Research ArticleCancer

Systems biology–based drug repositioning identifies digoxin as a potential therapy for groups 3 and 4 medulloblastoma

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Science Translational Medicine  24 Oct 2018:
Vol. 10, Issue 464, eaat0150
DOI: 10.1126/scitranslmed.aat0150
  • Fig. 1 Workflow of the systems biology–driven drug repositioning strategy.

    The systematic drug repositioning strategy included two major components: (A) uncovering driver signaling networks by integrating multiple genomic profiles through a NPBSA algorithm and (B) an integrative analysis of DFNs and driver signaling networks for systematic drug repositioning. DNA-seq, DNA sequencing; KEGG, Kyoto Encyclopedia of Genes and Genomes; CNV, copy number variation; NCI-PID, NCI Pathway Interaction Database.

  • Fig. 2 Correlation of driver network activity with patient with groups 3 and 4 MB overall survival.

    (A) Driver network activity– and (B) driver network gene–based clusters for group 3 MB correlated with differences in patient survival (P = 0.0232 and P = 0.0349, respectively). (C) Genome-wide mRNA expression–based clusters for group 3 MB were not associated with significant differences in patient survival (P = 0.290). (D) Driver network activity– and (E) driver network gene–based clusters for group 4 MB correlated with differences in patient survival (P = 0.00664 and P = 0.0077, respectively). (F) Genome-wide gene expression–based clusters for group 4 MB were not associated with significant differences in survival (P = 0.441).

  • Fig. 3 Cardiac glycosides as candidate drugs for groups 3 and 4 MB.

    (A) Comparison of in vitro inhibition of cell viability predicted by DSNI-DFN versus CMAP-GSEA. MED8A cells were incubated for 72 hours at 10 µM with predicted drugs, after which cell viability was assessed. (B) Details of a drug cluster predicted to strongly inhibit cell viability in groups 3 and 4 MB. This cluster contains known anticancer compounds and multiple members of the cardiac glycoside family. (C) Ranked order of cardiac glycosides predicted by DSNI-DFN in groups 3 and 4 SHH and WNT MB. FDA, U.S. Food and Drug Administration. (D) Schematic of drug-drug (red) and drug-target (dark blue) interactions for the cardiac glycoside digoxin derived from the STITCH database.

  • Fig. 4 Cardiac glycosides inhibit MB cell growth in vitro and prolong survival in vivo.

    (A) Dose-response curve for the cardiac glycosides (proscillaridin A, digoxin, lanatoside C, digitoxigenin, and digoxigenin) on MB-derived cell lines (MED8A and D283) and the control glioblastoma line (U87). Cell viability was assessed at 72 hours. (B) IC50 values for the dose titration experiment. (C) Schematic of orthotopic PDX model treatment with digoxin. Twenty-four days after tumor cell implantation, mice received digoxin (2 mg/kg ip daily) for 14 days, followed by 21 days without treatment, and followed by another 14 days of treatment. (D) Hematoxylin and eosin (H&E) staining examples of untreated tumors from orthotopic PDX models of group 3 (ICb-2555MB) and group 4 (ICb-1078MB) MB. (E and F) Kaplan-Meier curves showing digoxin-treated mice compared to untreated controls. (E) Digoxin treatment prolonged survival in an orthotopic PDX model of group 4 MB (ICb-1078MB) to 113 days (n = 7) versus 92 days (n = 6) for untreated controls (log-rank test, P = 0.001). Two long-surviving mice were euthanized at 219 days while asymptomatic. Gross-scale image of one long-surviving mouse with large tumor and H&E staining demonstrating no evidence of tumor in the two long-surviving mice, one with a tumor and one without is shown. (F) Digoxin treatment prolonged survival in an orthotopic PDX model of group 3 MB (ICb-2555MB) to 180 days (n = 10) versus 102 days (n = 8) for untreated controls (log-rank test, P < 0.001). H&E comparison of two long-term surviving mice, one with a microscopic tumor and one without is shown. Scale bars, 1 mm (black) and 0.1 mm (white).

  • Fig. 5 Comparison of the effects of digoxin and ionizing radiation alone and in combination on survival in an orthotopic PDX model of group 3 MB.

    (A) Treatment schematic for radiation alone. Seventeen days after implantation, mice received fractionated radiation to the craniospinal axis (2 Gy/day) for 5 days, followed by 2 days of recovery, and then 5 days focal radiation to the cerebellum (2 Gy/day). CSI, craniospinal irradiation. (B) Treatment schematic of combination therapy. Radiation therapy (XRT) was initiated on day 17 described in (A), with initiation of digoxin treatments 24 days after implantation, mice received digoxin (2 mg/kg ip daily) for 14 days, followed by 21 days with no treatment, and then an additional 14 days of treatment. (C) Kaplan-Meier curve comparing the median survival of ICb-2555MB tumor-bearing mice: untreated (n = 8, 102 days), digoxin-treated (n = 10, 180 days), and radiation alone (n = 10, 167 days). Both digoxin single-agent therapy (log-rank test, P = 0.007) and radiation alone (log-rank test, P < 0.001) showed significant prolongation of survival relative untreated controls but were comparable when compared to one another (log-rank test, P = 0.91) (D) Kaplan-Meier curve comparing the median survival of untreated (n = 8, 102 days), radiation only (n = 10, 167 days), and combination therapy–treated (n = 10, 219 days) mice. Combination therapy showed a significant prolongation of survival compared to both radiation alone (log-rank test, P = 0.04) and untreated controls (log-rank test, P < 0.001).

