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Enhancer Hijacking Activates GFI1 Family Oncogenes in Medulloblastoma

Paul A Northcott  1 Catherine Lee  2 Thomas Zichner  3 Adrian M Stütz  4 Serap Erkek  5 Daisuke Kawauchi  6 David J H Shih  7 Volker Hovestadt  8 Marc Zapatka  8 Dominik Sturm  6 David T W Jones  6 Marcel Kool  6 Marc Remke  7 Florence M G Cavalli  7 Scott Zuyderduyn  9 Gary D Bader  9 Scott VandenBerg  10 Lourdes Adriana Esparza  11 Marina Ryzhova  12 Wei Wang  8 Andrea Wittmann  6 Sebastian Stark  6 Laura Sieber  6 Huriye Seker-Cin  6 Linda Linke  6 Fabian Kratochwil  6 Natalie Jäger  13 Ivo Buchhalter  13 Charles D Imbusch  14 Gideon Zipprich  14 Benjamin Raeder  4 Sabine Schmidt  15 Nicolle Diessl  15 Stephan Wolf  15 Stefan Wiemann  15 Benedikt Brors  13 Chris Lawerenz  14 Jürgen Eils  14 Hans-Jörg Warnatz  16 Thomas Risch  16 Marie-Laure Yaspo  16 Ursula D Weber  8 Cynthia C Bartholomae  17 Christof von Kalle  18 Eszter Turányi  19 Peter Hauser  20 Emma Sanden  21 Anna Darabi  21 Peter Siesjö  21 Jaroslav Sterba  22 Karel Zitterbart  22 David Sumerauer  23 Peter van Sluis  24 Rogier Versteeg  24 Richard Volckmann  24 Jan Koster  24 Martin U Schuhmann  25 Martin Ebinger  25 H Leighton Grimes  26 Giles W Robinson  27 Amar Gajjar  28 Martin Mynarek  29 Katja von Hoff  29 Stefan Rutkowski  29 Torsten Pietsch  30 Wolfram Scheurlen  31 Jörg Felsberg  32 Guido Reifenberger  32 Andreas E Kulozik  33 Andreas von Deimling  34 Olaf Witt  33 Roland Eils  35 Richard J Gilbertson  27 Andrey Korshunov  34 Michael D Taylor  36 Peter Lichter  37 Jan O Korbel  38 Robert J Wechsler-Reya  11 Stefan M Pfister  39
Affiliations

Enhancer Hijacking Activates GFI1 Family Oncogenes in Medulloblastoma

Paul A Northcott et al. Nature.

Abstract

Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.

