Abstract

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific ‘MYC target gene signatures’ and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.

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Acknowledgements

We thank Z. Herbert for his expertise and guidance with next-generation sequencing. We thank R. Young and D. Hnisz for assistance with reagents. We thank P. Rahl, C. Ott, and J. Perry for helpful comments on the manuscript. C.Y.L. is supported by the Cancer Prevention Research Institute of Texas (RR150093) and by the NCI (1R01CA215452-01), and is a Pew-Stewart Scholar for Cancer Research (Alexander and Margaret Stewart Trust). R.Z. and J.E.B. are supported by the V Foundation for Cancer Research Translational Grant.

Author information

Affiliations

  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    • Rhamy Zeid
    • , Matthew A. Lawlor
    • , Jaime M. Reyes
    • , Mariateresa Fulciniti
    • , Michael A. Lopez
    • , Thomas G. Scott
    • , Behnam Nabet
    • , Michael A. Erb
    • , Georg E. Winter
    • , Zoe Jacobson
    • , Donald R. Polaski
    • , Nikhil P. Munshi
    • , Charles Y. Lin
    •  & James E. Bradner
  2. Division of Clinical Studies, The Institute of Cancer Research, London, UK

    • Evon Poon
    •  & Louis Chesler
  3. Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK

    • Evon Poon
    •  & Louis Chesler
  4. LeBow Institute for Myeloma Therapeutics and Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Boston, MA, USA

    • Mariateresa Fulciniti
    • , Michael A. Lopez
    •  & Nikhil P. Munshi
  5. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

    • Kristen L. Karlin
    • , Rachel A. Hirsch
    • , Thomas F. Westbrook
    •  & Charles Y. Lin
  6. Department of Medicine, Harvard Medical School, Boston, MA, USA

    • James E. Bradner
  7. Novartis Institute for Biomedical Research, Cambridge, MA, USA

    • James E. Bradner

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Contributions

R.Z., C.Y.L., and J.E.B. designed this study. R.Z. designed and performed biology experiments. C.Y.L., J.M.R., D.R.P., and R.A.H. performed data analysis. M.A. Lawlor assisted in ChIP–seq experiments. E.P. performed mouse experiments. M.F. and M.A. Lopez performed experiments in multiple myeloma. T.G.S., Z.J., and K.L.K. assisted in cellular assays. B.N. assisted in RNA-seq profiling. M.A.E. and G.E.W. assisted in exon-scanning CRISPR–Cas9. N.P.M., T.F.W., and L.C. supervised experiments. R.Z., C.Y.L., and J.E.B. analyzed results and wrote the manuscript with comments from all authors.

Competing interests

R.Z. is now an employee of C4 Therapeutics. C.Y.L. is a consultant of Jnana Therapeutics and is a shareholder and inventor of intellectual property licensed to Syros Pharmaceuticals. J.E.B. is a Scientific Founder of Syros Pharmaceuticals, SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics (now Roche), and C4 Therapeutics and is the inventor on intellectual property licensed to these entities. J.E.B. is now an executive and shareholder in Novartis AG.

Corresponding authors

Correspondence to Charles Y. Lin or James E. Bradner.

