Research ArticleCancer

Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening

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Science Translational Medicine  20 Nov 2019:
Vol. 11, Issue 519, eaaw0064
DOI: 10.1126/scitranslmed.aaw0064
  • Fig. 1 Drug candidates identified through high-throughput drug screening in DIPG.

    (A) Heat map representation of drug activities for four DIPG cell cultures (JHH-DIPG-1, SU-DIPG-XIII, SU-DIPG-XVII, and SU-DIPG-XXV) screened versus the MIPE 5.0 library. Activity scores are based on Z-AUC values. Tick marks on the right side of the plots highlight the relative ranking of proteasome and HDAC inhibitors, as well as the relative rankings of agents in active clinical evaluation in DIPG. (B) Mechanistic drug classes enriched among the 371 hits selected on the basis of consistent potency across DIPG cell cultures. Enrichment was defined as number of hits ≥2 and target coverage ≥35%. (C) Dose-response curves for selected agents from key enriched mechanistic classes (B) including panobinostat (HDAC), selumetinib (MEK), BMS-754807 (IGFR), and buparlisib (PI3K). (D) Potency (AC50) distribution for proteasome (red) and HDAC (blue) inhibitors compared with the 371 potency-selected hits (gray). (E) Potency (AC50) distribution for proteasome (red) and HDAC (blue) inhibitors compared with agents in active clinical evaluation in DIPG (mustard). (F) For the 371 potency-selected hits, Z-AUC values were compared to predicted CNS penetration using MPO scores. The color scheme is the same as in (E). (G) Distribution of MPO scores for proteasome inhibitors (red), HDAC inhibitors (blue), and agents in active clinical evaluation in DIPG (mustard). (H) Dose-response curves for the proteasome inhibitor marizomib.

  • Fig. 2 Synergistic drug-drug interactions in DIPG identified via HTS-enabled combination drug screening.

    (A) Schematic layout of the drug-versus-all screen for panobinostat and marizomib versus the entire MIPE 5.0 library. Top: Each drug pair was tested in a 6 × 6 matrix block, reflecting five doses plus DMSO control for each individual matrix block. Middle: An exemplar 1536 plate containing 6 × 6 blocks. Bottom: Examples of percent response and ΔBliss heat maps for additivity, synergy, or antagonism outcomes. (B) The entire panobinostat (left panels) and marizomib (right panels) drug-versus-all screen results were ranked by synergy, as assessed by the ExcessHSA metric (gray). Each panel highlights drugs from key mechanistic classes including HDAC, proteasome, MEK, IGFR, and PI3K inhibitors. (C) Left: ExcessHSA values for the combination of panobinostat with the proteasome inhibitor marizomib, the IGFR inhibitor BMS-754807, the MEK inhibitor selumetinib, and the PI3K inhibitor buparlisib. Right: %Response and ΔBliss heat maps for panobinostat with BMS-754807, selumetinib, or buparlisib. (D) Left: Thirty HDAC inhibitors from the MIPE 5.0 library were ranked on the basis of the ExcessHSA values when combined with marizomib. Class I (*) and class II/III (**) HDAC inhibitors are highlighted. Right: %Response and ΔBliss heat maps, for example, class I pan-HDAC inhibitors (romidepsin and dacinostat) or class II HDAC6 inhibitors (ACY-775 and tubastatin A). (E) Left: Correlation heat map of the 45-drug all-versus-all combination screen. Subgroups of drugs with similar combination profiles are highlighted (group 1: blue; group 2: brown; group 3: orange; group 4: light green; group 5: dark green). Right: Original ExcessHSA values for groups 1, 2, and 3. Ranking is based on the average ExcessHSA within each group. These figures including plots for groups 4 and 5 are expanded in fig. S2. (F) The 10 × 10 %Response heat maps and ΔBliss heat maps for the combinations of panobinostat with either marizomib or BMS-754807.

  • Fig. 3 In vitro validation of top drug combinations across representative DIPG cell cultures.

