SUPPLEMENTARY INFORMATION: Single cell derived clonal analysis of human glioblastoma links functional and genomic heterogeneity Mona Meyer*, Jüri Reimand*, Xiaoyang Lan, Renee Head, Xueming Zhu, Michelle Kushida, Jane Bayani, Jessica C. Pressey, Anath Lionel, Ian D. Clarke, Michael Cusimano, Jeremy Squire, Stephen Scherer, Mark Bernstein, Melanie A. Woodin, Gary D. Bader**, and Peter B. Dirks** * These authors contributed equally to this work. ** Correspondence: [email protected] or [email protected] Supplementary information - Meyer, Reimand et al.
Supplementary methods 4
Patient samples and fluorescence activated cell sorting (FACS)! 4!
Differentiation! 4!
Immunocytochemistry and EdU Imaging! 4!
Proliferation! 5!
Western blotting ! 5!
Temozolomide treatment! 5!
NCI drug library screen! 6!
Orthotopic injections! 6!
Immunohistochemistry on tumor sections! 6!
Promoter methylation of MGMT! 6!
Fluorescence in situ Hybridization (FISH)! 7!
SNP6 microarray analysis and genome segmentation! 7!
Calling copy number alterations! 8!
Mapping altered genome segments to genes! 8!
Recurrently altered genes with clonal variability! 9!
Global analyses of copy number alterations! 9!
Phylogenetic analysis of copy number alterations! 10!
Microarray analysis! 10!
Gene expression differences of TMZ resistant and sensitive clones of GBM-482! 10!
Reverse transcription-PCR analyses! 11!
Tumor subtype analysis of TMZ-sensitive and resistant clones! 11!
Pathway analysis of gene expression in the TMZ-sensitive clone of GBM-482! 11!
Supplementary figures and tables 13
2 Supplementary information - Meyer, Reimand et al.
Table S1: Individual clones from all patient tumors are tumorigenic. ! 14!
Fig. S1: clonal tumorigenicity.! 15!
Fig. S2: clonal heterogeneity of EGFR and PTEN expression.! 20!
Fig. S3: clonal heterogeneity of proliferation.! 21!
Fig. S4: clonal differentiation potential.! 22!
Fig. S5: correlation of TMZ resistance and proliferation. ! 27!
Fig. S6: doubling time of clones from tumor GBM-472. ! 28!
Fig. S7: clonal MGMT status.! 29!
Fig. S8: clonal drug response in NCI library.! 30!
Table S2: clonal drug response in NCI library.! 31!
Fig. S9: clonal dose response of bortezomib, daunorubicin, and romidepsin.! 37!
Fig. S10: PCA clustering of clonal copy number alterations.! 38!
Fig. S11: circos plots of clonal copy number alterations.! 39!
Fig. S12: Copy number of chromosome 3 in tumor GBM-482. ! 42!
Fig. S13: FISH validation of clonal chromosomal alterations. ! 43!
Table S3: genes with clonal copy number alterations.! 44!
Fig. S14: genes with consistent copy number alterations.! 49!
Fig S15: clonal copy number alterations observed in TCGA.! 50!
Fig. S16: pathway analysis of clonal copy number alterations.! 51!
Table S4: pathway analysis of clonal copy number alterations.! 52!
Fig S17: Subtype scoring of clones from tumor GBM-472, GBM-489, and GBM-498.! 57!
Table S5: genes with differential expression in TMZ sensitivity. ! 58!
Table S6: pathway analysis of TMZ expression profile. ! 67!
Bibliography! 69
3 Supplementary information - Meyer, Reimand et al.
Supplementary methods Patient samples and fluorescence activated cell sorting (FACS) Tumor samples were obtained from consenting patients, as approved by the Research Ethics Boards at The Hospital for Sick Children and Toronto Western Hospital (Toronto). Within 1 hour of surgical resection tumors were processed and acutely dissociated in oxygenated artificial cerebrospinal fluid (CSF) and subjected to enzymatic dissociation. Following dissociation tumor cells were stained for the cell surface markers CD15 (#347423, BD Bioscience) and human specific CD133/1 (AC133/1-PE, Miltenyi Biotech) and single cells from each population (CD negative, CD15 positive, CD133 positive and CD15/CD133 double positive) were sorted into a 96 well plate (BD FACSAria III, BD FACSAria II, Beckman Coulter MoFlo-XDP). Single cells were cultured in EGF/FGF conditions as previously described (1, 2) and grown as adherent monolayer on poly-L- ornithine (Sigma) and laminin (Sigma) coated plates. Cells were grown in serum-free Neurocult NS-A Basal media (Stemcell Technologies), supplemented with 2 mM L- glutamine, N2 and B27, 75 µg/mL BSA, 10 ng/mL rhEGF, 10 ng/mL bFGF and 2 µg/mL heparin. Cells were treated with Accutase (Sigma) for expansion or harvesting. Differentiation 10,000 cells from each clone were seeded onto poly-L-ornithine/laminin (Sigma) coated coverslips in 24-well plates. Clones were grown under EGF/FGF conditions. In parallel, clones were also grown under growth factor withdrawal conditions to promote neuronal differentiation. Briefly, cells were cultured overnight in NS media with B27 supplement, 10 ng/mL rhEGF, 10 ng/mL bFGF and 2 µg/mL heparin. To promote differentiation, the media was changed to Neurocult NS-A Basal media supplemented with FGF2 (5ng/mL) and 1X B27 supplement only for 7 days, then to a 1:1 mix of Neurocult NS-A Basal media with Neurobasal media (Invitrogen) with 1X B27 supplement for 14 days. Immunocytochemistry (IHC) was performed using antibodies against the epitopes Nestin (AB5922, rabbit polyclonal, Millipore), βIII-tubulin (MAB1637, mouse monoclonal, Millipore), GFAP (MAB360, mouse monoclonal, Millipore), MAP2 (MAB3418, mouse monoclonal, Millipore). Slides were visualized and collected using a Leica STP 6000 microscope. Representative images were taken at 20x magnification as merged images and their individual filter images. Immunocytochemistry and EdU Imaging Clones were grown on poly-L-ornithine/laminin–coated glass coverslips. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS, and permeabilized in 0.3% Triton X for 30min. Cells were stained with antibodies against human-specific Nestin (1:1,000; AB 5922; Millipore), human-specific GFAP (1:1,000; MAB 360, Millipore), and Map2 (1:MAB 3418, mouse monoclonal, Millipore). Secondary antibodies used include Alexa Fluor 488 Goat anti-Rabbit IgG (1:500; A11034, Invitrogen), Alexa Fluor 488 Goat anti-Mouse IgG (1:500, A21042, Invitrogen), and Alexa Fluor 568 Goat anti-Mouse IgG (1:500, A11004, Invitrogen). DNA staining was performed using DAPI (Sigma).
4 Supplementary information - Meyer, Reimand et al.
Proliferation All clones were seeded onto poly-L-ornithine/laminin (Sigma) coated 96-well plates at an initial density of 2000 cells per well. Relative cell proliferation was measured for each clone at days 0, 2, 4, 6, and 8 using an AlamarBlue assay (Life Technologies), as per manufacturer's protocol. Briefly, 10 μL of Alamar blue was added to 90 μL of culture medium in each well. After 6 hours, plates were read on an Spectramax Gemini EM microplate reader (Molecular Devices), with an excitation wavelength of 544 nm and emission wavelength of 595 nm. Data were acquired using SOFTmaxPRO and analysed using GraphPad Prism 5 software. Each data point is an average of at least 3 replicates, and error bars represent standard deviation. The population doubling level (PDL) for each clone from tumor 472 was calculated using the formular: PDL = X + 3.322 (log Y – log I), where, X = initial PDL; I = cell inoculum (number of cells plated); Y = final cell yield (number of cells at the end of the growth period). Doubling time assay for each clone was performed in triplicates. Western blotting Clonal protein expression of EGFR, EGFRvIII, and PTEN was analyzed with western blots using EGFRvIII-transfected human fetal brain cells (HF7450NS) as positive control and eGFP transfected cells as negative control. Western blotting was performed using the following primary antibodies: anti-EGFR (1:1,000; ab2430; Abcam), anti-EGFRvIII (1:1,000; Zymed; now discontinued), anti-PTEN (1:200; sc7974; Santa Cruz Biotechnology), anti-Actβ (1:5,000; A5441; Sigma-Aldrich), anti-GABRA3 (1:1000; HPA000839; Sigma-Aldrich), anti-phospho-H2A.X (Ser139) (1:1000; #2577; Cell Signaling). Secondary antibodies included goat anti-mouse IgG, HRP conjugate (1:100,000; A9044; Sigma-Aldrich), and goat anti-Rabbit IgG, HRP conjugate (1:40,000; A0545; Sigma-Aldrich). The human EGFR variant III cDNA [3] was cloned into the pIRES2-EGFP vector (#6029-1, Clontech Laboratories). The EGFRvIII construct was a gift from Dr. Abhijit Guha (The Hospital for Sick Children, Toronto). 5μg of pIRES2- EGFP and pIRES2-EGFRvIII were used to transfect approximately 1 million cells of the human fetal derived neural stem cell line HF7450. The transfection was performed using the Amaxa Nucleofector Kit (VPG-1004; Amaxa Biosystems) and the Nucleofector II Electroporator (Amaxa Biosystems) as per the manufacturer's instructions. Protein lysates were harvested for analysis 48 hours following transfection. Temozolomide treatment 3,000 cells of each clone were seeded into a well of a coated 96-well plate. All cells were treated once with temozolomide (TMZ) at a dose range of 0.39-100 µM. Cell viability was measured when DMSO treated control cells reached 70% confluence, using the AlamarBlue assay (Life Technologies), as per manufacturer's protocol, and read 6 hours later on an Spectramax Gemini EM microplate reader (Molecular Devices). Data were analysed with GraphPad Prism 5 software. Data points represent an average of three replicates, and error bars represent standard deviation. Unpaired T-test with Welch’s correction was used to calculate p-values of cell survival between relatively sensitive and resistant clones.
