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Targeted inhibition of histone H3K27 demethylation is effective in high-risk neuroblastoma

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Science Translational Medicine  16 May 2018:
Vol. 10, Issue 441, eaao4680
DOI: 10.1126/scitranslmed.aao4680
  • Fig. 1 Neuroblastomas are sensitive to the H3K27 demethylase inhibitor GSK-J4.

    (A) High-throughput drug screen of 773 solid tumor cell lines. Models are split into neuroblastomas (N; n = 31) and all other solid tumors (O; n = 742). The Mann-Whitney U test was used to assess statistical significance. LN, natural log; IC50, median inhibitory concentration. (B) Neuroblastoma cell lines were treated with 1 μM GSK-J4 for 72 hours, and cell viability was determined by CellTiter-Glo. Cell lines with less than 50% viable cells (red dashed line) under these conditions are termed “sensitive” (blue line), and those with more than 50% viable cells are termed “resistant” (red line). MYCN-amplified (Amp) cell lines are indicated with red bars. (C to F) Mice treated with GSK-J4 in CHLA20 (C) or IMR32 (D) xenograft models or FELIX (E) or COG-N-561 (F) PDX models. Black lines correspond to control mice, and red lines correspond to GSK-J4–treated mice. Data are shown as means ± SEM. Asterisks indicate a significant separation between control and GSK-J4 cohorts using Student’s t test (P < 0.05). For the experiments in (B), n = 4. For (C), n = 4 for both cohorts. For (D), cohorts are control (n = 5) and GSK-J4 (n = 4). For (E), n = 3 for both cohorts. For (F), cohorts are control (n = 7) and GSK-J4 (n = 6).

  • Fig. 2 RNA-seq of GSK-J4–sensitive neuroblastoma cell lines demonstrates gene expression changes.

    (A) Mean of normalized counts versus mean log2 ratio plots of differential gene expression analysis for IMR5, LAN5, SK-N-FI, and SK-N-BE(2) treated with GSK-J4 for 72 hours compared to untreated cells. Red dots indicate significantly altered genes [adjusted P (Padj) < 0.05]. (B) Log2 ratio for a panel of genes involved in neuroblastoma differentiation from the RNA-seq data in neuroblastoma cell lines treated with GSK-J4 for 72 hours. Asterisks indicate significance (Padj < 0.05). (C) Western blot of IMR5 and KELLY neuroblastoma cell lines treated with GSK-J4 for 24, 48, or 72 hours. Blot was probed with the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. No Rx, no treatment. (D) Log2 ratio for expression of apoptotic and ER stress–related genes from the RNA-seq data in neuroblastoma treated with GSK-J4 for 72 hours. Asterisks indicate significance (Padj < 0.05). Data are shown as the means ± SEM. (E) Western blot of IMR5 and KELLY lysates, probed for ER stress–related apoptosis proteins. Asterisks denote bands at about 23 kDa (top; PUMA-α) and 18 kDa (bottom; PUMA-β) (32). GAPDH was used as a loading control.

  • Fig. 3 GSK-J4 induces axonal growth and combines with RA to control the growth of GSK-J4–resistant neuroblastomas.

    (A) Venn diagram showing the overlap of significant gene expression changes in sensitive neuroblastomas compared to ATRA-treated SH-SY5Y cells from the study of Duffy et al. (39). (B) Axonal length was measured in the images similar to those shown in fig. S13 using ImageJ and graphed for no treatment (n = 215), GSK-J4 (n = 189), and RA (n = 177). Statistical significance was determined by Student’s t test. AU, arbitrary units. (C) CHLA90, SK-N-AS, and SK-N-BE(2) cell lines were treated with 1 μM GSK-J4, 20 μM RA, or a combination of both drugs and stained with crystal violet 6 days later. (D) Cell viability assays for CHLA90, SK-N-AS, and SK-N-BE(2) cell lines treated with 0 or 1 μM GSK-J4 and the indicated concentrations of RA for 6 days. (E) Western blot of CHLA90, SK-N-AS, and SK-N-BE(2) cell lines treated with 1 μM GSK-J4, 20 μM RA, or a combination of both drugs for 6 days. Blots were probed with the indicated antibodies. All bar graphs are shown as the means ± SEM. For the experiments in (D), n = 4. Asterisks denote bands at about 23 kDa (top) and 18 kDa (bottom), matching PUMA isoforms.