    (A) Dose-response curves of cell viability of patient-derived DIPG cell cultures, as measured by CellTiter-Glo and compared to DMSO control after 72-hour exposure to top candidate therapeutic agents marizomib, BMS-754807, buparlisib, and selumetinib alone (top) or with 25 nM panobinostat (bottom). Vertical line is at 1000 nM (1 μM), representing an approximate range for achievable concentrations in vivo. WT, wild type. (B) Cell viability compared to DMSO control after 72-hour exposure of six patient-derived DIPG cell cultures to varying doses of panobinostat (blue), marizomib (red), or both (purple). Similar measurements for the other top drug combination candidates can be found in fig. S4A. (C) Calculated median effect drug synergy CI scores (Biosoft CalcuSyn 2.0) across doses for each of the four candidate drug combinations [individual viability measurements in (B) and fig. S4A]. Horizontal dashed line indicates a CI = 1, where points below the line indicate synergy and points above the line indicate antagonism. (D) Cell proliferation as measured by flow cytometric analysis of EdU incorporation (left) and cell death as measured by surface labeling of Annexin V (middle) and permeability to DAPI (right) of six patient-derived DIPG cell cultures. For EdU analysis, cells were incubated with DMSO vehicle (control; gray), panobinostat (blue), marizomib (red), or combination of panobinostat and marizomib (combo; purple) for 16 hours and then exposed to 10 μM EdU for 24 hours before analysis. For Annexin V and DAPI analysis, cells were incubated for 48 hours before analysis. Individual flow cytometry histograms can be found in fig. S4B.

  • Fig. 4 Panobinostat and marizomib in xenograft models of DIPG and other DMGs.

    (A) In vivo bioluminescence imaging of SU-DIPG-VI GFP-luc xenografts after 4 weeks of treatment with marizomib at 150 μg/kg once every 2 weeks (top; n = 3 vehicle controls and n = 3 treated mice) or 150 μg/kg twice every 2 weeks (bottom; n = 5 vehicle controls and n = 3 treated mice; two-tailed t test). i.v., intravenously. (B) Overall survival of SU-DIPG-XIII-P* xenografted mice treated with vehicle, panobinostat alone (top: 5 mg/kg; bottom: 10 mg/kg; three times per week, every other week), marizomib alone (top: 150 μg/kg, one time per week; bottom: two times per week, every other week), or combination (panobinostat: 5 mg/kg, three times per week, every other week; marizomib: 150 μg/kg, one time per week, every other week in both cohorts; log-rank test). (C) In vivo bioluminescence imaging of QCTB-R059 GFP-luc xenografts after 4 weeks of treatment with vehicle control, panobinostat (5 mg/kg; three times weekly, every other week), marizomib (150 μg/kg; once weekly, every other week), or combination of panobinostat and marizomib (alternating every week). One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 and **P < 0.01.

  • Fig. 5 RNA sequencing analysis of the combination of panobinostat and marizomib in SU-DIPG-XIII cells.

    (A) Heat map representations of the log2 gene expression changes (log2FC) after treatment of SU-DIPG-XIII with 50 nM panobinostat, 20 nM marizomib, or two combination doses (50 nM + 20 nM or 100 nM + 50 nM, panobinostat and marizomib, respectively). Differentially expressed genes were selected on the basis of either panobinostat versus DMSO (left) or marizomib versus DMSO (right) comparisons, and heat maps depicting all four treatments were then generated by unsupervised hierarchical clustering. (B) Volcano plot of each individual treatment set with respect to the DMSO control. Significantly down- or up-regulated genes are highlighted in blue or red, respectively. (C) A fold change preranked list of each treatment versus DMSO was used to run GSEA against the Hallmark (shown here) and Reactome (fig. S9C) gene sets. Unsupervised hierarchical clustering of normalized enrichment scores (NES) was used to generate a comprehensive heat map visualization of the functional transcriptional outputs of the four treatment sets. (D) Cytoscape enrichment map visualization of top gene programs represented by significantly up-regulated genes present in combination-low treated cells but not either single-agent treated condition. Node size represents number of genes, node color represents significance [false discovery rate (FDR)], and edge thickness represents number of shared genes. Clustered gene programs are labeled. (E) Gene expression changes for the “leading-edge” genes from the Reactome-UPR gene set. Leading selection and ranking were based on the combo-high (h) treatment set. (F) Western blot analysis of the indicated ER stress/UPR or apoptosis biomarkers is shown for all four treatment sets. Black triangles denote cleaved PARP and CC3. β-Actin was used as a loading control. (G) %Response and ΔBliss heat maps are shown for the combination of either panobinostat or marizomib with the ER modulators eeyarestatin and bafilomycin A1.