5 Supplementary information - Meyer, Reimand et al.
NCI drug library screen Clones were seeded onto poly-L-ornithine/laminin (Sigma) coated 96-well plates at a density of 2000 cells per well. Drugs from the NCI anticancer library were added to the cells 24 hours later at a 500 nM concentration. Five days after treatment, cell viability was measured using the AlamarBlue assay (Life Technologies), as per the manufacturer's protocol. Six hours after adding AlamarBlue plates were read on an Spectramax Gemini EM microplate reader (Molecular Devices), with an excitation wavelength of 544 nm and emission wavelength of 595 nm. Data was acquired using SOFTmaxPRO. Dose response assays for bortezomib, daunorubicine, and romidepsin were performed at a dose range of 0.12-50 nM in 96-well plates and cell viability was measured using AlamarBlue assay (Life Technologies) when DMSO control treated cells reached 70% confluence. Orthotopic injections Intracranial cell transplantation of 100,000 cells re-suspended in 2 µl of phosphate buffered saline (PBS). Cell aliquots were injected stereotactically into the frontal cortices of 6- to 8-week-old NOD-SCID Gamma mice, following administration of general anaesthesia. The injection coordinates were 2 mm to the right of the midline, 4 mm anterior to lambda and 4 mm deep. Immunohistochemistry on tumor sections Paraformaldehyde-fixed, paraffin-embedded tissue sections were mounted on positively charged microscope slides. Tissue sections were baked overnight at 60°C, treated with epitope retrieval techniques, and blocked for endogenous peroxidase and biotin before the application of the primary antibody. Incubation of rabbit anti-human Nestin (Millipore AB5922), mouse anti-human GFAP (Sternberger SMI21), rabbit anti-human Ki67 (Thermo RM-9106-S) and mouse anti-mouse MAP2 (Millipore MAB3418) all at 1:500 dilution was performed at 4ºC overnight. Subsequent immunodetection was performed using the Alexa Fluor goat anti-rabbit A488 (Invitrogen A11034) and Alexa Fluor goat anti-mouse A568 (Invitrogen A21124). Samples were incubated at room temperature for an hour. Slides were mounted with Dako Fluorescent Mounting Medium (DAKO S2023). Promoter methylation of MGMT Promoter methylation status of MGMT was determined with methylation-specific PCR (MSP) assay. Sodium bisulfite conversion of purified DNA from clones was performed using EZ DNA Methylation-GoldTM Kit (Zymo Research). Control samples consisting of CpGenome Universal Methylated DNA (Millipore) as well as blood DNA from patients G472 and G482 were treated with the same method. MSP assays were performed using a nested two-stage PCR approach as described previously (3, 4). The first stage PCR amplified bisulfite converted DNA template regardless of methylation status. The primer sequences for first-stage PCR were 5’-GGA TAT GTT GGG ATA GTT-3’ (forward) and 5’-CCA AAA ACC CCA AAC CC-3’. First PCR reactions took place in a 20 µL volume with PCR buffer (10 mM Tris pH 8.3, 50 mM KCl), 1.5 mM MgCl2, dNTPs (250 mM), primers (0.3 µM), and 1 U Taq DNA Polymerase (Roche). PCR was carried out with an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30s and
6 Supplementary information - Meyer, Reimand et al. a final extension step at 72°C for 10 minutes. The second PCR reaction was specific to either methylated or unmethylated DNA. Primer sequences were 5’-TTT GTG TTT TGA TGT TTG TAG GTT TTT GT-3’ (forward) and 5’-AAC TCC ACA CTC TTC CAA AAA CAA AAC A-3’ (reverse) for the unmethylated reaction, 5’-TTT CGA CGT TCG TAG GTT TTC GC-3’ (forward) and 5’-GCA CTC TTC CGA AAA CGA AAC G-3’ (reverse) for the methylated reaction. Second PCR reactions took place in a 10 µL volume with PCR buffer (10 mM Tris pH 8.3, 50 mM KCl), 1.5 mM MgCl2, dNTPs (250 µM), primers (0.5 µM), and 0.5 U Taq DNA Polymerase (Roche). PCR was carried out with an initiatial denaturation step at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 50s, annealing at 59°C for 50 s, extension at 72°C for 50s and a final extension at 72°C for 2 minutes. PCR products were run on 4% agarose gels and visualized following ethidium bromide staining. Fluorescence in situ Hybridization (FISH) For clonal cultures, fixed metaphase and nuclear cytogenetic suspension was prepared according to standard methods (5). To enumerate copy-number changes of chromosomes 7, 3 and 17, the Urovision probe kit (Abbott Laboratories, Illinois, USA) was used according to manufacturer’s instructions. This probe set contains directly labelled DNAs for centromeres 3 (red), 7 (green) and 17 (aqua). Following post- hybridization washes, the slides were mounted in a DAPI-Antifade medium (Abbot Laboratories), and visualized and collected using a Zeiss Axioplan microscope (Carl Zeiss Canada). Normal human lymphocyte preparation was also used as a technical control. For each slide 50 nuclei were scored. For tissues embedded in paraffin section, 10 mm sections were pre-treated as described previously (6) and hybridized with the Urovision probes. Following post- hybridization washes, slides were mounted in a DAPI-Antifade medium (Abbot Laboratories) and visualized and collected using a Zeiss Axioplan microscope (Carl Zeiss Canada). At least 50 nuclei were scored. Representative images were taken at 60x magnification as merged images, and their individual filter images. SNP6 microarray analysis and genome segmentation Preprocessing of Affymetrix SNP6 microarrays and estimation of total copy numbers was carried out with the CRMAv2 pipeline (7). Full microarray annotation data for both single nucleotide polymorphism (SNP) and copy number variation (CV) probes of the human genome assembly HG37 were retrieved from the Aroma Project website (aroma- project.org/data/annotationData/chipTypes/GenomeWideSNP_6/). The set of 44 tumor samples, three matched blood samples, and 226 normal controls were analyzed in one single batch. The controls comprised blood samples collected as part of the Ontario Population Genomics Platform (OPGP) genetic epidemiological project from healthy adults of European ancestry from the Ontario population (8), genotyped using the same experimental protocols, microarray platform and facility as the tumor samples. Relative probe-level copy number calling was performed separately for matched tumors and one non-matched tumor. For three of four tumors (GBM-472, GBM-482, GBM-498), primary, unsorted and clonal populations were referenced to matched blood samples as probe-level ratios log2(tumor/blood). No matched blood sample was available for the fourth tumor (GBM-489). For the latter, a reference diploid genome was estimated from
7 Supplementary information - Meyer, Reimand et al.
the copy number profiles of 226 normal controls by median summarization of probe- level intensities as log2(tumor/median(controls)). Probes with missing reference values were removed before relative copy number calling. Identification of genomic copy number segments was performed with the Circular Binary Segmentation (CBS) algorithm of the DNAcopy R package (9). Prior to segmentation, relative copy numbers were smoothed with the DNAcopy function smooth.CNA() using default parameters. We averaged the signal for a small fraction genomic coordinates that had multiple corresponding probes (CV, SNP). We applied a weighted version of the CBS algorithm with tumor-specific probe weights that were inversely proportional to the variance of the probe across a particular tumor (1/var(probe)). The CBS algorithm was executed with split inversion settings (undo.splits="sdundo") and a conservative minimal segment size (min.width=5) to reduce the number of small segments that are likely artifacts of the segmentation algorithm. Calling copy number alterations All relative probe-level copy number calls were normalized for every sample independently and mean Z-scores were computed for CBS-derived segments. Segments were split into five classes of copy number based on their mean Z-scores: • balanced (no significant copy number alteration): -1.5
8 Supplementary information - Meyer, Reimand et al.
To verify our copy number analysis method, we compared our findings with the TCGA glioblastoma study (11). We used the Fisher’s exact test to assess the significance of overlap between genes with significant focal CNAs from this study and the frequently altered expression-correlated genes from the TCGA dataset. Pathway enrichment analysis of all frequency-ranked genes with copy number alterations was performed with the g:Profiler R package, using the same parameters as described below. Pathways and processes with less than three altered genes were discarded. To remove functional biases resulting from co-located gene families, the following four groups of genes were removed, keeping only the first gene as a representative example (PRAME family members - 21 genes discarded; olfactory receptors (OR5) - 22 genes discarded; interferons (IFN) - 7 genes discarded; haemoglobins (HB) - 4 genes discarded). Recurrently altered genes with clonal variability A gene was defined to have clonal variability of copy number if more than one copy number state (balanced, gain, loss, amplification, or deletion) was observed for that gene in the samples of a particular tumor (primary, unsorted and clonal populations). In contrast, a gene was defined to have consistent copy number alterations if all samples of a particular tumor had the gene in the same significant copy number state. Recurrent genes with clonal variability included all genes that were variable in at least one tumor and showed a significant copy number alteration (gain, loss, amplification, or deletion) in five or more samples across the entire dataset. Recurrent genes with clonal variability were visualized as a hierarchical clustering heatmap, using binary distance for similarity estimation (significant vs. non-significant copy number states) and complete agglomeration for clustering. To reduce redundancy, co-occurring and co-altered gene families were represented by single columns in the final heatmap. Smaller groups were fitted on the map using comma-separated symbols, while larger groups were labelled with pound signs - chr10 ORFs (C10ORF#: C10ORF68, C10ORF126) zinc finger-containing proteins (ZNF#: ZNF248, ZNF25, ZNF33A, ZNF37A, ZNF438), lipases (LIP#: LIPA, LIPF, LIPJ, LIPK, LIPM, LIPN), and interferons (IFN#: IFNA1, IFNA14, IFNA17, IFNA5, IFNA8, IFNE). Global analyses of copy number alterations Probe-level analyses were carried out on the smoothed set of 1.78M probes, either on relative copy numbers (log2 ratios) or normalized probe calls (Z-scores). The heatmap to visualize genetic heterogeneity of GBM is a summary of probe-level copy number ratios. All chromosomal regions covered by SNP6 probes were split into non-overlapping 1MB bins, and the median signal was computed in each bin. For color scaling, median values were capped at closest integer level copy number alterations (log2(1/2) for losses, log2(3/2) for gains). Probe-level unsupervised clustering with principal component analysis (PCA) was carried out with the prcomp() function in R, after discarding probes with missing values. Two PCA plots were produced - one with relative copy numbers (log2 ratios), and the other with normalized probe calls (Z-scores). Circos plots (12) were used to visualize genomic copy number segments for each individual primary tumor. Normalized probe values (Z-scores) were used for visualization. Scores were capped at z=abs(2) due to space constraints.