Integrated supplementary information

  1. Supplementary Figure 1 Deregulated MYCN binding at active promoters and enhancers in neuroblastoma.

    a, Cell-normalized steady-state mRNA transcript levels of MYCN in human neuroblastoma cell lines. b, Line plots of quantified western blot bands from increasing cell-number-normalized cropped western blots of MYCN protein levels. Error bars denote ±s.d. for four replicate blots. c, Scatterplots of average ranked MYCN occupancy across four cell lines (x axis) versus ranked MYCN occupancy in each respective cell line (y axis). Contour lines illustrate the density of correlation of MYCN occupancy and are color-coded from high density (red) to low density (yellow). d, Meta track representation of MYCN and H3K27ac ChIP–seq signal (RPM/bp) across four neuroblastoma cell lines at the RPL22 locus. e, Meta track representation of MYCN and H3K27ac ChIP–seq signal (RPM/bp) across four neuroblastoma cell lines at an upstream ID2 enhancer. f, Heatmap of MYCN (red) signal at promoters (left) and enhancers (right) in each respective neuroblastoma cell line. Each row shows the ±5-kb region centered on the TSS ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. g, Heatmap of H3K27ac (blue) signal at promoters (left) and enhancers (right) in each respective neuroblastoma cell line. Each row shows the ±5-kb region centered on the enhancer ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. h, Clustering of the indicated ChIP–seq signal at promoters in the BE(2)-C cell line. Color scaled intensities reflect pairwise similarity via Pearson correlation. i, Top, MYCN signal contribution in the BE(2)-C cell line at the top 20,000 promoters. Bottom, line plots of signal contribution at the top 20,000 promoters of the indicated marks. Error bars represent 95% confidence intervals of the mean. j, Line plots showing the correlation of ranked MYCN-enriched regions (x axis) versus average ChIP–seq signal (top), percentage overlap with a given genomic feature (middle), or E-box density (bottom) on the y axis. Error bars represent 95% confidence intervals of the mean.

  2. Supplementary Figure 2 Dynamic chromatin consequences of direct MYCN shutdown.

    a, ChIP-Rx signal (RPM/bp, before scaling) upon MYCN shutdown in the tet-off MYCN SHEP-21N cell line at the NPM1 locus. b, ChIP-Rx signal (RPM/bp) upon MYCN shutdown in the tet-off MYCN SHEP-21 cell line at the NPM1 locus. c, Heatmap and meta plot of MYCN signal at promoters (left) and enhancers (right) upon MYCN shutdown in the tet-off MYCN SHEP-21N cell line. Each row shows the ±5-kb region centered on the TSS or enhancer ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. d, Heatmap and meta plot of H3K27ac signal at promoters and enhancers upon MYCN shutdown in the tet-off MYCN SHEP-21N cell line. Each row shows the ±5-kb region centered on the TSS or enhancer ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. e, Box plots of MYCN and H3K27ac ChIP–seq signal at active promoters. MYCN and H3K27ac signal at 0, 2, and 24 h after MYCN shutdown for standard ChIP–seq; ChIP-Rx before/after scaling. Significant differences are denoted (Welch’s two-tailed t test): ***P < 1 × 10–9, **P < 1 × 10–6. f, Box plots of MYCN and H3K27ac ChIP–seq signal at active enhancers. MYCN and H3K27ac signal at 0, 2, and 24 h after MYCN shutdown for standard ChIP–seq; ChIP-Rx before/after scaling. Significant differences are denoted (Welch’s two-tailed t test): ***P < 1 × 10–9, **P < 1 × 10–6. g, Heatmap and meta plot of H3K4me3, RNA Pol II, and CTCF signal at promoters upon MYCN shutdown in the tet-off MYCN SHEP-21N cell line. Each row shows the ±5-kb region centered on the TSS ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. h, Cropped western blot of total histone H3 and H3K27ac levels at 0, 2, and 24 h after MYCN shutdown. The percentage of H3K27ac remaining versus 0 h is indicated. i, Distribution plots of RNA Pol II traveling ratios (TR) for all active genes in traditional normalized ChIP–seq data. Differences in the TR distribution at 0 h versus 2 h and 0 h versus 24 h are significant; Welch’s two-tailed t test: ***P < 1 × 10–9, **P < 1 × 10–6. j, Box plots of log2 fold changes in active gene expression (traditional normalization) at the indicated time points versus 0 h after MYCN shutdown. Significant differences are denoted (Welch’s two-tailed t test): **P < 1 × 10–6.

