Supplementary Online Content

Bredel M, Scholtens DM, Harsh GR, et al. A network model of a cooperative genetic landscape in brain tumors. JAMA. 2009;302(3):261-275.

eFigure 1. Genome-wide Dosage Maps of Gliomas

eFigure 2. Gene Set Enrichment Analysis of Gene Dosage Effects on

eFigure 3. Genome Dispersion of Gene Dosage Effects on Gene Expression in Gliomas

eFigure 4. Likelihood for Gene Dosage Effects

eFigure 5. Network Hub and Hub-Interacting and Their Relationships

eFigure 6. Prevalence of Cooperatively Tumorigenic Gene-Gene Network Relationships

This supplementary material has been provided by the authors to give readers additional information about their work.

© 2009 American Medical Association. All rights reserved.

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 1. Genome-wide Gene Dosage Maps of Gliomas

A Gains Losses 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 25%

0%

Frequency 25% O OA

n = 45 GBM A

33,245 cDNA clones mapped along genome position Gain Loss

B Gains Chromosome Losses 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

50% 0% 50% Frequency

n = 219 GBM

22,471 genes mapped along genome position Gain Loss

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 1 (continued): Gene dosage profiles were generated using circular binary segmentation (CBS) change point analysis of microarray-based genomic hybridization profiles. This analysis identified genomic regions of equal gene dosage. Heatmaps depict the gene dosage profiles for individual tumors across individual displayed according to genome order (x and y chromosomes omitted). Gene dosage gain and loss are denoted in red and green, respectively. Cartesian line diagrams depict the frequency of gene dosage alteration across the tumor population according to genome position along the chromosomes. Red lines indicate the frequency of gene dosage gains, green lines the frequency of gene dosage losses. The initial discovery set consisted of 45 gliomas from Stanford University (O, oligodendrocytic glioma; OA, oligoastrocytic glioma; GBM, glioblastoma; A, astrocytic glioma) (A). Data are plotted for 33,245 Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium cDNA clone objects (‘cDNA clones’) that assess the gene dosage profiles of individual genes across the genome. The validation set consisted of 219 glioblastomas from The Cancer Genome Atlas (TCGA) Pilot Project (B). Gene dosage data are here plotted for 22,471 genes. Both the discovery and the validation sets show consistently high frequencies of alterations involving chromosomes 1p, 7, 9p, 10, 12, 13, 19, 20, and 22.

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 2. Gene Set Enrichment Analysis of Gene Dosage Effects on Gene Expression

Genes with significant (q < 0.1) gene dosage effect on transcription (n = 1,562) Genes mapping to the interaction network of Figure 3A (n = 214) p < 0.05 Cancer Cell cycle Cellular growth and proliferation Cellular movement, migration, and invasion

omoting Tumor morphology Cell-to-cell signaling and interaction

functions Cell death

umor-p r Organismal survival T DNA replication, recombination, and repair Cell signaling

Embryonic development Cellular development Tissue development Organ development

functions Organismal development Nervous system development and function D ev elopmental

Integrin signaling signaling Pyrimidine metabolism Nucleotide excision repair pathway ays

ted PDGF signaling

th w EGF signalingg el a PI3K/AKT signaling Death receptor signaling ERK/MAPK signaling

Cancer- r Cell cycle: G1/S checkpoint regulation

signaling p a Cell cycle: G2/M DNA damage checkpoint regulation JAK/Stat signaling NF-κB signaling 8 6 4 2 Significance [-Log10(p)]

Bars represent the negative logarithm to the based 10 of the p value (according to right-tailed Fisher's exact test) indicating the significance of enrichment for tumor-promoting and developmental functions and cancer-related signaling pathways of two gene sets: all 1,562 genes with a significant (false discovery rate adjusted q < 0.1) gene dosage-gene expression relationship, and the 214 genes mapping to the interaction network of Figure 3A. The blue line indicates a p value cutoff corresponding to α = 0.05.

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 3. Genome Dispersion of Gene Dosage Effects on Gene Expression in Gliomas

123456789 10 11 12 13 14 15 16 17 18 19 20 21 22 X

Genetic landscape confirmed by TCGA Genome position of 10,351 expressed genes (glioma transcriptome) Genes with significant (q < 0.1) (hypomorphic) relationship between gene dosage loss/reduced gene expression Genes with significant (q < 0.1) (hypermorphic) relationship between gene dosage gain/increased gene expression