9 Supplementary information - Meyer, Reimand et al.
Phylogenetic analysis of copy number alterations Phylogenetic analysis of copy number alterations considered median copy number alteration (Z-score) in 1kb non-overlapping genomic windows. Median Z-scores were discretized into five classes as shown above as (abs(z)<1.5 - balanced copy number; 1.5
Gene expression differences of TMZ resistant and sensitive clones of GBM-482 Heterogeneous TMZ resistance phenotype in GBM-482 was correlated to changes in gene expression. We compared seven TMZ-resistant clones with one TMZ-sensitive clone (482_9) by transforming all normalised gene expression values to Z-scores and identifying genes with absolute(Z)>=2 in the clone 482_9, corresponding to 822 differentially expressed genes at significance cutoff p=0.05. Differentially expressed genes in the TMZ-sensitive were plotted in the order of log fold change relative to mean expression in seven TMZ-resistant clones, and canonical cancer genes from earlier review papers (16-19) were highlighted. One-tailed Fisher’s exact test was used to evaluate the enrichment of cancer genes in the list of differentially expressed genes using the gene universe represented on the microarray. We were unable to evaluate variation of gene-specific expression in TMZ-sensitive cells due to the single clone and therefore lack statistical power. Several further strategies were tested to extract TMZ-related gene signatures. Exploratory correction of p-values from Z-scores with Benjamini-Hochberg False Discovery Rate (FDR) was tested but it did not yield significant results. Expression analysis of sensitive and resistant clones from multiple tumors with confounding factors on clonal origin was also tested but failed
10 Supplementary information - Meyer, Reimand et al. to retrieve significant TMZ-sensitivity gene signatures, probably as inter-tumor differences in gene expression exceeded and masked differences reflecting TMZ response. Reverse transcription-PCR analyses Total RNA (500 ng) was used to do reverse transcription-PCR (RT-PCR). The following primers were used: human c-Met forward, 5’-GTTTCCCAATTTCTGACC-3’, and reverse, 5’-TATATCAAAGGTGTTTAC-3’; human EGFR forward, 5’- CGGGACATAGTCAGCAGTGA-3’, and reverse, 5’-GGGACAGCTTGGATCACACT-3’; glyceraldehyde-3-phosphate dehydroge- nase (GAPDH) forward, 5’- GAAGGTGAAGGTCGGAGT CA-3’, and reverse, 5’-GACAAGCTTCCCGTTCTCAG-3’. Expression levels of c-Met and EGFR mRNA were quantified using quantitative fluorescent real- time PCR. RNA samples were reverse-transcribed using random hexamers as described by the manufacturer (Roche, Quebec, Canada). PCR assays were done with SsoFast™ EvaGreen® Supermix (Bio-Rad, Bio-Rad Laboratories, CA) in a PTC-200 Peltier Thermal Cycler (Bio-Rad, Bio-Rad Laboratories, CA) and analyzed with Opticon Monitor 3 Analysis Software (Bio-Rad, Bio-Rad Laboratories, CA). Levels of c-Met and EGFR mRNA were normalized against GAPDH mRNA. Tumor subtype analysis of TMZ-sensitive and resistant clones Subtype analysis of gene expression data was based on GBM subtype predictor gene sets developed by the TCGA consortium (20) and retrieved from the website [http://tcga- data.nci.nih.gov/docs/publications/gbm_exp/]. We studied three subtypes (classical, proneural, mesenchymal). We used normalized gene expression data following the preprocessing steps described above. We implemented a single-sample classification strategy similar to the one used in (21). For a given classifier gene set and one sample, the mean expression value (intensity of probesets corresponding to signature genes) in that sample was divided by the mean expression of the entire array (intensities of all probesets), resulting in a raw subtype score for that sample and subtype. To establish confidence intervals of raw scores, the scores were bootstrapped by randomly drawing genes with replacement from the subtype signature. In each bootstrap iteration, some signature genes would be absent and others present multiple times, smoothing out the effect of any individual gene over the analysis. To normalize the bootstrapped raw scores to the relative background of the array and signature size, we also computed scores corresponding to 1,000 random signatures of genes of the same size as the true subtype signature. Bootstrapped scores were then normalized by subtracting the 95th percentile of random scores. Pathway analysis of gene expression in the TMZ-sensitive clone of GBM-482 Pathway analysis of gene expression was carried out on the genes differentially expressed in the TMZ-sensitive clone (cutoff absolute(Z)>2), using ordered enrichment analysis of the g:Profiler R package (15). The ordered list strategy helped emphasize pathway genes with the strongest differential expression and gain additional power through integration of signals from multiple genes.
11 Supplementary information - Meyer, Reimand et al.
Genes with increased and decreased expression were ranked by significance of differential expression (Z-score) and subjected to separate pathway analyses. We used g:Profiler parameters for FDR multiple testing correction and an upper limit of 500 genes for functional gene sets and pathways. Gene Ontology (22) categories, Reactome (23) and KEGG (24) pathways, and CORUM (25) protein complexes were included to the enrichment analysis, while other data sources were excluded. Enriched categories were further filtered: pathways with less than three altered genes were discarded, and pathways corresponding to alterations in less than two clones were also discarded. Functional enrichments were visualized as an Enrichment Map (26) with Cytoscape (27).
12 Supplementary information - Meyer, Reimand et al.
Supplementary figures and tables
13 Supplementary information - Meyer, Reimand et al.
Table S1: Individual clones from all patient tumors are tumorigenic. Five clones from each patient tumor were orthotopically injected into mice. Numbers indicate ratio of tumor engraftment to injections. GBM-472 GBM-482 GBM-489 GBM-498
Naïve Recurrent Naïve Recurrent Cells # Cells # Cells # Cells #
unsorted 3 / 4 unsorted N/D unsorted 4 / 4 unsorted 3 / 4
472_18 3 / 4 482_11 3 / 4 489_8 2 / 4 498_1 4 / 4
472_17 2 / 4 482_1 2 / 4 489_5 4 / 4 498_3 3 / 4
472_13 2 / 4 482_ 4 1 / 4 489_7 4 / 4 498_2 3 / 4
472_4 3 / 3 482_9 3 / 4 489_13 4 / 4 498_6 4 / 4
472_9 2 / 4 482_3 2 / 4 489_10 1 / 4 498_7 4 / 4
14 Supplementary information - Meyer, Reimand et al. Fig. S1: clonal tumorigenicity. Orthotopic injections of clones from all four tumors lead to diffusely infiltrative tumor growth in mice. Immunostaining of paraffin sections of GBM-472 (S1A), GBM-482 (S1B), GBM-489 (S1C) and GBM-498 (S1D) against human-specific Nestin (green),
15 Supplementary information - Meyer, Reimand et al. human specific GFAP (red) and Map2 (red). 100,000 cells of the unsorted cell population, as well as five clones from each GBM tumor were intracranially injected into the forebrain of NSG mice. The images show tumor cells infiltrated throughout the mouse brain; a consistent phenotype of glioblastoma cells in situ. Staining against Ki67 (green) demonstrates proliferation of tumor cells in all xenografts. Pictures on the right show DAPI (blue) staining of cells stained against Ki67/ Map2. Leica STP 6000 microscope, 40x magnification.
16 Supplementary information - Meyer, Reimand et al.
Fig. S1B. Immunostaining of paraffin sections of GBM-482 xenografts against human specific Nestin (green) and human specific GFAP (red) and Map2 (red).
17 Supplementary information - Meyer, Reimand et al.
Fig. S1C. Immunostaining of paraffin sections of GBM-489 xenografts against human specific Nestin (green) and human specific GFAP (red) and Map2 (red).
18 Supplementary information - Meyer, Reimand et al. Fig. S1D. Immunostaining of paraffin sections of GBM-498 xenografts against human specific Nestin (green) and human specific GFAP (red) and Map2 (red).
19 Supplementary information - Meyer, Reimand et al. GBM-489 Neg. ctl. Pos. ctl. 489 Primary tissue 489_unsorted 489_13 489_1 489_3 489_5 489_6 489_7 489_8 489_12 EGFR EGFRvIII EGFRvIII PTEN Act Negative control - HF7450NS pIRES2-eGFP Positive control - HF7450NS pIRES2-EGFRvIII Fig. S2: clonal heterogeneity of EGFR and PTEN expression. Western blotting confirms variable expression of EGFR, EGFRvIII, and PTEN in tumor GBM-489.
20 Supplementary information - Meyer, Reimand et al.
Fig. S3: clonal heterogeneity of proliferation. Individual clones of the same tumor have different proliferative capacity. Relative proliferation of clones from tumors GBM-472 (S3A), GBM-482 (S3B), GBM-489 (S3C), and GBM-498 (S3D) was measured over a 8-day interval. AlamarBlue assays were performed at days 0, 2, 4, 6, and 8 to assess cell growth.
21 Supplementary information - Meyer, Reimand et al.
Fig. S4: clonal differentiation potential. Diverse ability of individual clones to express differentiation markers. Clones from GBM-482 (S4A), GBM-489 (S4B) and GBM-498 (S4C) were grown under EGF/FGF withdrawal conditions. Immunohistochemistry was performed with antibodies against human Nestin (green), human GFAP (red). Asterisks indicate differentiation markers (βIII-tubulin - red, Map2 - green). EdU (red) staining after EdU labelling for 2h. DAPI (blue) staining for Map2/EdU stained cells is shown on the right. Images were taken at 20x magnification with a Leica microscope.
22 Supplementary information - Meyer, Reimand et al.
hu-Nestin / hu-GFAP/ DAPI BIII-tubulin* / DAPI Map2*/ EdU DAPI
EGF/FGF
50 µm 50 µm 50 µm 50 µm
489_unsorted EGF/FGF withdrawal
50 µm 50 µm 50 µm 50 µm
EGF/FGF
50 µm 50 µm 50 µm 50 µm 489_1
EGF/FGF withdrawal
50 µm 50 µm 50 µm 50 µm
EGF/FGF
50 µm 50 µm 50 µm 50 µm 489_6
EGF/FGF withdrawal
50 µm 50 µm 50 µm 50 µm Fig. S4B. Differentiation assay, GBM-489. Immunohistochemistry: human Nestin (green), human GFAP (red). Asterisks indicate differentiation markers. EdU (red) staining after EdU labelling for 2h. DAPI (blue) staining for Map2/EdU stained cells is shown on the right. Asterisks indicate differentiation markers.
23 Supplementary information - Meyer, Reimand et al.
hu-Nestin / hu-GFAP/ DAPI BIII-tubulin*/ DAPI Map2*/ EdU DAPI
EGF/FGF
50 µm 50 µm 50 µm 50 µm 489_7
EGF/FGF withdrawal
50 µm 50 µm 50 µm 50 µm
EGF/FGF
50 µm 50 µm 50 µm 50 µm 489_13
EGF/FGF withdrawal
50 µm 50 µm 50 µm 50 µm
Fig. S4B, continued. Differentiation assay of GBM-489.
24 Supplementary information - Meyer, Reimand et al. Fig. S4C. Differentiation assay of GBM-498. Innmunohistochemistry: human Nestin (green), human GFAP (red). Asterisks indicate differentiation markers. EdU (red) staining after EdU labelling for 2h. DAPI (blue) staining for Map2/EdU stained cells is shown on the right. Asterisks indicate differentiation markers.