  • Fig. 4 GSK-J4 combines with venetoclax and induces tumor regression.

    (A) Seventy-two–hour CellTiter-Glo assay of the indicated cell lines with GSK-J4 alone or combined with 1 μM venetoclax. Data are graphed as the fraction of viable cells relative to no treatment. (B) Apoptosis assay in IMR5, LAN5, SMS-SAN, and KELLY cells treated with 1 μM GSK-J4, 1 μM venetoclax, or the combination for 48 hours. Bars plotted represent only apoptotic annexin V–positive cells. (C) Nu/Nu mice injected with LAN5 cells (left) were treated daily with GSK-J4 (100 mg/kg), venetoclax (100 mg/kg), or the combination of both drugs. Nonobese diabetic severe combined immunodeficient gamma (NSG) mice injected with SK-N-DZ (center) or IMR5 (right) cells were treated with GSK-J4 (50 mg/kg) for 4 days a week, venetoclax (100 mg/kg) for 5 days a week, or the combination of both drugs. (D) Tumors shown in (C) were followed until the end of the experiment or until the humane limit of tumor volume was reached. The percentage change from initial tumor size is plotted for individual tumors. Student’s t test was used to determine significance of differences between groups within the waterfall cohorts. Data are presented as means ± SEM. For (A), n = 4. For (B), n = 3. For (C) and (D), LAN5 data are presented as control (n = 4), GSK-J4 (n = 6), venetoclax (n = 5), and combination (n = 6); SK-N-DZ data are presented as control (n = 5), GSK-J4 (n = 5), venetoclax (n = 4), and combination (n = 5). For IMR5, n = 5 for all cohorts.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/441/eaao4680/DC1

    Fig. S1. Sensitivity to GSK-J4 is independent of MYCN amplification or p53 functionality.

    Fig. S2. Sensitivity to GSK-J4 is independent of H3K27 methyltransferase or demethylase expression.

    Fig. S3. Neuroblastoma cell lines show similar sensitivity to structurally distinct demethylase inhibitors, and JMJD3 is necessary for GSK-J4 sensitivity.

    Fig. S4. GSK-J4 induces H3K27me3 accumulation in tumors and is well tolerated.

    Fig. S5. GSK-J4 increases H3K27 methylation in neuroblastoma cell lines.

    Fig. S6. Multiple differentially expressed genes overlap between cell lines treated with GSK-J4.

    Fig. S7. GSK-J4 increases differentiation and ER stress markers in the CHLA20 cell line.

    Fig. S8. GSK-J4 induces apoptosis and cell death.

    Fig. S9. GSK-J4 induces caspase-3 activity.

    Fig. S10. Inhibition of caspase cleavage protects against GSK-J4–mediated apoptosis.

    Fig. S11. BBC3 and ER stress silencing protects from GSK-J4–induced cell death.

    Fig. S12. Statistical analysis shows gene expression overlap between GSK-J4 and RA treatment of neuroblastoma cell lines.

    Fig. S13. GSK-J4 induces axonal outgrowth in LAN5 cells.

    Fig. S14. GSK-J4 synergizes with RA in GSK-J4– and RA-resistant cell lines.

    Fig. S15. GSK-J4 combines with venetoclax.

    Fig. S16. Combination of GSK-J4 and venetoclax is tolerated in xenograft models.

    Table S1. Cell line information and GSK-J4 screen data.

    Table S2. Genetic information for neuroblastoma cell lines from GSK-J4 screen.

    Table S3. Significant differentially expressed genes from GSK-J4 RNA-seq.