  3. Supplementary Figure 3 Replicative analysis of the SHEP-21N system and the dynamics of the tet-on MYCN SHEP system.

    a, Left, differential analysis volcano plot of two ChIP–seq replicates comparing the log2 fold change at 2 h versus log10 P value. Blue and red circles denote distal and TSS MYCN sites, respectively. Right, box plot representation of differential regions (P < 0.1, fold change > log2 0.5) showing the log2 fold change at MYCN TSSs and distal sites. b, Left, differential analysis volcano plot of two ChIP–seq replicates comparing the log2 fold change at 24 h versus log10 P value. Blue and red circles denote distal and TSS MYCN sites, respectively. Right, box plot representation of differential regions (P < 0.1, fold change > log2 0.5) showing the log2 fold change at MYCN TSSs and distal sites. c, Scatter of MYCN peak AUC between MYCN ChIP–seq replicates at 0, 2, and 24 h after MYCN shutdown. Pearson correlation is noted. d, Box plots of the log2 fold change of MYCN load at the TSSs and distal enhancers of the top 5,000 genes ranked by proximal MYCN signal. Left, ChIP-Rx before scaling. Right, ChIP-Rx after scaling. e, Heatmap and meta plot of MYCN signal at promoters (left) and enhancers (right) upon MYCN induction in the tet-on MYCN SHEP cell line. Each row shows the ±5-kb region centered on the TSS or enhancer ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. f, Heatmap and meta plot of H3K27ac signal at promoters (left) and enhancers (right) upon MYCN induction in the tet-on MYCN SHEP cell line. Each row shows the ±5-kb region centered on the TSS or enhancer ranked by average H3K27ac signal. Color scaled intensities are in units of RPM/bp. g, Box plots of MYCN (red) and H3K27ac (blue) ChIP–seq signal (RPM/bp) at active promoters and enhancers at 0, 2, and 6 h after MYNC induction. Significant differences are denoted (Welch’s two-tailed t test): ***P < 1 × 10–9.

  4. Supplementary Figure 4 Deregulated MYCN binding at promoters and enhancers in the TH-MYCN genetically engineered neuroblastoma mouse model.

    a, Immunohistochemistry staining of MYCN and a corresponding no-stain control in celiac ganglia, TH-MYCN tumors, and spleen. b, Meta track representation of H3K27ac ChIP–seq signal (RPM/bp) in ganglia (celiac and superior cervical) and TH-MYCN tumors as well as MYCN ChIP–seq signal in TH-MYCN tumors at the indicated loci. c, Meta track representation of H3K27ac ChIP–seq signal (RPM/bp) in ganglia (celiac and superior cervical) and TH-MYCN tumors as well as MYCN ChIP–seq signal in TH-MYCN tumors at an upstream Id2 enhancer. d, Meta track representation of H3K27ac ChIP–seq signal (RPM/bp) in ganglia (celiac and superior cervical) and TH-MYCN tumors as well as MYCN ChIP–seq signal in TH-MYCN tumors at the indicated loci. e, Meta track representation of MYCN and H3K27ac ChIP–seq signal (RPM/bp) across four neuroblastoma cell lines at the indicated loci. f, Pie chart showing the percentage of MYCN binding sites in human neuroblastoma cell lines that exhibit H3K27ac signal in TH-MYCN tumors and/or ganglia at promoters (left) and enhancers (right).