25 Supplementary information - Meyer, Reimand et al. Fig. S4C, continued. Differentiation assay of GBM-498.
26 Supplementary information - Meyer, Reimand et al.
Lack of correlation between proliferation and drug resistance
● 12
●
y 8 tumor ● ● X472 ● ● ● ● ● X482 ation, d a 8 ● X489 e r ● ● ● X498 ● Proli f ● ● ● ●
●
● ● ● ● 4 ● ● ● ●
● ● ● ●
● ●
0.00 0.25 0.50 0.75 1.00 Drug resistance Cell survival at 100ul TMZ Fig. S5: correlation of TMZ resistance and proliferation. Comparsion of cell proliferation and TMZ-resistance of clones and unsorted populations reveals no significant correlation between proliferation and drug resistance. Pearson correlation was computed for mean cell survival and proliferation values for three or more replicates. Error bars denote s.e.m..
27 Supplementary information - Meyer, Reimand et al. 400" 350" 300" 250" 200" 150"
doubling)*me)[hrs]) 100" 50" 0" 472_4" 472_7" 472_8"
472_13" 472_14" 472_17" 472_18" Clones Doubling time [hrs] S.D. 472_4 156 64 472_7 195 79 472_8 139 44 472_13 275 81 472_14 69 15 472_17 136 21 472_18 81 6 Fig. S6: doubling time of clones from tumor GBM-472. The resistant clones (472_8, 472_18) differ in their doubling time (81 hrs vs 139 hrs). This suggests that temozolomide resistance is cell intrinsic and not due to a slower proliferation index of the resistant clones. Error bars denote s.d..
28 Supplementary information - Meyer, Reimand et al.
GBM-472 100 bp U M U M U M U M U M U M U M U M U M U M U M H O (+)* (-)* Un 13 4 7 8 14 17 18 2 (++) (+) Un 13 4 7 8 9 14 17 18 MGMT Act (++) - posititve control for MGMT expression, 489_13 (+) - positive control for MGMT expression, 489_unsorted GBM-489 100 bp U M U M U M U M U M U M U M U M U M U M U M U M H O (+)* (-)* Un 13 1 3 5 6 7 8 12 2 Un 13 1 3 5 6 7 8 12 MGMT Act GBM-498 100 bp U M U M U M U M U M U M U M U M U M
H2O (+)* (-)* Un 6 7 1 2 3 (+) Un 6 7 1 2 3 MGMT Act (+) - positive control for MGMT expression, 489_6 *(+) - posititve control, universally methylated DNA *(-) - negative control, blood DNA from the corresponding tumour Fig. S7: clonal MGMT status. Methylation specific PCR for MGMT promoter and MGMT protein expression in clones from tumors GBM-472, GBM-489 and GBM-498. All clones and the unsorted population from tumor GBM-472 are methylated and do not express MGMT protein. Clones from GBM-489 and GBM-498 show a heterogeneous methylation pattern and MGMT protein expression. Universally methylated DNA served as positive control, and DNA from blood was used as a control for unmethylated DNA.
29 Supplementary information - Meyer, Reimand et al.
a. Mitoxantrone Vincristine sulfate Paclitaxel Clofarabine Ixabepilone Plicamycin % growth Docetaxel inhibition Gemcitabine HCl 80 Cabazitaxel ug
D r Vinorelbine tartrate 60 Mitomycin C Vinblastine sulfate 40 Dactinomycin Daunorubicin HCl Doxorubicin HCl Topotecan HCl Romidepsin Bortezomib 489_1 489_5 489_6 489_7 489_8 489_10 489_12 489_13 clone b. Exemestane Dacarbazine Dasatinib Cladribine Mitomycin C Valrubicin Mitoxantrone % growth Gemcitabine HCl inhibition Topotecan HCl Doxorubicin HCl 75 Vinblastine sulfate ug Docetaxel D r 50 Vinorelbine tartrate Plicamycin 25 Dactinomycin Paclitaxel 0 Romidepsin Vincristine sulfate Bortezomib Cabazitaxel Ixabepilone Daunorubicin HCl
498_1 498_2 498_3 498_4 498_6 498_7 clone Fig. S8: clonal drug response in NCI library. NCI drug library screen reveals clones with variable drug response within the same tumor. Clones were subjected to 98 drugs from the NCI drug library. Cells were treated once with 500 nM of each drug. Cell growth inhibition was measured five days after treatment using AlamarBlue assay. All drugs with at least 50% cell growth inhibition are shown in order of decreasing variance. Drug screens for tumors GBM-498 (S8A) and GBM-498 (S8B) are shown.
30 Supplementary information - Meyer, Reimand et al.
Table S2: clonal drug response in NCI library. a. NCI drug screen for GBM-482. Cell growth inhibition of each drug of the NCI anticancer library on individual clones is shown. Growth inhibition was calculated relative to DMSO control treated cells. Drug 482_2 482_16 482_3 482_4 482_7 482_9 482_11 482_13 Allopurinol 0 0 0 2.47 0 0 0 3.166 Fluorouracil 24.555 5.74 0 2.777 0 0 0 0 Hydroxyurea 7.514 4.92 0 4.375 0 0 0 11.489 Thioguanine 37.077 0 0 0 0 2.522 0 0 Mercaptopurine 16.753 0 0 9.347 8.464 0 0 1.275 Nitrogen mustard 13.293 6.804 0 9.333 0 6.618 0 12.427 Thiotepa 7.936 12.379 2.002 12.092 10.071 0 0 8.475 Aminolevulinic acid 5.704 0.23 0 5.378 10.25 0 2.11 3.572 Dacarbazine 0.683 4.036 0 0 0 0 0 0 Arsenic trioxide 33.301 0 0 2.777 0 0 7.521 0 Temozolomide 6.552 0 0.125 15.677 0 0 0 0 Busulfan 11.51 0 0 1.984 0 0 0 11.799 Altretamine 0 0 0 0 0 0 13.921 0 Floxuridine 3.875 0 0 2.893 0 0.218 13.13 0 Methoxsalen 14.611 0 0 2.546 0 0 0 2.256 Lomustine, CCNU 2.799 0 0 5.323 12.468 0 0 1.823 Carmustine 0 0 0 0 0 0 20.336 0 Cyclophosphamide 0.407 0 0 0.405 0 0 12.086 0 Uracil mustard 13.481 0 1.72 3.204 0 0 4.326 0 Cytarabine HCl 30.865 6.194 61.377 62.139 9.577 42.897 29.162 69.763 Thalidomide 0 0 0 0 0 0 7.501 0 Procarbazine 0 0 0 0 0 0 15.499 0 Streptozocin 0 0 0 5.403 0 0 0 0 Cladribine 61.871 26.895 54.611 32.86 33.941 73.351 50.587 78.498 Ifosfamide 40.063 0 0 7.713 0 0 0 7.14 Cisplatin 17.603 0 0 0 0 0 1.404 0 Tretinoin 57.664 0 0.011 18.344 0 0 5.888 0 Dexrazoxone 5.528 0 0 0 0 0 0 0 Pentostatin 10.056 0 0 17.042 0 0 0 0 Gemcitabine HCl 60.689 45.415 62 79.646 45.22 65.515 67.649 78.703 Chlorambucil 45.416 0 0 5.115 5.562 0 0 21.963 Mitomycin C 82.304 37.597 44.098 81.097 59.521 65.173 73.607 78.375 Mitotane 0 0 0 5.579 0 0 0 10.863 Daunorubicin HCl 87.408 88.31 79.096 92.822 88.837 91.784 90.4 88.097 Etoposide 21.501 4.576 11.786 36.28 11.466 19.708 22.607 41.014 Tamoxifen citrate 0 1.408 0 0 0 0 0 0 Estramustine disodium 5.029 0 0 0 0 0 0 2.315 phosphate Pemetrexed 0 0 0 1.979 0 0 0 16.046 Teniposide 50.02 20.192 34.283 46.08 37.265 25.151 43.228 58.647 Irinotecan HCl 8.431 0 0 10.533 0 0 0 9.886 Pipobroman 8.984 0 0 5.445 0 0 2.581 13.752 Megestrol acetate 0 0 0 1.778 0 10.642 0 0 Carboplatin 0 0 0 9.058 0 0 0 0 Oxaliplatin 0.372 0 0 0 0 3.259 0 0 Methotrexate 0 0 0 0 0 0 0 3.411 Acrichine 0 0 0 0.074 0 0.987 0 0 Topotecan HCl 59.118 46.289 40.884 84.778 53.2 45.817 66.158 76.237 Docetaxel 63.186 64.476 73.576 83.241 68.122 90.346 73.741 79.059 Rapamycin 18.716 23.821 6.467 34.501 13.47 16.499 28.812 36.839 Dactinomycin 79.26 75.055 65.834 86.327 78.808 67.312 86.217 83.436 Vinorelbine tartrate 61.128 62.82 65.531 80.519 60.403 80.204 76.162 74.994 Azacitidine 0 0 0 0.502 0 0 0 23.842 Decitabine 0 0 0 8.703 0 3.604 0 27.643 Amifostine 0 0 0 0 0 0 7.018 19.657 Nelarabine 5.355 6.943 0 7.743 3.691 0 0 0 Vorinostat 36.206 0 0 28.22 26.007 0 1.9 0 Exemestane 1.194 0 0 0 0 0 0 0