    Table S4. Differentially expressed gene overlaps in cell lines from GSK-J4 RNA-seq.

    Table S5. Functional pathways from IPA analysis.

    Table S6. Genes reported from neuritogenesis IPA pathway.

    Table S7. Gene names from Fisher’s exact test and GSEA comparisons.

    Table S8. Common affected genes between GSK-J4 and EPZ-6438 treatment in the IMR5 cell line.

    Table S9. Primary data.

  • Supplementary Material for:

    Targeted inhibition of histone H3K27 demethylation is effective in high-risk neuroblastoma

    Timothy L. Lochmann, Krista M. Powell, Jungoh Ham, Konstantinos V. Floros, Daniel A. R. Heisey, Richard I. J. Kurupi, Marissa L. Calbert, Maninderjit S. Ghotra, Patricia Greninger, Mikhail Dozmorov, Madhu Gowda, Andrew J. Souers, C. Patrick Reynolds, Cyril H. Benes,* Anthony C. Faber*

    *Corresponding author. Email: acfaber{at}vcu.edu (A.C.F.); cbenes{at}mgh.harvard.edu (C.H.B.)

    Published 16 May 2018, Sci. Transl. Med. 10, eaao4680 (2018)
    DOI: 10.1126/scitranslmed.aao4680

    This PDF file includes:

    • Fig. S1. Sensitivity to GSK-J4 is independent of MYCN amplification or p53 functionality.
    • Fig. S2. Sensitivity to GSK-J4 is independent of H3K27 methyltransferase or demethylase expression.
    • Fig. S3. Neuroblastoma cell lines show similar sensitivity to structurally distinct demethylase inhibitors, and JMJD3 is necessary for GSK-J4 sensitivity.
    • Fig. S4. GSK-J4 induces H3K27me3 accumulation in tumors and is well tolerated.
    • Fig. S5. GSK-J4 increases H3K27 methylation in neuroblastoma cell lines.
    • Fig. S6. Multiple differentially expressed genes overlap between cell lines treated with GSK-J4.
    • Fig. S7. GSK-J4 increases differentiation and ER stress markers in the CHLA20 cell line.
    • Fig. S8. GSK-J4 induces apoptosis and cell death.
    • Fig. S9. GSK-J4 induces caspase-3 activity.
    • Fig. S10. Inhibition of caspase cleavage protects against GSK-J4–mediated apoptosis.
    • Fig. S11. BBC3 and ER stress silencing protects from GSK-J4–induced cell death.
    • Fig. S12. Statistical analysis shows gene expression overlap between GSK-J4 and RA treatment of neuroblastoma cell lines.
    • Fig. S13. GSK-J4 induces axonal outgrowth in LAN5 cells.
    • Fig. S14. GSK-J4 synergizes with RA in GSK-J4– and RA-resistant cell lines.
    • Fig. S15. GSK-J4 combines with venetoclax.
    • Fig. S16. Combination of GSK-J4 and venetoclax is tolerated in xenograft models.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Cell line information and GSK-J4 screen data.
    • Table S2 (Microsoft Excel format). Genetic information for neuroblastoma cell lines from GSK-J4 screen.
    • Table S3 (Microsoft Excel format). Significant differentially expressed genes from GSK-J4 RNA-seq.
    • Table S4 (Microsoft Excel format). Differentially expressed gene overlaps in cell lines from GSK-J4 RNA-seq.
    • Table S5 (Microsoft Excel format). Functional pathways from IPA analysis.
    • Table S6 (Microsoft Excel format). Genes reported from neuritogenesis IPA pathway.
    • Table S7 (Microsoft Excel format). Gene names from Fisher’s exact test and GSEA comparisons.
    • Table S8 (Microsoft Excel format). Common affected genes between GSK-J4 and EPZ-6438 treatment in the IMR5 cell line.
    • Table S9 (Microsoft Excel format). Primary data.

    [Download Tables S1 to S9]

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