A human ISCN-850 chromosome ideogram is shown and the chromosomal mapping of genes with a significant (false discovery rate adjusted q value < 0.1) relationship between gene dosage and gene expression. Green bars indicate the chromosomal position of genes with a significant hypomorphic gene-transcript relationship, i.e. gene dosage loss that reduces gene expression; red bars indicate the position of hypermorphic relationships, i.e. gene dosage gain that increase gene expression. Gray bars denote the chromosomal positions of all 10,351 genes --with combined availability of gene dosage and gene expression data in the discovery set (Stanford) --used in the gene dosage/expression analysis. The TCGA-validated nonrandom genetic landscape is denoted by the dark red background boxes. Despite a widespread association of changes in gene dosage and expression across the glioma genome, there was a greater likelihood for a gene dosage effect on the expression of genes mapping to the genetic landscape compared to genes mapping to random genetic alterations (p < 0.001, Pearson's chi-square test; Odds ratio [OR]: 7.7, 95%-confidence interval [CI] = 6.9 -8.6). Within the genetic landscape, there was a greater likelihood for alterations to act hypomorphic rather than hypermorphic (OR hypomorphic vs. hypermorphic: 8.4 [95%-CI: 7.3 -9.6] vs. 3.5 [95%-CI: 2.8 -4.2]; both p < 0.001).

Downloaded From: https://jamanetwork.com/ on 09/24/2021

eFigure 4. Likelihood for Gene Dosage Effects

Likelihood of Gene Dosage Effect in Genetic Landscape (LS) vs. Genome-wide:

Gene-dosage effect (q < 0.1) yes no n = Value 95%-CI yes 976 1563 2539 Odds Ratio (OR) 7.700 6.856-8.648 LS no 586 7226 7812 Chi-square 1431.531 n = 1562 8789 10351 p value < 0.001

Likelihood of Hypomorphic vs. Hypermorphic Effect within the Genetic Landscape:

Hypomorphic (q < 0.1) yes no n = Value 95%-CI yes 751 1788 2539 Odds Ratio (OR) 8.400 7.343-9.610 LS no 372 7440 7812 Chi-square 1220.132 n = 1123 9228 10351 p value < 0.001

Hypermorphic (q < 0.1) yes no n = Value 95%-CI yes 225 2314 2539 Odds Ratio (OR) 3.452 2.847-4.186 LS no 214 7598 7812 Chi-square 176.857 n = 439 9912 10351 p value < 0.001

Analysis based on 10,351 genes with combined availability of gene dosage and expression data in the Stanford data set.

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 5. Network Hub and Hub-interacting Genes and their Relationships

A B FDR < 0.05 FDR < 0.05 gain/loss gain/gain or loss/loss Score

SMARCB1 Sign. association (+/+ or -/-) Sign. association (+/-)

CASP3 Non-sign. association (FDR > 0.05) CDC42 EP300 IGF1R EGFR PTEN

RAF1 Sign. interaction within LS (+/+ or -/-) CSF1 BAX Sign. interaction within LS (+/-) Sign. interaction outside LS (+/+ or -/-) Sign. interaction outside LS (+/-) Non-sign. interaction (FDR > 0.05) CDC42 No molecular interaction CSF1 RAF1 CASP3 EGFR MYC MYC PTEN PTEN Frequency (no. of genes) IGF1R IGF1R BAX BAX

EP300 / IGF1R CSF1 BAX / RAF1 SMARCB1 CDC42 SMARCB1 95%-quantile EP300 EP300 BAX PTEN MYC EGFR CASP3 RAF1 CDC42 EGFR CASP3 PTEN MYC

Connectivity (no. of interactions)

Sign. association (+/+ or -/-) C Sign. association (+/-) 92 hub-interacting genes with tumor-related ontology Non-sign. association (FDR > 0.5) Sign. interaction within LS (+/+ or -/-) Sign. interaction within LS (+/-) Sign. interaction outside LS (+/+ or -/-) FDR < 0.05 FDR < 0.05 Sign. interaction outside LS (+/-) gain/loss gain/gain or loss/loss Non-sign. interaction (FDR > 0.05) Score No molecular interaction ENO1 FRAP1 FBXO2 MFN2 CAPZB RPL11 AK2 MACF1 PRDX1 FUBP1 PRKACB CYR61 BIF1 PKN2 GTF2B GBP2 BCAR3 VCAM1 RAP1A GPX1 PAK2 CASP6 PPID TPD52L1 CYCS TAX1BP1 RALA POLD2 MGAT3 CUL1 FASTK TNFRSF10B BNIP3L PLEC1 SMARCA2 JAK2 TOPORS BAG1 RAPGEF1 AKR1C3 PRKCQ STAM MLLT10 YME1L1 ANXA7 CHUK SLK MXI1 PDCD4 SMPD1 APLP2 FKBP4 NOL1 MDM2 PPP1CC DACH1 ABCC4 CUL4A FBN1 FURIN CRK YES1 CDC37 GDF15 CAPNS1 ACTN4 PAK4 FBL RPS19 CEACAM1 PVR PPP1R15A POLD1 TRIM28 SIRPA PCNA EIF6 RBL1 PLCG1 MYBL2 PTPN1 BID SLC25A1 CRKL EWSR1 TIMP3 MKL1 RANGAP1 TIMP1 OGT LAMP2 BCAP31 CDC42 1p36.12 CSF1 1p13.3 RAF1 3p25.2 CASP3 4q35.1 EGFR 7p11.2 MYC 8q24.21 PTEN 10q23.31 IGF1R 15q26.3 BAX 19q13.33 SMARCB1 22q11.23 EP300 22q13.2