31 Supplementary information - Meyer, Reimand et al.
Drug 482_2 482_16 482_3 482_4 482_7 482_9 482_11 482_13 Anastrozole 0 0 0 0 18.026 0 0 0 Letrozole 0 0 0 0 0 0 0 0 Zolendronic acid 0 0 0 5.41 0 0 0 0 Lenalidomide 0.526 0 0 0 0 0 0 0 Clofarabine 55.083 28.235 53.365 75.863 53.516 69.094 64.805 78.247 Fludarabine 19.122 0 0 0 0 0 0 0 Bortezomib 92.408 89.21 90.267 93.853 89.19 94.367 91.215 89.049 Capecitabine 0 11.707 0 4.535 3.845 0 0 8.472 Celecoxib 0 0 0 3.79 16.777 0 0 0 Sunitinib 0 0 0 0 0 0 0 0 Mitoxantrone 61.107 53.863 55.037 77.553 64.718 76.834 67.009 79.762 Gefitinib 0 0 0 0 0 0 0 11.135 Erlotinib HCl 0 0 0 0.832 0 0 0 0.903 Dasatinib 69.038 64.817 22.035 78.058 68.756 20.283 79.778 74.95 Imatinib 9.232 0 0 1.243 0 0 0 0 Sorafenib 0.229 6.24 0 9.211 12.191 0 0 5.223 Raloxifene HCl 0 0 0 0 0 0 0 0 Fulvestrant 0 0 0 0 0 0 0 0 Plicamycin 73.954 77.286 61.433 84.762 77.262 67.449 83.777 82.058 Vinblastine sulfate 70.333 67.5 62.44 84.782 70.975 77.98 80.441 78.989 Imiquimod 0 0 0 0 4.671 0 0 0 Triethylenemelamine 35.161 0 9.753 32.944 17.799 21.82 4.641 47.884 Melphalan 4.812 0 0 7.772 16.275 0 0 8.011 Vincristine sulfate 74.632 65.616 65.688 84.235 67.937 78.359 82.478 78.036 Bleomycin 37.027 0 14.79 43.134 16.444 31.124 67.926 61.953 Paclitaxel 57.175 33.668 73.521 79.706 40.004 84.856 67.816 71.532 Valrubicin 46.514 23.006 62.614 40.103 33.779 78.955 48.477 57.063 Everolimus 0 0 17.736 28.642 0 23.106 18.48 29.659 Nilotinib 0 0 0 0 0 0 0 0 Ixabepilone 52.007 27.599 69.575 75.157 55.523 79.169 62.261 72.812 Doxorubicin HCl 81.253 56.064 90.303 76.97 72.119 93.088 68.973 83.914 Lapatinib 0 0 0 0 0 0 0 0 Bendamustine HCl 0 0 0 0 0 0 0 0 Pazopanib HCl 0 0 0 0 0 0 0 0 Romidepsin 86.977 88.938 89.989 93.369 88.747 93.809 90.337 88.335 Pralatrexate 0.736 0 0 0 0 0 0 0 Vandetanib 35.668 0 0 0 11.503 0 0 34.893 Vemurafenib 15.538 1.803 0 0 0 0 0 0 Cabazitaxel 65.658 68.311 74.024 82.665 62.019 80.289 74.302 77.513 Crizotinib 0 2.929 0 0.133 2.142 6.117 0 16.596
32 Supplementary information - Meyer, Reimand et al.
Table S2B. NCI drug screen for GBM-489. Drug 489_1 489_5 489_6 489_7 489_8 489_10 489_12 489_13 Allopurinol 0 0 10.973 0 0 1.087 0 4.602 Fluorouracil 0 0 0 0 7.149 0 0.365 0 Hydroxyurea 0 0 0 0 11.652 0 6.708 2.169 Thioguanine 2.221 8.673 6.144 0 6.587 7.022 14.441 0 Mercaptopurine 0 0 1.988 0 9.589 0.762 4.317 8.134 Nitrogen mustard 0 0 0 0 11.251 8.999 1.287 5.872 Thiotepa 0 0 5.107 0 13.667 17.22 0 0 Aminolevulinic acid 0 0 3.671 0 11.452 0 0 0 Dacarbazine 5.504 0 0 0 1.623 0 0 0 Arsenic trioxide 0 0 0 0 0 0 0 0 Temozolomide 0 0 0 0 0 0 0 0 Busulfan 0 0 0 0 4.165 0 11.331 0 Altretamine 0 0 0 0 4.794 0 0 0 Floxuridine 10.639 0 0 0 14.065 0 0 0 Methoxsalen 0 0 0 0 6.457 8.129 3.099 0.379 Lomustine, CCNU 11.235 0.371 10.125 0 2.456 0 0.192 0.689 Carmustine 1.685 0.677 0 0 0 2.859 0 3.945 Cyclophosphamide 0 0 0 0 2.104 0 0 0 Uracil mustard 0.091 0 0 0 7.103 0 0 4.624 Cytarabine HCl 11.299 36.098 11.31 20.511 22.601 11.369 23.285 27.537 Thalidomide 0 0 0 0 1.453 0 0 0 Procarbazine 0 0 0 0 2.268 0 0 0 Streptozocin 0 0 2.186 0 7.58 2.582 0 0 Cladribine 47.452 47.424 45.371 32.565 33.185 30.19 21.177 34.399 Ifosfamide 17.421 0 0 0 0 0 0 0 Cisplatin 5.686 0 0 0 0 0 0 0 Tretinoin 14.912 0 5.858 0 2.049 0 0 13.283 Dexrazoxone 4.861 0 0 0 1.237 0 0 0 Pentostatin 0 0 0 0 0 -9.681 0 0 Gemcitabine HCl 57.174 47.828 27.369 34.88 47.827 39.117 38.867 44.597 Chlorambucil 0 0 0 0 11.547 3.691 0 0 Mitomycin C 44.062 48.306 50.136 43.663 43.913 47.502 30.239 48.338 Mitotane 8.192 0 0 0 0 0 0 1.226 Daunorubicin HCl 91.437 89.899 80.259 76.532 82.886 87.146 86.841 88.352 Etoposide 13.039 11.087 14.036 0 17.654 9.632 4.896 7.205 Tamoxifen citrate 0 0 0 0 0 0 0 0 Estramustine disodium 0.347 0 0 0 0 0 0 0 phosphate Pemetrexed 0 0 0 0 0 0 0 0 Teniposide 43.904 37.734 22.333 21.975 34.9 46.126 27.817 33.9 Irinotecan HCl 1.108 6.464 26.927 0 6.981 0 2.614 0.977 Pipobroman 2.903 0 5.184 0 1.436 0 18.292 0 Megestrol acetate 0 0 0 0 0 0 0 0 Carboplatin 0 0 0 0 0 0 0 0 Oxaliplatin 3.213 0 0 0 0 0 0 0 Methotrexate 2.377 0 0 0 1 0 0 0 Acrichine 0 0 0 0 0 0 0 0 Topotecan HCl 55.005 52.54 47.007 42.184 56.456 54.914 50.849 49.139 Docetaxel 71.038 71.668 53.391 63.704 66.2 55.238 50.667 75.624 Rapamycin 35.336 32.579 6.448 11.619 32.584 25.022 23.537 25.271 Dactinomycin 92.334 91.36 82.957 75.803 83.232 88.048 90.336 88.808 Vinorelbine tartrate 60.006 58.521 51.01 49.098 58.256 51.641 49.837 71.458 Azacitidine 7.777 0.679 0 0 0 0 0 0 Decitabine 9.655 0 0 0 2.289 0 0 0 Amifostine 3.936 0 0 0 0 0 0 0 Nelarabine 3.602 0 0 0 10.111 5.403 0 0 Vorinostat 17.187 4.559 12.739 4.629 7.733 3.25 0 2.519 Exemestane 0 0 0 0 0 0 0.197 0 Anastrozole 0 0 0 0 0 0 0 0
33 Supplementary information - Meyer, Reimand et al.
Drug 489_1 489_5 489_6 489_7 489_8 489_10 489_12 489_13 Letrozole 0 0 0 0 0 0 0 0 Zolendronic acid 0 0 0 0 0 0 0 0 Lenalidomide 3.133 0 0 0 0 0 0 0 Clofarabine 56.279 41.827 30.045 30.685 37.013 35.312 24.867 42.056 Fludarabine 0 0 0 0 1.483 0 0 0 Bortezomib 94.237 92.915 89.632 89.964 90.972 92.776 94.808 93.405 Capecitabine 12.73 0.54 0 0 0 0 0.832 0 Celecoxib 0 0 0 0 0 0 0 0 Sunitinib 0 0 0 0 0 0 0 0 Mitoxantrone 56.175 71.02 21.983 49.84 51.489 54.242 59.429 64.271 Gefitinib 8.835 0.647 0 0 0 0 0 0 Erlotinib HCl 0 0 0 0 4.162 0.704 0 0 Dasatinib 28.316 10.087 2.821 23.363 28.265 24.337 7.468 9.173 Imatinib 0 0 0.619 0 0 0 0 0 Sorafenib 0 0 0 0.928 0 0 0 0 Raloxifene HCl 0 0 0 0 0 0 0 0 Fulvestrant 0 0 0 0 0 0 0 0 Plicamycin 86.845 86.316 59.252 77.561 79.788 82.085 88.058 86.174 Vinblastine sulfate 71.889 66.052 71.441 57.953 71.245 56.959 61.382 62.643 Imiquimod 0 0 0 0 0.444 0 0 0 Triethylenemelamine 0 20.827 14.898 0 15.315 23.626 0 8.855 Melphalan 3.612 0 7.664 0 0.261 0 0 0 Vincristine sulfate 73.154 58.898 79.115 59.828 68.403 39.048 53.465 58.644 Bleomycin 0 0 0 0 1.774 0 0 0 Paclitaxel 62.509 67.195 42.996 51.859 55.167 52.584 33.458 67.705 Valrubicin 35.061 48.354 28.991 27.023 33.967 44.014 27.596 31.701 Everolimus 15.222 12.703 6.103 0 26.003 14.379 0 0 Nilotinib 0 0 0 0 0 0 0 0 Ixabepilone 49.863 54.005 34.712 46.762 54.022 58.952 32.837 55.3 Doxorubicin HCl 72.338 79.418 78.027 65.844 76.716 74.093 69.566 79.921 Lapatinib 0 0 0 0 0 0 0 0 Bendamustine HCl 0 0 0 0 0 0 0 0 Pazopanib HCl 0 0 0 0 0 0 0 0 Romidepsin 90.563 87.891 84.37 84.822 87.666 87.954 91.664 89.845 Pralatrexate 0 0 0 0 0 0 0 0 Vandetanib 0 0 0 0 0 0 0 0 Vemurafenib 0 0 0 0 0 0 0 0 Cabazitaxel 64.22 66.222 49.585 62.828 64.324 55.456 47.662 72.331 Crizotinib 13.611 0.975 0 0 2.82 0 0 0