Figures

Extended Data Figure 1
Extended Data Figure 1. Recurrent somatic copy-number aberrations target a common region on 9q34
Affymetrix SNP6 copy-number output for 22 primary MBs from the published MAGIC series exhibiting focal somatic copy-number aberrations within the 9q34 region of interest defined by WGS in the current study. Of the affected samples, MB subgroup information was available for 15/22 cases: SHH (n=1*), Group 3 (n=11), and Group 4 (n=3). Close examination of the single non-Group 3/Group 4 MB affected by a focal copy-number event in the region (MB-1318, SHH), revealed that this sample exhibits a homozygous deletion (in the context of broad chr9q deletion) specifically overlapping TSC1 and is therefore unlikely to be related to the events which target GFI1B for transcriptional activation. Indicated coordinates are based on the hg18 reference genome (NCBI Build 36.1) that was used in the original MAGIC study.
Extended Data Figure 2
Extended Data Figure 2. Non-functional DDX31:GFI1B fusion transcripts detected by RNA-seq
(a) A complex SV on 9q34 in ICGC_MB9 resulted in expression of DDX31 (exon 19) fused to GFI1B (intron 2, antisense orientation). Note the intronic reads in GFI1B after the fusion breakpoint. (b) 9q34 inversions in ICGC_MB247 resulted in expression of DDX31 (exon 19) fused to GFI1B (exon 2, sense orientation). This fusion transcript included a frameshift, inferred to generate a C-terminal-truncated DDX31 protein and no GFI1B protein from this fused allele.
Extended Data Figure 3
Extended Data Figure 3. Expression and correlation of 9q34 genes in MB subgroups
(a–c) Boxplots summarizing expression of BARHL1, DDX31, and GTF3C4 according to MB subgroup. Dataset includes 375 MBs profiled on the Affymetrix U133plus2 array. (d) Pearson correlation analysis showing correlated expression of DDX31 with BARHL1 and GTF3C4 in Group 3 and Group 4 MBs. DDX31 expression is positively correlated with both BARHL1 (r=0.741) and GTF3C4 (r=0.622). (e) PRRC2B expression in MB subgroups. Samples are from the same series summarized in (a–c). (f) Distribution of H3K27Ac ChIP-seq signal at predicted enhancers in Group 3 MBs (data for MAGIC_MB360 is shown). Enhancer regions are plotted in increasing order based on their input-normalized H3K27Ac signal. SEs are defined as the population of enhancers above the inflection point of the curve (horizontal dashed grey line). Positions of the predicted BARHL1/DDX31 and PRRC2B SEs described in the text are highlighted.
Extended Data Figure 4
Extended Data Figure 4. Frequency and distribution of GFI1/GFI1B activation in MB subgroups
(a) Stacked bar graph indicates the proportion of GFI1/GFI1B-expressing cases in each of the four MB subgroups, as determined by Affymetrix gene expression profiling of two independent cohorts (n=727). (b) Stacked bar graph indicates the proportion of GFI1/GFI1B-positive cases in each of the four MB subgroups, as determined by IHC performed with α-GFI1 and α-GFI1B antibodies on FFPE sections derived from a MB clinical trial cohort (HIT2000, NCT00303810; n=156). (c–f) Representative positive and negative IHC results for Group 3 MBs stained with α-GFI1 (c, d) and α-GFI1B (e, f) antibodies, respectively.
Extended Data Figure 5
Extended Data Figure 5. Demographic and clinical characteristics of GFI1/GFI1B-activated Group 3 MB
(a, b) Unsupervised hierarchical clustering of Group 3 MB samples profiled by Affymetrix gene expression array (a) or Illumina 450K DNA methylation array (b). (c) Patient characteristics, including age, gender, histological subtype (histology), and metastatic status (M-stage) for Group 3 MBs stratified according to GFI1/GFI1B expression status. Both gene expression and IHC cohorts are summarized. (d, e) Overall survival of Group 3 MBs stratified by GFI1/GFI1B expression status for both our gene expression (d) and IHC series (e).
Extended Data Figure 6
Extended Data Figure 6. Summary of GFI1 SVs detected by WGS in Group 3 MB
(a) Schematics depicting the six different GFI1 translocations detected by large-insert paired-end sequencing of our GFI1-activated validation series. (b) WGS coverage plots showing SVs affecting the GFI1 locus in GFI1-activated MBs sequenced in our series. (c) Fluorescence in situ hybridization (FISH) analysis of MAGIC_MB1338 validating the unbalanced t(1:9) translocation (shown in (a)) predicted by WGS.
Extended Data Figure 7
Extended Data Figure 7. Chromatin states proximal to SVs observed in GFI1-activated Group 3 MBs
(a–d) ChIP-seq (H3K27Ac and H3K9Ac) and WGBS data respectively highlighting the active chromatin and methylation states present in the regions proximal to SV breakpoints identified in GFI1 translocation cases. (e) Schematic summarizing the series of focal tandem duplications observed approximately ~45 kb downstream of GFI1 in Group 3 MBs (n=3; ICGC_MB18 is shown as a representative case). Activating and repressive histone marks overlapping the region of interest are shown for a non-GFI1-activated Group 3 MB (MAGIC_MB360) and the tandem duplication case (ICGC_MB18).
Extended Data Figure 8
Extended Data Figure 8. Association between GFI1/GFI1B activation and MYC in Group 3 MB