  • Fig. 6 Targeted metabolic profiling of the combination of panobinostat and marizomib in SU-DIPG-XIII cells.

    (A) Cytoscape enrichment map visualization of top gene programs represented by significantly down-regulated genes present in combination-low treated cells but not either single-agent treated condition. Node size represents number of genes, node color represents significance (false discovery rate), and edge thickness represents number of shared genes. Clustered gene programs are labeled. (B) Gene expression changes for the leading-edge genes from the Hallmark-Oxidative Phosphorylation gene set. Leading selection and ranking were based on the combo-high treatment set. Complex I gene family members (NDUFs) are bolded. (C) %Response and ΔBliss heat maps highlighting the “synthetic lethality” for the combination of ETC inhibitors (the complex I inhibitor rotenone or the H+-ATP synthase inhibitor oligomycin A) with glycolytic flux inhibitors (the GLUT1 inhibitor BAY-876 or the MCT2 inhibitor AZD-3965). (D) ExcessHSA values for well-resolved subclusters of ETC (group 4) or glycolytic flux (group 5) inhibitors originally identified in the correlation heat map for the 45-drug all-versus-all screen (Fig. 2E). Ranking is based on the average ExcessHSA within each group. This plot is expanded in fig. S2C. (E) Heat map displaying unsupervised hierarchical clustering of fold change of metabolites with respect to DMSO control, quantified by liquid chromatography/mass spectrometry. (F) Relative abundance of NAD+ and 3-phosphoglycerate in each treatment set with respect to the DMSO control. (G) Basal respiration and spare respiratory capacity, as assessed by Seahorse experiments, are reported for each treatment set. (H) Relative NAD+ concentration in combination-treated (24 hours) cells with respect to the DMSO control, in the presence or absence of the NAD+ precursor NMN, the NAMPT inhibitor daporinad, or both. (I) Cell death in combination-treated (24 hours) cells with respect to DMSO control in the presence or absence of NMN, daporinad, or both. **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

  • Table 1 Cellular cytotoxic response to agents in active clinical evaluation in DIPG (excluding biologics).

    Drug nameMechanism of
    action
    Clinical trial
    identifier
    MPO
    score*
    CNS
    penetrant
    JHH-DIPG-1SU-DIPG-XIIISU-DIPG-XVIISU-DIPG-
    XXV
    DoxorubicinTopo II
    inhibitor
    NCT02758366§2.79Limited1.048 μM0.934 μM0.235 μM1.176 μM
    IrinotecanTopo I inhibitorNCT030866162.94YesI.C.I.C.0.263 μMI.C.
    LenalidomideCereblon
    inhibitor
    NCT01222754#5.42YesI.C.I.C.I.C.I.C.
    MebendazoleTub. depol.
    inhibitor
    NCT01837862**5.09Yes0.468 μM0.417 μM0.093 μM0.331 μM
    VincristineTub. pol.
    inhibitor
    NCT01837862**2.88Limited0.029 μM0.331 μM0.030 μM0.030 μM
    AbemaciclibCDK4/6
    inhibitor
    NCT026444603.11YesI.C.I.C.I.C.I.C.
    RibociclibCDK4/6
    inhibitor
    NCT03355794††4.30YesI.C.I.C.I.C.I.C.
    ErlotinibEGFR inhibitorNCT02233049‡‡4.25LimitedI.C.I.C.I.C.I.C.
    DasatinibMultikinase
    inhibitor
    NCT01644773§§3.54YesI.C.I.C.I.C.I.C.
    CrizotinibMultikinase
    inhibitor
    NCT01644773§§3.02Limited0.590 μM0.332 μM0.468 μM0.468 μM
    EverolimusmTOR inhibitorNCT03355794††1.94LimitedI.C.I.C.I.C.I.C.
    TemsirolimusmTOR inhibitorNCT02420613‖‖2.06YesI.C.I.C.I.C.I.C.
    AdavosertibWee1 inhibitorNCT01922076#3.67YesI.C.I.C.I.C.I.C.
    ONC-201Multiple
    reports
    NCT034165305.66UnknownI.C.I.C.I.C.I.C.
    Valproic acidHDAC inhibitorNCT00879437¶¶5.62YesI.C.I.C.I.C.I.C.
    VorinostatHDAC inhibitorNCT01189266#,##4.82Unknown1.481 μM1.176 μM1.864 μM0.833 μM
    PanobinostatHDAC inhibitorNCT02717455***5.25Unknown0.166 μM0.166 μM1.176 μM0.148 μM