34 Supplementary information - Meyer, Reimand et al.
Table S2C. NCI drug screen for GBM-498. Drug 498_6 498_7 498_1 498_2 498_3 498_4 Allopurinol 1.026 0 0.705 0 0.549 0 Fluorouracil 3.311 4.347 0 3.245 0 0 Hydroxyurea 2.362 0.478 0 0 0 0 Thioguanine 7.712 6.859 3.345 12.712 1.781 0 Mercaptopurine 4.023 4.742 4.842 6.204 0.39 0 Nitrogen mustard 3.846 4.365 1.329 0 0 0 Thiotepa 1.881 5.237 0 0 0 0 Aminolevulinic acid 1.786 5.646 2.641 0 1.703 0 Dacarbazine 0 0 0 0 99.912 0 Arsenic trioxide 0.73 0 0 0 0 0 Temozolomide 1.529 2.331 0 0 0 0 Busulfan 1.087 0 0 5.918 0 0 Altretamine 0.841 0.918 0 0 0 0 Floxuridine 17.744 13.54 1.32 10.538 7.857 0 Methoxsalen 2.011 3.829 0 6.525 0 0 Lomustine, CCNU 1.061 2.573 0 3.023 0 0 Carmustine 0 0 0 0 0 0 Cyclophosphamide 0 1.957 0 0 0 0 Uracil mustard 0.022 0.004 0 0 0 0 Cytarabine HCl 2.691 13.88 0 0.548 2.85 3.018 Thalidomide 0.025 2.07 0 0 0 0 Procarbazine 1.493 2.63 0 1.139 0 0 Streptozocin 1.473 1.083 0 0 0 0 Cladribine 9.204 69.279 58.434 0 19.543 34.937 Ifosfamide 0.309 0 0 0 0 0 Cisplatin 0 0 0 0 0 0 Tretinoin 0 0 0 0 0 0 Dexrazoxone 0 2.2 0 0 0 0 Pentostatin 0.863 1.856 0 1.689 0 0 Gemcitabine HCl 45.506 69.131 34.126 67.112 44.935 60.535 Chlorambucil 1.626 4.32 0 6.322 0 0 Mitomycin C 28.595 74.246 33.21 73.148 79.904 71.863 Mitotane 0 0 0 0 0 1.614 Daunorubicin HCl 88.62 92.979 93.389 92.119 90.262 93.875 Etoposide 2.715 15.165 0.624 23.002 6.137 18.305 Tamoxifen citrate 0.029 2.418 0 0 0 0 Estramustine disodium 0 0 0 0 0 0 phosphate Pemetrexed 0 0 0 1.655 0 0.482 Teniposide 17.291 42.225 5.708 45.284 32.405 49.911 Irinotecan HCl 0 0.877 0 3.021 0.056 0 Pipobroman 0 0 0 0.994 0 0 Megestrol acetate 0 0 0 0 0 0 Carboplatin 0 0 0 0 0 0 Oxaliplatin 0 6.049 0 0 0 8.477 Methotrexate 18.049 3.463 8.722 7.42 0 2.815 Acrichine 0 1.615 0 0 0 21.875 Topotecan HCl 31.04 62.439 43.994 50.674 48.849 60.075 Docetaxel 79.5 88.715 88.348 88.713 85.974 75.934 Rapamycin 6.59 28.537 13.044 48.807 19.226 8.817 Dactinomycin 88.934 93.648 94.561 92.342 85.764 95.103 Vinorelbine tartrate 74.962 83.893 79.539 87.551 85.518 78.902 Azacitidine 0 0 0 0 0 0 Decitabine 0 0.763 0 0 0 3.605 Amifostine 0 0 0 0 0 0.848 Nelarabine 0 0 0 0 0 4.226 Vorinostat 0 5.147 2.367 4.853 0 9.755 Exemestane 0 0 0 0 99.95 0 Anastrozole 0 0 0 0 0 0
35 Supplementary information - Meyer, Reimand et al.
Drug 498_6 498_7 498_1 498_2 498_3 498_4 Letrozole 0 0 0 0 0 20.756 Zolendronic acid 0 0 0 0 0 0 Lenalidomide 0 0 0 2.82 0 0 Clofarabine 12.703 32.103 7.418 2.763 48.029 39.022 Fludarabine 0 0 0 0 0 0 Bortezomib 94.582 95.875 95.857 96.008 95.989 89.592 Capecitabine 0 0 0 6.598 0 0 Celecoxib 0 0 0 0 0 0 Sunitinib 0 0 0 0 0 8.557 Mitoxantrone 61.808 86.052 42.583 72.542 74.691 74.824 Gefitinib 0 0 0 0 0 0 Erlotinib HCl 0 0 0 0 0 0 Dasatinib 2.672 48.795 0 76.388 35.125 55.258 Imatinib 0 0 0 0.79 0 0 Sorafenib 0 0 0 3.718 0 8.392 Raloxifene HCl 0 0 0 0 0 0 Fulvestrant 0 0 0 0 0 0 Plicamycin 84.179 92.949 84.755 90.137 89.318 94.647 Vinblastine sulfate 76.086 88.19 75.517 91.138 86.095 83.799 Imiquimod 0 0.764 0 1.607 0 12.249 Triethylenemelamine 0 3.883 0 4.122 0 2.04 Melphalan 0 0 0 9.425 0 6.932 Vincristine sulfate 84.905 85.104 81.812 90.324 86.208 85.683 Bleomycin 8.638 3.878 3.568 0 2.653 0 Paclitaxel 89.627 85.238 88.953 88.209 87.975 82.013 Valrubicin 74.636 67.144 74.507 48.21 32.739 61.978 Everolimus 26.521 20.037 18.431 35.992 12.573 10.846 Nilotinib 0 0 0.68 0 0 0 Ixabepilone 84.659 85.331 85.611 86.435 85.37 80.15 Doxorubicin HCl 80.597 90.624 95.963 78.813 72.908 80.33 Lapatinib 1.173 0 2.448 0 0 0 Bendamustine HCl 1.874 0 0 0 0 0 Pazopanib HCl 6.822 0 0 0 0 0 Romidepsin 95.394 91.087 86.554 89.912 89.748 90.655 Pralatrexate 21.638 0 12.368 18.782 0 0 Vandetanib 4.197 6.005 1.172 0 0 5.033 Vemurafenib 0 0 1.716 0 0 0 Cabazitaxel 86.693 87.7 88.222 89.1 86.042 82.421 Crizotinib 3.651 0 1.622 0 2.698 0
36 Supplementary information - Meyer, Reimand et al. Fig. S9: clonal dose response of bortezomib, daunorubicin, and romidepsin. Dose response assays on clones from tumor GBM-482 and control cell lines (human fetal neuronal cell line hf5281, BJ fibroblast cell line). All three drug assays show a broad cytotoxic drug response in the two control cell lines, resulting in extensive cell death with IC50 values of each drug in the low nanomolar range. This unfortunately indicates that the three drugs are unsuitable candidates of alternative drugs in GBM treatment due to their highly cytotoxic effect on non-cancer cells.
37 Supplementary information - Meyer, Reimand et al.
Relative copy number (log2 ratio) Normalized copy number (Z score)
primary primary unsorted unsorted clone clone 489 489
498 498
Fig. S10: PCA clustering of clonal copy number alterations. Unsupervised clustering of probe-level copy number calls with principal component analysis (PCA) confirms heterogeneity of clonal populations. Colors denote different patient tumors (asterisks - primary samples; diamonds - unsorted populations).
38 Supplementary information - Meyer, Reimand et al.
Fig. S11: circos plots of clonal copy number alterations. a. Circos plot of segmented and normalized copy number alterations (Z-scores) in tumor GBM-472. Inner circle represents balanced copy number (z=0) in chromosomes ordered clockwise from top. Segments expanding towards periphery denote copy number gains, and segments expanding towards the centre denote copy number losses. Line color represents primary tumor samples (black), unsorted populations (grey) and clones (colors). Dashed lines represent landmarks on the Z-score axis.
39 Supplementary information - Meyer, Reimand et al.
Fig. S11B. Circos plot of segmented and normalized copy number alterations (Z-scores) in tumor GBM-489.
40 Supplementary information - Meyer, Reimand et al.
Fig. 11C. Circos plot of segmented and normalized copy number alterations (Z-scores) in tumor GBM-498.
41 Supplementary information - Meyer, Reimand et al.
atio r
v Fig. S12: Copy number of chromosome 3 in tumor GBM-482. The clone 482_3 (red) shows a sub-threshold chromosomal gain, while other samples have no CN alterations (black).
42 Supplementary information - Meyer, Reimand et al.
Fig. S13: FISH validation of clonal chromosomal alterations. S13A. Fluorescence in situ Hybridization (FISH) quantification of primary tissue samples of GBM-482 shows that the major clone (~70%) contains two copies of chr3, three copies of chr7 and two copies of chr17. A minor clone (~30%) carries three copies of chr3, three copies of chr7 and two copies of chr17. Cells with only one signal per cell occur due to truncation of the nucleus. S13B. FISH quantification of four clonal cultures of GBM-482. Three clones are homogeneous in all chromosomes, while 482_1 shows a minority of cells with chr7 amplification whose numerical changes are typical for GBM. S13C. FISH confirms chr3 gain in clone 482_3.