(a) MYC expression in Group 3 MBs (n=168) according to GFI1/GFI1B activation status. (b) Genesets with significant enrichment in GFI1/GFI1B associated genes from the MSigDB c2 gene set collection. The collection highlighted in red is the only result found that shows a significant enrichment in both GFI1 and GFI1B associated genes and a clear connection to a known pathway. (c) Heatmap of the expression values for the 50 genes in the KIM_MYC_AMPLIFICATION_TARGETS_UP gene set with the most significant association with GFI1 or GFI1B expression (the complete gene set contains 187 profiled genes). Genes are ordered top to bottom from most to least significant. A set of 90 Group 3 MBs included in the analysis are displayed. Sample-wise hierarchical clustering was performed only to enhance the visual organization of the heatmap. (d) Affymetrix SNP6 copy-number output for 82 primary Group 3 MBs from the published MAGIC series, highlighting the incidence of MYC amplification in the context of GFI1/GFI1B-activation. MYC amplification was found at a comparable frequency in both GFI1-activated (n=2/14, 14.3%) and non-GFI1/GFI1B-activated (n=10/57, 17.5%) Group 3 MBs. Indicated coordinates are based on the hg18 (NCBI Build 36.1) reference genome that was used in the original MAGIC study.
Extended Data Figure 9
Extended Data Figure 9. Phenotypic characteristics of novel Gfi1/Gfi1b orthotopic mouse models
(a, b) Bioluminescent imaging of animals injected with either Gfi1- (a) or Gfi1b-expressing (b) neural stem cells at the indicated time points. No tumour signal was detectable in these animals. (c) H&E staining of cerebellar sections derived from MGB tumour-bearing mice. (d) Immunofluorescence imaging of cerebellar sections from MGB tumours stained with the indicated antibodies.
Figure 1
Figure 1. Recurrent SVs activate the GFI1B proto-oncogene in MB
(a) Genome-wide SVs identified by WGS in a discovery cohort of Group 3 and Group 4 MBs (n=137). (b) Summary of SVs affecting a common locus of aberration on 9q34. (c) Expression boxplots (n=96) for the 7 genes contained within the 9q34 region of interest. (d) GFI1B expression across MB subgroups (n=727). Dashed line delineates the threshold for detectable expression (see Online Methods). (e) GFI1B expression for Group 3 and Group 4 MBs (n=119) coloured according to 9q34 SV state. Dashed line indicates the threshold for detectable expression (see Online Methods).
Figure 2
Figure 2. Summary of recurrent SVs identified in GFI1B activated MBs
(a, b) Representative WGS coverage plots and associated schematics summarizing the different mechanisms of SV observed in GFI1B-activated MBs.
Figure 3
Figure 3. Recurrent SVs juxtapose GFI1B proximal to active enhancers on 9q34
(a) SV breakpoints (n=18), enhancer-histone marks (H3K27Ac and H3K9Ac; n=6), and whole-genome DNA methylation data (n=6) overlapping the 9q34 locus in a subset of analysed MBs. (b) Allelic analysis of RNA-seq and enhancer ChIP-seq reads overlapping GFI1B. (c) Luciferase reporter activity for regions encompassed within the predicted enhancers indicated in panel (a) compared to empty vector. Error bars represent standard deviation from 3–4 independent experiments.
Figure 4
Figure 4. Mutually exclusive activation of GFI1 and GFI1B in MB
(a, b) GFI1 expression is largely restricted to Group 3 (a) and is mutually exclusive from GFI1B expression (b). (c) GFI1 expression for Group 3 and Group 4 MBs (n=119) coloured according to underlying SV state. Dashed line indicates the threshold for detectable gene expression (see Online Methods). (d) Summary of GFI1 translocations (n=6) observed in Group 3 MB. (e) Schematic of the reciprocal t(1:21) translocation observed in GFI1-activated MAGIC_MB359. Histone marks overlapping the breakpoints proximal to GFI1 and the partner chr21 translocation region are shown for a non-GFI1-activated case (MAGIC_MB399) and the translocation case (MAGIC_MB359).
Figure 5
Figure 5. GFI1 and GFI1B cooperate with MYC to promote MB in mice
(a) Strategy for evaluating Gfi1/Gfi1b as putative MB oncogenes. (b) Whole-mount images of GFP-expressing MG (MYC + GFI1) and MGB (MYC + GFI1B) tumours. (c) Survival curves for animals receiving 1×105 cells infected with viruses carrying the indicated transgenes. (d) Bioluminescent imaging of recipient animals at the indicated time points. X’s denote animals necessitating sacrifice prior to reaching the indicated time point. (e) H&E staining of cerebellar sections derived from MG tumour-bearing mice. (h) Immunofluorescence imaging of MG tumours stained with the indicated antibodies. (i) Subgroup probabilities for Ptch1+/−, MG, and MGB models based on cross-species molecular classification.
Figure 6
Figure 6. Summary of inferred mechanisms underlying GFI1/GFI1B activation in MB
Predominant mechanisms of SV and corresponding genomic redistribution of strong enhancers, including SEs, observed in GFI1/GFI1B-activated MBs. Activation of GFI1/GFI1B occurs in a mutually exclusive manner in either Group 3 or Group 4 and both oncogenes can cooperate with MYC to promote MB pathogenesis.

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