    *The CNS MPO scores are based on the design algorithm defined by Wager et al. (18).

    †Outcome definitions: “Yes” means that published results confirmed CNS exposure in animal or human studies. “Limited” means that published results suggest no or limited CNS exposure in animal or human studies. “Unknown” means that published results were not available to the best of our knowledge.

    ‡AC50 values are provided for agents with Z-AUC values < −0.85 and are based on the NCATS curve generator and are only provided for agents with a curve class designation of −1.1, −1.2, or −2.1 (otherwise noted as I.C. or “incomplete curve”) [see (43) for curve class definitions].

    §NCT02758366 doses doxorubicin using a prolonged infusion alone or in combination with temozolomide or radiation.

    ‖In NCT02758366, doxorubicin is being dosed using a prolonged, slow infusion, which may increase the CNS exposure.

    ¶NCT03086616 doses irinotecan using a liposomal formulation administered using convection enhanced delivery.

    #NCT01189266, NCT01222754, and NCT01922076 are trials combining the named drug with radiation therapy.

    **NCT01837862 examines mebendazole alone or in combination with vincristine, carboplatin, irinotecan, bevacizumab, and/or temozolomide.

    ††NCT03355794 examines a combination therapy involving ribociclib and everolimus after radiation therapy.

    ‡‡NCT02233049 examines combinations of erlotinib, dasatinib, and everolimus.

    §§NCT01644773 examines a combination therapy involving dasatinib and crizotinib.

    ‖‖NCT02420613 examines a combination therapy involving temsirolimus and vorinostat.

    ¶¶NCT00879437 examines a combination therapy involving valproic acid and bevacizumab and radiation.

    ## Vorinostat is also being explored in NCT02420613.

    ***NCT03566199 doses panobinostat using a nanoparticle formulation administered using convection enhanced delivery.

    • Table 2 Cellular cytotoxic response to agents with likely CNS exposure and cellular activity in DIPG.

      Drug nameMechanism
      of action
      Development
      phase*
      MPO
      score
      CNS
      penetrant
      JHH-DIPG-1§SU-DIPG-XIII§SU-DIPG-XVII§SU-DIPG-XXV§
      ColchicineTub. pol. inhibitorApproved5.47Unknown0.029 μM0.052 μM0.030 μM0.041 μM
      PlinabulinTub. pol. inhibitorPhase 35.03Yes0.052 μM0.166 μM0.037 μM0.047 μM
      Combretastatin A-4Tub. pol. inhibitorPhase 25.36Yes0.010 μM0.018 μM0.007 μM0.010 μM
      AzixaTub. pol. inhibitorPhase 25.08Yes0.011 μM0.009 μM0.008 μM0.006 μM
      SepantroniumSurvivin inhibitor.Preclinical5.63Unknown0.104 μM0.002 μM0.009 μM0.026 μM
      MarizomibProteasome
      inhibitor
      Phase 15.50Yes0.029 μM0.014 μM0.033 μM0.018 μM
      PI-103PI3K inhibitorPreclinical5.40Unknown5.254 μM0.332 μM0.525 μM0.186 μM
      DaporinadNAMPT inhibitorPhase 15.09Yes0.004 μM0.002 μM0.012 μM0.005 μM
      NAMPT-IN-1NAMPT inhibitorPreclinical4.45Unknown0.047 μM0.037 μM0.263 μM0.083 μM
      GMX-1778NAMPT inhibitorPreclinical5.16Unknown0.015 μM0.008 μM0.030 μM0.013 μM
      GDC-0623MEK inhibitorPhase 14.49Yes0.105 μM0.148 μM0.148 μM0.331 μM
      AZD-8330MEK inhibitorPhase 24.44Unknown0.059 μM0.132 μM0.166 μM0.296 μM
      TriptolideXBP inhibitorPhase 25.75Yes0.083 μM0.105 μM0.186 μM0.209 μM
      TriptonideXBP inhibitorPreclinical6.00Yes0.132 μM0.263 μM0.296 μM0.372 μM
      GanetespibHSP90 inhibitorPhase 25.16Yes0.066 μM0.042 μM0.037 μM0.037 μM
      HSP-990HSP90 inhibitorPreclinical4.92Unknown0.037 μM0.042 μM0.030 μM0.030 μM
      NSC-319726Oxidative stressPreclinical5.75Unknown0.026 μM0.024 μM0.009 μM0.013 μM
      ElesclomolOxidative stressPhase 15.21Unknown0.005 μM0.009 μM0.019 μM0.019 μM
      DinaciclibCDK inhibitorPhase 15.20Yes0.042 μM0.037 μM0.037 μM0.030 μM
      CGP-60474CDK inhibitorPreclinical5.16Unknown0.118 μM0.083 μM0.132 μM0.132 μM
      SB-1317CDK inhibitorPhase 24.54Unknown0.166 μM0.132 μM0.083 μM0.093 μM
      PodofiloxTopo II inhibitorApproved5.27Unknown0.030 μM0.030 μM0.042 μM0.047 μM