43 Supplementary information - Meyer, Reimand et al. Table S3: genes with clonal copy number alterations. All genes with clonal copy number alterations, ordered by total count of events (B - balanced; L - loss; D - deletion; G - gain; A - amplification). No Gene CN tot 472 482 489 498 No Gene CN tot 472 482 489 498 1 C9orf53 B;D 42 10 12 11 9 232 PRAMEF14 B;L 2 0 0 2 0 2 CDKN2A B;D 42 10 12 11 9 233 PRAMEF15 B;L 2 0 0 2 0 3 CDKN2B B;D 42 10 12 11 9 234 PRAMEF16 B;L 2 0 0 2 0 4 MTAP B;D 42 10 12 11 9 235 PRAMEF17 B;L 2 0 0 2 0 5 IFNA1 B;D 23 0 12 11 0 236 PRAMEF18 B;L 2 0 0 2 0 6 IFNA14 B;D 23 0 12 11 0 237 PRAMEF19 B;L 2 0 0 2 0 7 IFNA17 B;D 23 0 12 11 0 238 PRAMEF2 B;L 2 0 0 2 0 8 IFNA5 B;D 23 0 12 11 0 239 PRAMEF20 B;L 2 0 0 2 0 9 IFNA8 B;D 23 0 12 11 0 240 PRAMEF21 B;L 2 0 0 2 0 10 IFNE B;D 23 0 12 11 0 241 PRAMEF22 B;L 2 0 0 2 0 11 KLHL9 B;D 23 0 12 11 0 242 PRAMEF3 B;L 2 0 0 2 0 12 DMRTA1 B;D 20 0 0 11 9 243 PRAMEF4 B;L 2 0 0 2 0 13 ELAVL2 B;D 20 0 0 11 9 244 PRAMEF5 B;L 2 0 0 2 0 14 IZUMO3 B;D 20 0 0 11 9 245 PRAMEF6 B;L 2 0 0 2 0 15 TUSC1 B;D 20 0 0 11 9 246 PRAMEF7 B;L 2 0 0 2 0 16 EGFR A;B;G 18 6 1 9 2 247 PRAMEF8 B;L 2 0 0 2 0 17 SEC61G A;B;G 16 6 1 7 2 248 PRAMEF9 B;L 2 0 0 2 0 18 LANCL2 A;B;G 14 6 1 7 0 249 PRDM2 B;L 2 0 0 2 0 19 VOPP1 A;B;G 13 6 1 6 0 250 PTCHD2 B;L 2 0 0 2 0 20 KIAA1217 B;D 12 5 0 0 7 251 RBP7 B;L 2 0 0 2 0 21 NFIA B;D 12 0 12 0 0 252 RERE B;L 2 0 0 2 0 22 C9orf11 B;D 11 0 0 11 0 253 RNF207 B;L 2 0 0 2 0 23 CAAP1 B;D 11 0 0 11 0 254 RPL22 B;L 2 0 0 2 0 24 IFNA10 B;D 11 0 0 11 0 255 RSC1A1 B;L 2 0 0 2 0 25 IFNA13 B;D 11 0 0 11 0 256 RSG1 B;L 2 0 0 2 0 26 IFNA16 B;D 11 0 0 11 0 257 SLC25A33 B;L 2 0 0 2 0 27 IFNA2 B;D 11 0 0 11 0 258 SLC25A34 B;L 2 0 0 2 0 28 IFNA21 B;D 11 0 0 11 0 259 SLC2A5 B;L 2 0 0 2 0 29 IFNA4 B;D 11 0 0 11 0 260 SLC2A7 B;L 2 0 0 2 0 30 IFNA6 B;D 11 0 0 11 0 261 SLC45A1 B;L 2 0 0 2 0 31 IFNA7 B;D 11 0 0 11 0 262 SPATA21 B;L 2 0 0 2 0 32 IFT74 B;D 11 0 0 11 0 263 SPEN B;L 2 0 0 2 0 33 LRRC19 B;D 11 0 0 11 0 264 SPSB1 B;L 2 0 0 2 0 34 MOB3B B;D 11 0 0 11 0 265 SRM B;L 2 0 0 2 0 35 PLAA B;D 11 0 0 11 0 266 SZRD1 B;L 2 0 0 2 0 36 TEK B;D 11 0 0 11 0 267 TARDBP B;L 2 0 0 2 0 37 INADL B;D 8 0 8 0 0 268 TAS1R1 B;L 2 0 0 2 0 38 VSTM2A A;B;G 7 0 1 6 0 269 THAP3 B;L 2 0 0 2 0 39 ANKRD30A B;D;L 5 0 0 5 0 270 TMEM201 B;L 2 0 0 2 0 40 ARHGAP12 B;D;L 5 0 0 5 0 271 TMEM51 B;L 2 0 0 2 0 41 ARMC4 B;D;L 5 0 0 5 0 272 TMEM82 B;L 2 0 0 2 0 42 BAMBI B;D;L 5 0 0 5 0 273 TNFRSF1B B;L 2 0 0 2 0 43 C10orf126 B;D;L 5 0 0 5 0 274 TNFRSF25 B;L 2 0 0 2 0 44 C10orf68 B;D;L 5 0 0 5 0 275 TNFRSF8 B;L 2 0 0 2 0
44 Supplementary information - Meyer, Reimand et al.
45 CCDC7 B;D;L 5 0 0 5 0 276 TNFRSF9 B;L 2 0 0 2 0 46 CCNY B;D;L 5 0 0 5 0 277 UBE4B B;L 2 0 0 2 0 47 CREM B;D;L 5 0 0 5 0 278 UBIAD1 B;L 2 0 0 2 0 48 CUL2 B;D;L 5 0 0 5 0 279 UTS2 B;L 2 0 0 2 0 49 EPC1 B;D;L 5 0 0 5 0 280 VAMP3 B;L 2 0 0 2 0 50 FZD8 B;D;L 5 0 0 5 0 281 VPS13D B;L 2 0 0 2 0 51 GJD4 B;D;L 5 0 0 5 0 282 ZBTB17 B;L 2 0 0 2 0 52 ITGB1 B;D;L 5 0 0 5 0 283 ZBTB48 B;L 2 0 0 2 0 53 KIAA1462 B;D;L 5 0 0 5 0 284 ACTA2 B;D 1 0 0 0 1 54 KIF5B B;D;L 5 0 0 5 0 285 ADCY8 A;B 1 0 0 1 0 55 LRP1B B;D;L 5 0 0 5 0 286 AKAP9 A;B 1 0 0 1 0 56 LYZL1 B;D;L 5 0 0 5 0 287 ANKIB1 A;B 1 0 0 1 0 57 LYZL2 B;D;L 5 0 0 5 0 288 ANKRD22 B;D 1 0 0 0 1 58 MAP3K8 B;D;L 5 0 0 5 0 289 ANXA13 B;G 1 0 0 1 0 59 MKX B;D;L 5 0 0 5 0 290 ARL11 B;L 1 0 0 1 0 60 MPP7 B;D;L 5 0 0 5 0 291 ATAD1 B;D 1 0 0 0 1 61 MTPAP B;D;L 5 0 0 5 0 292 ATAD2 B;G 1 0 0 1 0 62 MTRNR2L7 B;D;L 5 0 0 5 0 293 B3GAT2 B;L 1 0 0 1 0 63 MYC A;B;G 5 0 0 5 0 294 C10orf11 B;D 1 0 1 0 0 64 NAMPTL B;D;L 5 0 0 5 0 295 C2orf91 B;G 1 0 1 0 0 65 NRP1 B;D;L 5 0 0 5 0 296 C6orf57 B;L 1 0 0 1 0 66 PARD3 B;D;L 5 0 0 5 0 297 C8orf76 B;G 1 0 0 1 0 67 POU5F1B A;B;G 5 0 0 5 0 298 CAB39L B;L 1 0 0 1 0 68 PTCHD3 B;D;L 5 0 0 5 0 299 CCDC122 B;L 1 0 0 1 0 69 RAB18 B;D;L 5 0 0 5 0 300 CCDC132 A;B 1 0 0 1 0 70 SEPT7L B;D;L 5 0 0 5 0 301 CD109 B;L 1 0 0 1 0 71 SVIL B;D;L 5 0 0 5 0 302 CDADC1 B;L 1 0 0 1 0 72 TMEM75 A;B;G 5 0 0 5 0 303 CDK6 A;B 1 0 0 1 0 73 WAC B;D;L 5 0 0 5 0 304 CH25H B;D 1 0 0 0 1 74 ZEB1 B;D;L 5 0 0 5 0 305 CNBD1 B;D 1 0 0 0 1 75 ZNF248 B;D;L 5 0 0 5 0 306 COL12A1 B;L 1 0 0 1 0 76 ZNF25 B;D;L 5 0 0 5 0 307 COL19A1 B;L 1 0 0 1 0 77 ZNF33A B;D;L 5 0 0 5 0 308 COL9A1 B;L 1 0 0 1 0 78 ZNF37A B;D;L 5 0 0 5 0 309 CRISPLD1 A;B 1 0 0 1 0 79 ZNF438 B;D;L 5 0 0 5 0 310 CSMD1 B;L 1 0 0 1 0 80 ANKRD65 B;L 3 0 0 3 0 311 CYP51A1 A;B 1 0 0 1 0 81 ATAD3A B;L 3 0 0 3 0 312 CYSLTR2 B;L 1 0 0 1 0 82 ATAD3B B;L 3 0 0 3 0 313 DCAF4L2 B;D 1 0 0 0 1 83 ATAD3C B;L 3 0 0 3 0 314 DDX43 B;L 1 0 0 1 0 84 BORA B;L 3 0 0 3 0 315 DERL1 B;G 1 0 0 1 0 85 C1orf233 B;L 3 0 0 3 0 316 DLEU1 B;L 1 0 0 1 0 86 CCNL2 B;L 3 0 0 3 0 317 DLEU7 B;L 1 0 0 1 0 87 CDK11A B;L 3 0 0 3 0 318 DNAJC15 B;L 1 0 0 1 0 88 CDK11B B;L 3 0 0 3 0 319 DPPA5 B;L 1 0 0 1 0 89 CLN5 B;L 3 0 0 3 0 320 EBPL B;L 1 0 0 1 0 90 COMMD6 B;L 3 0 0 3 0 321 EEF1A1 B;L 1 0 0 1 0 91 DACH1 B;L 3 0 0 3 0 322 EFR3A A;B 1 0 0 1 0 92 DIS3 B;L 3 0 0 3 0 323 ENOX1 B;L 1 0 0 1 0 93 EDNRB B;L 3 0 0 3 0 324 EPHA3 B;L 1 0 0 1 0