  5. Supplementary Figure 5 Enhancer invasion shapes transcriptional sensitivity to MYCN perturbation in neuroblastoma.

    a, Top, plot showing the top 5,000 genes in SHEP-21N ranked by total proximal MYCN signal. Bottom, dot plot of the percentage of enhancer contribution, the size of contributing MYCN binding, the density of contributing MYCN, and the area under the curve of contributing MYCN binding with a best fit line superimposed (loess correlation). b, Top, plot showing the top 5,000 genes across four neuroblastoma cell lines ranked by total proximal MYCN signal. Bottom, dot plot of the percentage of enhancer contribution, the size of contributing MYCN binding, the density of contributing MYCN, and the area under the curve of contributing MYCN binding with a best fit line superimposed (loess correlation). c, Standard (left) and cell count (right) normalized levels of the RPL22, HAND2, and ID2 transcripts during MYCN shutdown. Units are FPKM and cell-count-normalized FPKM, respectively, for triplicate biological replicates. Error bars represent s.d. d, Standard normalized log2 fold change (versus 0 h) of gene expression changes during MYCN shutdown in the SHEP-21N system. Genes are grouped according to rank-ordered MYCN proximal load (promoters and enhancers). Error bars represent the 95% confidence interval (CI) of the mean. e, Box plots of the log2 fold change (versus 0 h) of the amount of RNA Pol II (ChIP-Rx, before scaling) at the TSS (left) and gene body (right) of genes grouped according to rank-ordered MYCN proximal load. Significance is denoted by Welch’s two-tailed t test: ***P < 1 × 10–9, **P < 1 × 10–6, *P < 1 × 10–3. f, Box plots of the log2 fold change (versus 0 h) of the amount of RNA Pol II (ChIP–seq) at the TSS (left) and gene body (right) of genes grouped according to rank-ordered MYCN proximal load. Significance is denoted by Welch’s two-tailed t test: ***P < 1 × 10–9, **P < 1 × 10–6, *P < 1 × 10–3.

  6. Supplementary Figure 6 BET bromodomain inhibition in MYCN-driven neuroblastoma.

    a, Cropped western blot of vinculin and MYCN levels at 0, 1, 2, 4, 8, and 24 h of treatment with the BET inhibitor JQ1 (1 μM). The percentage of MYCN remaining versus 0 h is indicated. b, Box plots of the log2 mRNA fold change (versus 0 h) at active genes (defined by H3K27ac ChIP–seq signal at TSSs and expressed) with JQ1 (1 μM). Cell-normalized and traditional normalized log2 mRNA fold change levels are shown. Significance is denoted by Welch’s two-tailed t test: ***P < 1 × 10–9, **P < 1 × 10–6, *P < 1 × 10–3. c, Cell-normalized (left) and standard normalized (right) levels of the RPL22, HAND2, and ID2 transcripts during MYCN shutdown. Units are cell FPKM for triplicate biological replicates. Error bars represent s.d. d, Cell-normalized and standard normalized log2 fold change of gene expression changes ranked by total MYCN load during JQ1 treatment. Genes are grouped according to rank-ordered MYCN proximal load (promoters and enhancers). Error bars represent the 95% confidence interval (CI) of the mean. e, log2 fold change of gene expression changes of the top 5,000 genes ranked by total MYCN load during JQ1 treatment. Genes are grouped according to rank-ordered MYCN proximal load at promoters (left) or enhancers (right). Error bars represent the 95% confidence interval (CI) of the mean.