      Notes: Agents with an MPO score above 4.0 and an average Z-AUC below −1.9.

      *Highest achieved clinical phase in any indication at the time of publication.

      †The CNS MPO scores are based on the design algorithm defined by Wager et al. (18).

      ‡Outcome definitions: “Yes” means that published results confirmed CNS exposure in animal or human studies. “Unknown” means that published results were not available to the best of our knowledge.

      §AC50 values are provided for agents with Z-AUC values < −0.85 and are based on the NCATS curve generator and are only provided for agents with a curve class designation of −1.1, −1.2, or −2.1 [see (43) for curve class definitions].

      ‖This agent is in active clinical evaluation as a prodrug named Minnelide.

      Supplementary Materials

      • stm.sciencemag.org/cgi/content/full/11/519/eaaw0064/DC1

        Materials and Methods

        Fig. S1. MIPE library annotations, correlation analysis of MIPE 5.0 screens, and additional potency distributions.

        Fig. S2. MoA similarities defined by clustering analyses of combination assessments.

        Fig. S3. Combination assessments of drugs in active DIPG clinical evaluations.

        Fig. S4. DIPG cell viability after treatment with candidate combinations.

        Fig. S5. Flow cytometry analysis of BMS-754807– and selumetinib-treated DIPG cells.

        Fig. S6. BMS-754807 and selumetinib in vivo alone or in combination with panobinostat and assessment of the effect of panobinostat and marizomib on brain viability.

        Fig. S7. Efficacy of panobinostat alone and with marizomib on nonpontine DMG.

        Fig. S8. Effect of extended treatment with panobinostat and marizomib on normal and malignant cell viability.

        Fig. S9. Transcriptional responses to panobinostat and marizomib treatment in SU-DIPGXIII cells.

        Fig. S10. GSEA of panobinostat and marizomib treatment in SU-DIPG-XIII cells.

        Fig. S11. Transcriptional analysis of panobinostat and marizomib treatments in SU-DIPGVI and QCTB-R059 cells.

        Fig. S12. Effects of panobinostat and marizomib treatment on ER stress and the UPR pathway in DIPG cells.

        Fig. S13. Down-regulated transcriptional programs in SU-DIPG-VI and QCTB-R059 cells.

        Fig. S14. Drug-induced metabolic rewiring and collapse in DIPG.

        Fig. S15. Lack of rescue by the ROS mitigator N-acetylcysteine from proteasome inhibition and HDAC inhibitor–induced toxicity in DIPG.

        Fig. S16. Effects of metabolic and NAD+ perturbations on DIPG.

        Data file S1 contains tables S1 to S11.

        Table S1. Listing of all PubChem AIDs for all single-agent screening data.

        Table S2. Complete listing of agents, MoAs, clinical phase, and CAS numbers for the MIPE 5.0 library.