CDC42 1p36.12 CSF1 1p13.3 RAF1 3p25.2 CASP3 4q35.1 EGFR 7p11.2 MYC 8q24.21 PTEN 10q23.31 1 network hub genes 15q26.3 1 IGF1R BAX 19q13.33 SMARCB1 22q11.23 EP300 22q13.2 Xq28 7p13 3q29 4q25 Xq24 12q15 20p13 20q12 13q34 7p14.1 9p21.1 1p35.1 1p34.3 1p34.1 1p31.1 1p31.1 1p22.3 1p22.3 1p22.2 1p22.2 1p22.2 1p22.1 1p21.2 1p13.2 4q32.1 7p15.3 7p15.2 7q22.1 7q36.1 7q36.1 8p21.2 8p21.2 8q24.3 9p24.3 9p24.1 9p13.3 Xp11.3 Xq13.1 1p36.23 1p36.22 1p36.22 1p36.22 1p36.13 1p36.11 3p21.31 6q22.31 9q34.13 10p15.1 10p15.1 10p12.1 10q22.2 10q25.1 10q25.2 10q25.2 13q32.1 19q13.2 19q13.2 19q13.2 19q13.2 19q13.2 20p12.3 22q12.2 22q12.3 22q13.1 22q13.2 11p15.4 11q24.3 15q21.1 15q26.1 17p13.3 19p13.2 20q13.12 10p12.33 10p12.31 10q24.31 13q21.33 19q13.12 19q13.32 19q13.33 19q13.33 19q13.43 20q11.22 20q11.23 20q13.13 12p13.33 12p13.31 12q24.11 18p11.32 19p13.11 22q11.21 22q11.21 22q11.21

(A) Histogram showing the distribution of connectivity (i.e., number of interactions with other genes) of all genes in the network shown in Figure 3. Normal curve shown in red. Genes with connectivity above the 95%-quantile (α = 0.05) of the overall distribution of gene connectivity (3 9 connections) were deemed network `hub' genes and are labeled. (B, C) Association maps for 11 network hub genes (B) and the 11 network hub and 92 hub-interacting genes with tumor-related functions (C). Hub genes mapping to the genetic landscape (LS) are labeled in green and red according to hypomorphic (i.e., gene is deleted and under- expressed) and hypermorphic (i.e., gene is gained and over-expressed) dosage effect, respectively. Hub genes mapping outside the genetic landscape are labeled in black. Above diagonal in B and above horizontal in C: significant (false discovery rate < 0.05) associations of hub gene alterations, modeled using a similar computational approach as in Figure 2. Red indicates a significant coincidence of gene dosage gain/gain (+/+) or loss/loss ( -/ -) events; blue a significant coincidence of gene dosage gain/loss events (+/ -). Color intensities indicate the level of significance of the associations. White squares denote non-significant (FDR > 0.05) associations. Below diagonal in B and below horizontal in C: integration of the association data shown above the diagonal with information about the presence of physical and functional gene interactions reveals significant gene interactions (i.e., interactions between genes whose gene dosage alterations are significantly associated). Significant interactions between genes mapping to the genetic landscape are indicated by filled red (+/+ or -/ - events) and blue (+/ - events) squares (no color gradation according to level of significance); significant interactions between genes outside the landscape are indicated by squares with red (+/+ or -/ - events) and blue (+/ - events) crosses. Black squares indicate physical or functional gene-gene interactions that are not significantly associated (FDR > 0.05).

Downloaded From: https://jamanetwork.com/ on 09/24/2021 eFigure 6. Prevalence of Cooperatively Tumorigenic Gene-Gene Network Relationships

11 hub genes plus 92 hub-interacting genes with tumor-related functions Interaction cooperative non-cooperative total MYC 28 10 38 EGFR 12 9 21 PTEN 16 4 20 CASP3 19 5 24 Paired t-test CDC42 7 1 8 T value 3.913161 11 hub genes CSF1 7 5 12 p value < 0.003 RAF1 5 7 12 Degrees of Freedom 10 IGF1R 6 3 9 BAX 11 2 13 SMARCB1 7 1 8 EP300 7 2 9 Sum, n = 125 49 174 Percentage 71.8 28.2

Downloaded From: https://jamanetwork.com/ on 09/24/2021