  7. Supplementary Figure 7 Enhancer invasion accounts for tumor-specific MYCN signatures in neuroblastoma.

    a, Differential MYCN signal contribution in the BE(2)-C cell line for promoters (red) and enhancers (blue) of associated genes (y axis) of the top 5,000 proximal MYCN-bound regions are shown ranked by difference in MYCN enhancer to promoter contribution (x axis). b, GSEA plots of MYCN-bound promoter- (red) versus enhancer- (blue) dominant gene sets defined by leading edge analysis. c, Normalized enrichment scores (NESs) of target gene signatures (Molecular Signature Database) are plotted on the x axis versus the FDR (false discovery rate) on the y axis. d, Highly significant gene signatures from promoter (red) and enhancer (blue) bias gene sets are highlighted and tabulated. e, Differential H3K27ac signal contribution in the BE(2)-C cell line for promoters (red) and enhancers (blue) of associated genes (y axis) of the top 5,000 proximal H3K27ac-bound regions are shown ranked by difference in H3K27ac enhancer to promoter contribution (x axis). f, GSEA plots of H3K27ac-bound promoter- (red) versus enhancer- (blue) dominant gene sets defined by leading edge analysis. g, Normalized enrichment scores (NESs) of target gene signatures (Molecular Signature Database) are plotted on the x axis versus the FDR (false discovery rate) on the y axis. h, Highly significant gene signatures from promoter (red) and enhancer (blue) bias gene sets are highlighted and tabulated. i, Overall survival of patients ranked by expression of the top 25 genes defined by total MYCN load at promoter-dominant genes (left) or enhancer-dominant genes (right) for all patients with neuroblastoma. Significance is denoted by a chi-squared test, and P values are shown. j, Overall survival of patients ranked by expression of the top 25 genes defined by total MYCN load at promoter-dominant genes (left) or enhancer-dominant genes (right) for patients with neuroblastoma without MYCN amplification. Significance is denoted by a chi-squared test, and P values are shown. k, Heatmap of gene signatures enriched in promoter- (red) or enhancer- (blue) dominant pathways as defined by MYCN or H3K27ac in the BE(2)-C cell line. Selected signatures are annotated at an FDR < 0.01 and NES > 2 cutoff. l, Normalized enrichment scores (NESs) of target gene signatures (Molecular Signature Database) are plotted on the x axis versus the FDR (false discovery rate) on the y axis at 0, 2, and 6 h after MYCN induction.

  8. Supplementary Figure 8 MYC-bound enhancer axes reflect tumor-specific pathways in deregulated-MYC-driven cancers.

    a, Top, plot showing the top 5,000 genes (x axis) in SHEP-21N ranked by total proximal MYCN signal. Bottom, dot plot of the percentage of enhancer contribution (enhancer/total MYC signal) sampled across bins (100 genes/bin) with a best fit line superimposed (loess correlation). b, Differential MYC signal contribution in the H2171 cell line for promoters (red) and enhancers (blue) of associated genes (y axis) of the top 5,000 proximal MYC-bound regions are shown ranked by difference in MYC enhancer to promoter contribution (x axis). c, GSEA plots of MYC-bound promoter- (red) versus enhancer- (blue) dominant gene sets defined by leading edge analysis. d, Normalized enrichment scores (NESs) of target gene signatures (Molecular Signature Database) are plotted on the x axis versus the FDR (false discovery rate) on the y axis. e, Highly significant gene signatures from promoter (red) and enhancer (blue) bias gene sets are highlighted and tabulated. fi, Same as in ae (respectively) in the U87 cell line. ko, Same as in ae (respectively) in the MM1.S cell line. pt, Same as in ae (respectively) in the P493-6 cell line.