        Table S3. MoA distribution for all MIPE 5.0 library members.

        Table S4. Complete listing of 371 potency-selected agents.

        Table S5. Complete listing of agents in current or recently completed clinical trials for DIPG.

        Table S6. Complete listing of agents with average Z-AUC < −2.0 and predicted CNS exposure (MPO score > 4.4).

        Table S7. Complete listing of outcomes from the panobinostat “drug-versus-all” experiment in SU-DIPG-XXV.

        Table S8. Complete listing of outcomes from the marizomib drug-versus-all experiment in SU-DIPG-XXV.

        Table S9. Complete listing of outcomes from the 45-agent all-versus-all experiment in SU-DIPG-XXV.

        Table S10. Characteristics of patient-derived high-grade glioma cell cultures.

        Table S11. Original numerical data for experiments presented as composite graphs.

        Reference (42)

      • The PDF file includes:

        • Materials and Methods
        • Fig. S1. MIPE library annotations, correlation analysis of MIPE 5.0 screens, and additional potency distributions.
        • Fig. S2. MoA similarities defined by clustering analyses of combination assessments.
        • Fig. S3. Combination assessments of drugs in active DIPG clinical evaluations.
        • Fig. S4. DIPG cell viability after treatment with candidate combinations.
        • Fig. S5. Flow cytometry analysis of BMS-754807– and selumetinib-treated DIPG cells.
        • Fig. S6. BMS-754807 and selumetinib in vivo alone or in combination with panobinostat and assessment of the effect of panobinostat and marizomib on brain viability.
        • Fig. S7. Efficacy of panobinostat alone and with marizomib on nonpontine DMG.
        • Fig. S8. Effect of extended treatment with panobinostat and marizomib on normal and malignant cell viability.
        • Fig. S9. Transcriptional responses to panobinostat and marizomib treatment in SU-DIPGXIII cells.
        • Fig. S10. GSEA of panobinostat and marizomib treatment in SU-DIPG-XIII cells.
        • Fig. S11. Transcriptional analysis of panobinostat and marizomib treatments in SU-DIPGVI and QCTB-R059 cells.
        • Fig. S12. Effects of panobinostat and marizomib treatment on ER stress and the UPR pathway in DIPG cells.
        • Fig. S13. Down-regulated transcriptional programs in SU-DIPG-VI and QCTB-R059 cells.
        • Fig. S14. Drug-induced metabolic rewiring and collapse in DIPG.
        • Fig. S15. Lack of rescue by the ROS mitigator N-acetylcysteine from proteasome inhibition and HDAC inhibitor–induced toxicity in DIPG.
        • Fig. S16. Effects of metabolic and NAD+ perturbations on DIPG.
        • Reference (42)

        [Download PDF]

        Other Supplementary Material for this manuscript includes the following:

        • Data file S1 contains tables S1 to S11.
        • Table S1 (Microsoft Excel format). Listing of all PubChem AIDs for all single-agent screening data.
        • Table S2 (Microsoft Excel format). Complete listing of agents, MoAs, clinical phase, and CAS numbers for the MIPE 5.0 library.
        • Table S3 (Microsoft Excel format). MoA distribution for all MIPE 5.0 library members.
        • Table S4 (Microsoft Excel format). Complete listing of 371 potency-selected agents.
        • Table S5 (Microsoft Excel format). Complete listing of agents in current or recently completed clinical trials for DIPG.
        • Table S6 (Microsoft Excel format). Complete listing of agents with average Z-AUC < −2.0 and predicted CNS exposure (MPO score > 4.4).
        • Table S7 (Microsoft Excel format). Complete listing of outcomes from the panobinostat “drug-versus-all” experiment in SU-DIPG-XXV.
        • Table S8 (Microsoft Excel format). Complete listing of outcomes from the marizomib drug-versus-all experiment in SU-DIPG-XXV.
        • Table S9 (Microsoft Excel format). Complete listing of outcomes from the 45-agent all-versus-all experiment in SU-DIPG-XXV.
        • Table S10 (Microsoft Excel format). Characteristics of patient-derived high-grade glioma cell cultures.
        • Table S11 (Microsoft Excel format). Original numerical data for experiments presented as composite graphs.

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