of MKI67 mRNA expression in single GBM1 cells 5 days after DMSO (D; 0.1%) or KHS101 (K; 7.5 μM) treatment. (D) Clonal growth capacity of individual GBM1 cells in the presence of DMSO (D; 0.1%) or KHS101 (K; 1 or 7.5 μM). (E) Quantification of acellular/pyknotic areas in anterior GBM1 tumor sections; V, vehicle; K, KHS101. Dots represent individual tumor measurements. (F) GBM1 xenograft tumor size (percent tumor area of the sectioned brain) in vehicle- or KHS101-treated animals assessed by hematoxylin and eosin staining in sequential brain areas (frontal to caudal; scale bar, 2 mm). Dots represent individual tumors. (G) Imaging and quantification of vimentin-positive GBM1 xenograft tumor cells infiltrating the corpus callosum (CC) of the hemisphere contralateral to the injection site in animals of the vehicle (n = 5) or KHS101 (n = 6) treatment groups (using ≥3 sections per xenograft tumor). Dotted line indicates border of CC and striatum (S). Scale bar, 300 μm. All boxplots show the 10 to 90 percentile and median. *P < 0.05 and **P < 0.01, Mann-Whitney U test (one-tailed).

  • Fig. 8 KHS101 treatment increases survival in the GBMX1 in vivo model.

    (A) Survival analysis of the specified glioma subtype categories and median preset thresholds for HSPD1 mRNA expression (using default settings available from www.betastasis.com/glioma/rembrandt/kaplan_meier_survival_curve/. (B) Kaplan-Meier (log-rank test) analysis of GBMX1 tumor–carrying animals. Tumors were established over a 2-week period followed by 10 weeks of vehicle (n = 8) or KHS101 (n = 8) treatment. (C) Kaplan-Meier (log-rank test) analysis of GBMX1 tumors that were allowed to establish over a 6-week period followed by continuous vehicle (n = 4) or KHS101 (n = 5) treatment until the endpoint (arrowhead). (D) GBMX1 xenograft tumor size (percent tumor area of the sectioned brain) in vehicle- or KHS101-treated brains at their respective endpoints [shown in (C)] assessed by hematoxylin and eosin staining (using ≥4 sections per specimen; scale bar, 2 mm). Boxplot shows the 10 to 90 percentile and median, and dots represent individual (brain section) values. **P < 0.01, Mann-Whitney U test (one-tailed).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/454/eaar2718/DC1

    Materials and Methods

    Fig. S1. KHS101 reduces GBM cell viability in different culture conditions.

    Fig. S2. TACC3 down-regulation is not causally linked to KHS101-induced GBM cell degradation.

    Fig. S3. KHS101 disrupts ATP production in GBM cells.

    Fig. S4. Schematic representation of glucose carbon tracing through glycolysis, the TCA cycle, malic enzyme, and pyruvate carboxylase reactions.

    Fig. S5. KHS101-BP and KHS101 show comparable bioactivities in GBM cells.

    Fig. S6. RNA interference–mediated knockdown of HSPD1 results in reduced oxidative capacity and proliferation in GBM cells.

    Fig. S7. MC recapitulates KHS101 cytotoxicity in GBM cells.

    Fig. S8. Time-dependent changes in protein abundances in KHS101-treated GBM1 cells.

    Fig. S9. KHS101 induces NAD(P)H/NAD+ imbalance in GBM1 cells and xenograft tumors.

    Fig. S10. Antitumor effects of KHS101 in xenograft tumor experiments.

    Table S1. Overview of patient-derived GBM cell model characterization.

    Table S2. List of aggregated proteins (and their known functions) that were identified within the mitochondrial fraction of KHS101-treated GBM1 cells.

    Table S3. List of Delta-gene assays (Fluidigm) used for single-cell qRT-PCR analysis.

    Table S4. Chemical structures of the indicated compounds.

    Table S5. List of TaqMan probes used for high-throughput qRT-PCR on bulk cell populations.

    File S1. Report of the bioinformatics (R) analysis using single-cell gene expression data.

    File S2. Study data in tabular format and organized by figure.

    File S3. Supplementary lists of up- and down-regulated proteins.

    File S4. Description of chemical synthesis and compound characterization.

    Movie S1. Live cell imaging of GBM1 cells comparing DMSO (0.1%; left) with KHS101 treatment (7.5 μM; right).

    Reference (60)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. KHS101 reduces GBM cell viability in different culture conditions.
    • Fig. S2. TACC3 down-regulation is not causally linked to KHS101-induced GBM cell degradation.
    • Fig. S3. KHS101 disrupts ATP production in GBM cells.
    • Fig. S4. Schematic representation of glucose carbon tracing through glycolysis, the TCA cycle, malic enzyme, and pyruvate carboxylase reactions.
    • Fig. S5. KHS101-BP and KHS101 show comparable bioactivities in GBM cells.
    • Fig. S6. RNA interference–mediated knockdown of HSPD1 results in reduced oxidative capacity and proliferation in GBM cells.
    • Fig. S7. MC recapitulates KHS101 cytotoxicity in GBM cells.
    • Fig. S8. Time-dependent changes in protein abundances in KHS101-treated GBM1 cells.
    • Fig. S9. KHS101 induces NAD(P)H/NAD+ imbalance in GBM1 cells and xenograft tumors.
    • Fig. S10. Antitumor effects of KHS101 in xenograft tumor experiments.
    • Table S1. Overview of patient-derived GBM cell model characterization.
    • Table S2. List of aggregated proteins (and their known functions) that were identified within the mitochondrial fraction of KHS101-treated GBM1 cells.
    • Table S3. List of Delta-gene assays (Fluidigm) used for single-cell qRT-PCR analysis.
    • Table S4. Chemical structures of the indicated compounds.
    • Table S5. List of TaqMan probes used for high-throughput qRT-PCR on bulk cell populations.
    • Legends for files S1 to S4
    • Legend for movie S1
    • Reference (60)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • File S1 (.pdf format). Report of the bioinformatics (R) analysis using single-cell gene expression data.
    • File S2 (Microsoft Excel format). Study data in tabular format and organized by figure.
    • File S3 (Microsoft Excel format). Supplementary lists of up- and down-regulated proteins.
    • File S4 (.pdf format). Description of chemical synthesis and compound characterization.
    • Movie S1 (.mov format). Live cell imaging of GBM1 cells comparing DMSO (0.1%; left) with KHS101 treatment (7.5 μM; right).

    [Download Files S1 to S4]

Stay Connected to Science Translational Medicine

Navigate This Article