  9. Supplementary Figure 9 TWIST1 co-occupies enhancers with MYCN and is a MYCN-specific dependency in neuroblastoma.

    a, Schematic of the computational approach to identifying a core regulatory circuit based on TF enhancer binding by identifying transcription factor motifs within ATAC–seq-defined nucleosome-free regions. The ‘in’ and ‘out’ degrees of transcription factor binding are defined. b, Network depiction of the conserved enhancer regulatory transcription factor network in the indicated neuroblastoma cell lines. TF nodes are denoted, and predicted binding interactions with super-enhancers driving other TFs are shown as lines (edges). Enhancer-regulated bHLH TFs are highlighted in orange. c, Heatmap of enhancer-regulated TFs in neuroblastoma cell lines (rows) clustered by similarity of regulatory degree. The bHLH TFs HAND2 and TWIST1 are highlighted in orange. d, Network depiction of the conserved enhancer regulatory transcription factor network across the four neuroblastoma cell lines. TF nodes are denoted, and predicted binding interactions with super-enhancers driving other TFs are shown as lines (edges). e, Top, waterfall plot of log2 MYCN fold change at 2 versus 0 h after MYCN shutdown. Bottom, line plot of log2 TWIST1 fold change at 2 h after MYCN shutdown. Regions ranked by change in MYCN levels 2 h after MYCN shutdown. f, CRISPR scan of the NSD1 locus. Top, Illumina sequencing readout of log2 fold enrichment/depletion (early versus late time point) of 3,351 sgRNAs. Bottom, simple moving average of log2 fold enrichment/depletion is shown. g, ChIP–seq signal of H3K27ac (blue), ATAC–seq (green), MYCN (red), and TWIST1 (orange) at the NSD1 locus with respect to CRISPR sgRNAs. h, Left, cropped western blot of TWIST1, MYCN, and vinculin protein levels upon siRNA-mediated knockdown of TWIST1 in the indicated cell lines. Right, corresponding viable cell counts 72 h after siRNA transfection. Error bars denote ±s.d. i, Left, cropped western blot of TWIST1, MYCN, and vinculin protein levels upon siRNA-mediated knockdown of TWIST1 in the MYCN ‘on’ and MYCN ‘off’ states in the SHEP21-N cell line. Right, corresponding viable cell counts at 72 h after siRNA transfection. Error bars denote ±s.d. j, log2 fold change of gene expression changes upon MYCN shutdown of genes ranked by MYCN promoter load (top) or MYCN distal enhancer load (bottom). Genes are grouped according to rank-ordered MYCN proximal load (promoters and enhancers). Error bars represent the 95% CI.

  10. Supplementary Figure 10 TCF3 is a TF dependency in IgH/MYC-translocated multiple myeloma.

    a, Network depiction of the conserved enhancer regulatory transcription factor network in the MM1.S cell line. TF nodes are denoted, and predicted binding interactions with super-enhancers driving other TFs are shown as lines (edges). Enhancer-regulated bHLH TFs are highlighted in orange. b, Histogram plot ranking core regulatory circuit TFs based on motif co-occupancy with MYC. c, Left axis, quantified mRNA levels of TCF3 upon shRNA-mediated knockdown in the MM.1S cell line. Right axis, corresponding viability defect observed upon TCF3 knockdown as measured by ATP levels. Shown is the average of three experiments performed in triplicate. d, Line plot of 3H thymidine uptake at days 1 and 3 after shRNA cell lines expressing selection for each respective shRNA in the MM.1S cell line. Thymidine uptake is shown normalized to day 1 levels. e, Bar plot of the fraction of live shRNA-expressing cells as measured by GFP positivity. Data are represented as fold change as compared to day 1 after infection. f, Percentage of early (Annexin V) and late (Annexin V and DAPI) apoptotic and live (unstained) cells four days after shRNA infection in the MM.1S cell line. The MM1.S cell line (left) and U266 cell line (right) are shown. gj, Same as in cf (respectively) in the U266 cell line.

  11. Supplementary Figure 11 Full scan (uncropped) western blots from main figures

    Uncropped full scans of western blots from the corresponding cropped western blots shown within the main text. Molecular weight markers are indicated. Figure subpanel is indicated for each respective blot.

  12. Supplementary Figure 12 Full scan (uncropped) western blots from supplementary figures

    Uncropped full scans of western blots from the corresponding cropped western blots shown within the supplementary figures. Molecular weight markers are indicated. Figure subpanel is indicated for each respective blot.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–12 and Supplementary Note.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Quantified levels of MYCN in neuroblastoma cell lines.

  4. Supplementary Table 2

    Overall patient survival stratification.

  5. Supplementary Table 3

    CRISPR scan of TWIST1 and inducible knockdown of TWIST1.

  6. Supplementary Table 4

    CRISPR scan of NSD1 and siRNA-mediated knockdown of TWIST1.

  7. Supplementary Table 5

    Characterization of TCF3 in multiple myeloma.

  8. Supplementary Table 6

    Microarray sample detail summary.

  9. Supplementary Table 7

    ChIP–seq, ChIP-Rx, and ATAC–seq sample detail summary.

  10. Supplementary Table 8

    RNA-seq sample detail summary.

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DOI

https://doi.org/10.1038/s41588-018-0044-9

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