  • Fig. 6 Cellular responses to digoxin in orthotopic PDX models of MB.

    (A) TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining of tumors from group 3 (ICb-2555MB) and group 4 (ICb-1078MB) models before digoxin treatment, during treatment, or at time of recurrence. Scale bar, 25 mM. (B and C) Quantitation of TUNEL staining from group 3 (ICb-2555MB) (B) and group 4 (ICb-1078MB) (C). hpf, high-power field. (D and E) Heatmaps depicting changes gene expression in group 3 tumor (D) (ICb-2555 MB) and group 4 (E) (ICb-1078MB) tumors before and after digoxin treatment. Red text denotes genes that are differentially expressed in both groups 3 and 4 MB. For the heatmap, red indicates up-regulation, and green indicates down-regulation.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/464/eaat0150/DC1

    Materials and Methods

    Fig. S1. Cardiac glycosides do not inhibit the growth of the triple-negative breast cancer cell line MDA-MB231 in vitro.

    Fig. S2. Establishment of ICb-2555MB, an orthotopic PDX model of group 3 MB.

    Fig. S3. Assessment of long-surviving ICb-2555MB digoxin-treated mice for tumor formation.

    Fig. S4. Digoxin plasma trough concentrations in tumor-bearing mice.

    Fig. S5. Comparison of alternative single-agent digoxin treatment regimen with ionizing radiation alone in a group 3 model of MB.

    Fig. S6. Comparison of a combination of digoxin and radiation with single-agent digoxin alone in a group 3 model of MB.

    Fig. S7. Quality assessment of DFNs.

    Table S1. Summary of patient data used to generate driver networks.

    Table S2. Driver genes that consist of somatic mutated genes, copy number variation genes, and differentially expressed genes in group 3 MB.

    Table S3. Driver genes that consist of somatic mutated genes, copy number variation genes, and differentially expressed genes in group 4 MB.

    Table S4. Identified driver signaling networks for group 3 MB.

    Table S5. Identified driver signaling networks for group 4 MB.

    Table S6. Comparison of drug prediction performance between the two computational methods (DSNI-DFN versus CMAP-GSEA).

    Table S7. Ranking order of drugs in DSNI-DFN for group 3 MB.

    Table S8. Ranking order of drugs in CMAP-GSEA for group 3 MB.

    Table S9. Digoxin plasma trough concentrations in individual tumor-bearing mice.

    Table S10. Thirty most differentially expressed genes after digoxin treatment of an orthotopic PDX model (ICb-2555MB) of group 3 MB.

    Table S11. Thirty most differentially expressed genes after digoxin treatment of an orthotopic PDX model (ICb-1078MB) of group 4 MB.

    Table S12. Pathway enrichment analysis results using differentially expressed genes in digoxin-treated orthotopic PDX tumors of group 3 MB.

    Table S13. Pathway enrichment analysis results using differentially expressed genes in digoxin-treated orthotopic PDX tumors of group 4 MB.

    References (4853)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Cardiac glycosides do not inhibit the growth of the triple-negative breast cancer cell line MDA-MB231 in vitro.
    • Fig. S2. Establishment of ICb-2555MB, an orthotopic PDX model of group 3 MB.
    • Fig. S3. Assessment of long-surviving ICb-2555MB digoxin-treated mice for tumor formation.
    • Fig. S4. Digoxin plasma trough concentrations in tumor-bearing mice.
    • Fig. S5. Comparison of alternative single-agent digoxin treatment regimen with ionizing radiation alone in a group 3 model of MB.
    • Fig. S6. Comparison of a combination of digoxin and radiation with single-agent digoxin alone in a group 3 model of MB.
    • Fig. S7. Quality assessment of DFNs.
    • Table S1. Summary of patient data used to generate driver networks.
    • Table S6. Comparison of drug prediction performance between the two computational methods (DSNI-DFN versus CMAP-GSEA).
    • Table S9. Digoxin plasma trough concentrations in individual tumor-bearing mice.
    • Table S10. Thirty most differentially expressed genes after digoxin treatment of an orthotopic PDX model (ICb-2555MB) of group 3 MB.
    • Table S11. Thirty most differentially expressed genes after digoxin treatment of an orthotopic PDX model (ICb-1078MB) of group 4 MB.
    • References (4853)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Driver genes that consist of somatic mutated genes, copy number variation genes, and differentially expressed genes in group 3 MB.
    • Table S3 (Microsoft Excel format). Driver genes that consist of somatic mutated genes, copy number variation genes, and differentially expressed genes in group 4 MB.
    • Table S4 (Microsoft Excel format). Identified driver signaling networks for group 3 MB.
    • Table S5 (Microsoft Excel format). Identified driver signaling networks for group 4 MB.
    • Table S7 (Microsoft Excel format). Ranking order of drugs in DSNI-DFN for group 3 MB.
    • Table S8 (Microsoft Excel format). Ranking order of drugs in CMAP-GSEA for group 3 MB.
    • Table S12 (Microsoft Excel format). Pathway enrichment analysis results using differentially expressed genes in digoxin-treated orthotopic PDX tumors of group 3 MB.
    • Table S13 (Microsoft Excel format). Pathway enrichment analysis results using differentially expressed genes in digoxin-treated orthotopic PDX tumors of group 4 MB.

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