45 Supplementary information - Meyer, Reimand et al.
94 ELTD1 B;L 3 0 0 3 0 325 EPSTI1 B;L 1 0 0 1 0 95 FBXL3 B;L 3 0 0 3 0 326 ERVW-1 A;B 1 0 0 1 0 96 GNB1 B;L 3 0 0 3 0 327 FAM133B A;B 1 0 0 1 0 97 IFI44 B;L 3 0 0 3 0 328 FAM135A B;L 1 0 0 1 0 98 IFI44L B;L 3 0 0 3 0 329 FAM216B B;L 1 0 0 1 0 99 IRG1 B;L 3 0 0 3 0 330 FAM83A B;G 1 0 0 1 0 100 KCTD12 B;L 3 0 0 3 0 331 FAM84B B;G 1 0 0 1 0 101 KLF12 B;L 3 0 0 3 0 332 FAM91A1 B;G 1 0 0 1 0 102 KLF5 B;L 3 0 0 3 0 333 FAS B;D 1 0 0 0 1 103 KLHL1 B;L 3 0 0 3 0 334 FAXC B;L 1 0 0 1 0 104 LMO7 B;L 3 0 0 3 0 335 FBXL4 B;L 1 0 0 1 0 105 LPHN2 B;L 3 0 0 3 0 336 FBXO32 B;G 1 0 0 1 0 106 MIB2 B;L 3 0 0 3 0 337 FER1L6 B;G 1 0 0 1 0 107 MMP23B B;L 3 0 0 3 0 338 FNDC3A B;L 1 0 0 1 0 108 MRPL20 B;L 3 0 0 3 0 339 GATAD1 A;B 1 0 0 1 0 109 MYCBP2 B;L 3 0 0 3 0 340 GPR63 B;L 1 0 0 1 0 110 MZT1 B;L 3 0 0 3 0 341 HBB B;L 1 0 0 1 0 111 NADK B;L 3 0 0 3 0 342 HBD B;L 1 0 0 1 0 112 NDFIP2 B;L 3 0 0 3 0 343 HBE1 B;L 1 0 0 1 0 113 PCDH9 B;L 3 0 0 3 0 344 HBG1 B;L 1 0 0 1 0 114 PIBF1 B;L 3 0 0 3 0 345 HBG2 B;L 1 0 0 1 0 115 POU4F1 B;L 3 0 0 3 0 346 HEPACAM2 A;B 1 0 0 1 0 116 PTGFR B;L 3 0 0 3 0 347 HHLA1 A;B 1 0 0 1 0 117 RBM26 B;L 3 0 0 3 0 348 IFIT1B B;D 1 0 0 0 1 118 RNF219 B;L 3 0 0 3 0 349 IFIT2 B;D 1 0 0 0 1 119 SCEL B;L 3 0 0 3 0 350 IFIT3 B;D 1 0 0 0 1 120 SLAIN1 B;L 3 0 0 3 0 351 JAK2 B;L 1 0 0 1 0 121 SLC35E2 B;L 3 0 0 3 0 352 KCNMA1 B;D 1 0 1 0 0 122 SLC35E2B B;L 3 0 0 3 0 353 KCNQ3 A;B 1 0 0 1 0 123 SLITRK1 B;L 3 0 0 3 0 354 KCNQ5 B;L 1 0 0 1 0 124 SPRY2 B;L 3 0 0 3 0 355 KCNRG B;L 1 0 0 1 0 125 SSU72 B;L 3 0 0 3 0 356 KHDC1 B;L 1 0 0 1 0 126 TBC1D4 B;L 3 0 0 3 0 357 KHDC1L B;L 1 0 0 1 0 127 TMEM240 B;L 3 0 0 3 0 358 KHDC3L B;L 1 0 0 1 0 128 TMEM88B B;L 3 0 0 3 0 359 KIAA0196 B;G 1 0 0 1 0 129 UCHL3 B;L 3 0 0 3 0 360 KLHL32 B;L 1 0 0 1 0 130 VWA1 B;L 3 0 0 3 0 361 KLHL38 B;G 1 0 0 1 0 131 AADACL3 B;L 2 0 0 2 0 362 KLLN B;D 1 0 0 0 1 132 AADACL4 B;L 2 0 0 2 0 363 KPNA3 B;L 1 0 0 1 0 133 ACOT7 B;L 2 0 0 2 0 364 KRIT1 A;B 1 0 0 1 0 134 AGMAT B;L 2 0 0 2 0 365 LACC1 B;L 1 0 0 1 0 135 AGTRAP B;L 2 0 0 2 0 366 LCE3B B;D 1 0 0 1 0 136 AJAP1 B;L 2 0 0 2 0 367 LIPA B;D 1 0 0 0 1 137 ANGPTL7 B;L 2 0 0 2 0 368 LIPF B;D 1 0 0 0 1 138 ANO7L1 B;L 2 0 0 2 0 369 LIPJ B;D 1 0 0 0 1 139 APITD1 B;L 2 0 0 2 0 370 LIPK B;D 1 0 0 0 1 140 APITD1-CORT B;L 2 0 0 2 0 371 LIPM B;D 1 0 0 0 1 141 ARHGEF19 B;L 2 0 0 2 0 372 LIPN B;D 1 0 0 0 1 142 ASAP1 A;B;G 2 0 0 2 0 373 LRRC6 A;B 1 0 0 1 0
46 Supplementary information - Meyer, Reimand et al.
143 BAI3 B;L 2 0 0 2 0 374 LRRD1 A;B 1 0 0 1 0 144 C1orf127 B;L 2 0 0 2 0 375 MB21D1 B;L 1 0 0 1 0 145 C1orf134 B;L 2 0 0 2 0 376 MLNR B;L 1 0 0 1 0 146 C1orf158 B;L 2 0 0 2 0 377 MMP26 B;L 1 0 0 1 0 147 C1orf167 B;L 2 0 0 2 0 378 MMS22L B;L 1 0 0 1 0 148 C1orf195 B;L 2 0 0 2 0 379 MTERF A;B 1 0 0 1 0 149 C1orf196 B;L 2 0 0 2 0 380 MTO1 B;L 1 0 0 1 0 150 C1orf200 B;L 2 0 0 2 0 381 MTSS1 B;G 1 0 0 1 0 151 C1orf64 B;L 2 0 0 2 0 382 NDRG1 A;B 1 0 0 1 0 152 CA6 B;L 2 0 0 2 0 383 NDUFAF4 B;L 1 0 0 1 0 153 CAMTA1 B;L 2 0 0 2 0 384 NDUFB9 B;G 1 0 0 1 0 154 CASP9 B;L 2 0 0 2 0 385 NSMCE2 B;G 1 0 0 1 0 155 CASZ1 B;L 2 0 0 2 0 386 OC90 A;B 1 0 0 1 0 156 CCDC144A B;D 2 0 0 2 0 387 OGFRL1 B;L 1 0 0 1 0 157 CELA2A B;L 2 0 0 2 0 388 OOEP B;L 1 0 0 1 0 158 CELA2B B;L 2 0 0 2 0 389 OR51A2 B;L 1 0 0 1 0 159 CHD5 B;L 2 0 0 2 0 390 OR51A4 B;L 1 0 0 1 0 160 CLCN6 B;L 2 0 0 2 0 391 OR51A7 B;L 1 0 0 1 0 161 CLCNKA B;L 2 0 0 2 0 392 OR51B2 B;L 1 0 0 1 0 162 CLCNKB B;L 2 0 0 2 0 393 OR51B4 B;L 1 0 0 1 0 163 CLSTN1 B;L 2 0 0 2 0 394 OR51B5 B;L 1 0 0 1 0 164 CORT B;L 2 0 0 2 0 395 OR51B6 B;L 1 0 0 1 0 165 CTNNBIP1 B;L 2 0 0 2 0 396 OR51G1 B;L 1 0 0 1 0 166 CTRC B;L 2 0 0 2 0 397 OR51G2 B;L 1 0 0 1 0 167 DDI2 B;L 2 0 0 2 0 398 OR51H1P B;L 1 0 0 1 0 168 DFFA B;L 2 0 0 2 0 399 OR51I1 B;L 1 0 0 1 0 169 DHRS3 B;L 2 0 0 2 0 400 OR51I2 B;L 1 0 0 1 0 170 DNAJC11 B;L 2 0 0 2 0 401 OR51J1 B;L 1 0 0 1 0 171 DNAJC16 B;L 2 0 0 2 0 402 OR51L1 B;L 1 0 0 1 0 172 DRAXIN B;L 2 0 0 2 0 403 OR51M1 B;L 1 0 0 1 0 173 EFHD2 B;L 2 0 0 2 0 404 OR51Q1 B;L 1 0 0 1 0 174 ENO1 B;L 2 0 0 2 0 405 OR51T1 B;L 1 0 0 1 0 175 EPHA2 B;L 2 0 0 2 0 406 OR51V1 B;L 1 0 0 1 0 176 ERRFI1 B;L 2 0 0 2 0 407 OR52A1 B;L 1 0 0 1 0 177 ESPN B;L 2 0 0 2 0 408 OR52A5 B;L 1 0 0 1 0 178 EXOSC10 B;L 2 0 0 2 0 409 OR52D1 B;L 1 0 0 1 0 179 FAM131C B;L 2 0 0 2 0 410 OR52E2 B;L 1 0 0 1 0 180 FAM49B A;B;G 2 0 0 2 0 411 OR52J3 B;L 1 0 0 1 0 181 FBLIM1 B;L 2 0 0 2 0 412 PEX1 A;B 1 0 0 1 0 182 FBXO2 B;L 2 0 0 2 0 413 PHF11 B;L 1 0 0 1 0 183 FBXO42 B;L 2 0 0 2 0 414 PHF20L1 A;B 1 0 0 1 0 184 FBXO44 B;L 2 0 0 2 0 415 PIK3CA B;G 1 1 0 0 0 185 FBXO6 B;L 2 0 0 2 0 416 POTEM B;L 1 0 0 1 0 186 FHAD1 B;L 2 0 0 2 0 417 POU3F2 B;L 1 0 0 1 0 187 GPR153 B;L 2 0 0 2 0 418 PTEN B;D 1 0 0 0 1 188 GPR157 B;L 2 0 0 2 0 419 RBM48 A;B 1 0 0 1 0 189 GSDMC A;B;G 2 0 0 2 0 420 RCBTB1 B;L 1 0 0 1 0 190 H6PD B;L 2 0 0 2 0 421 RIMS1 B;L 1 0 0 1 0 191 HES2 B;L 2 0 0 2 0 422 RNASEH2B B;L 1 0 0 1 0
47 Supplementary information - Meyer, Reimand et al.
192 HES3 B;L 2 0 0 2 0 423 RNF139 B;G 1 0 0 1 0 193 HNRNPCL1 B;L 2 0 0 2 0 424 RNLS B;D 1 0 0 0 1 194 HSPB7 B;L 2 0 0 2 0 425 SAMD9 A;B 1 0 0 1 0 195 ICMT B;L 2 0 0 2 0 426 SAMD9L A;B 1 0 0 1 0 196 KAZN B;L 2 0 0 2 0 427 SERP2 B;L 1 0 0 1 0 197 KCNAB2 B;L 2 0 0 2 0 428 SETDB2 B;L 1 0 0 1 0 198 KIAA2013 B;L 2 0 0 2 0 429 SFMBT1 B;L 1 0 0 1 0 199 KIF1B B;L 2 0 0 2 0 430 SLA A;B 1 0 0 1 0 200 KLHL21 B;L 2 0 0 2 0 431 SLC17A5 B;L 1 0 0 1 0 201 LMBRD1 B;L 2 0 0 2 0 432 SMAP1 B;L 1 0 0 1 0 202 LRRC38 B;L 2 0 0 2 0 433 SMIM2 B;L 1 0 0 1 0 203 LZIC B;L 2 0 0 2 0 434 SPRYD7 B;L 1 0 0 1 0 204 MAD2L2 B;L 2 0 0 2 0 435 SQLE B;G 1 0 0 1 0 205 MASP2 B;L 2 0 0 2 0 436 ST8SIA1 B;L 1 0 0 1 0 206 MFN2 B;L 2 0 0 2 0 437 STAMBPL1 B;D 1 0 0 0 1 207 MIIP B;L 2 0 0 2 0 438 TATDN1 B;G 1 0 0 1 0 208 MMP16 A;B;D 2 0 0 1 1 439 TG A;B 1 0 0 1 0 209 MTHFR B;L 2 0 0 2 0 440 TMEM65 B;G 1 0 0 1 0 210 MTOR B;L 2 0 0 2 0 441 TMEM71 A;B 1 0 0 1 0 211 NECAP2 B;L 2 0 0 2 0 442 TRIB1 B;G 1 0 0 1 0 212 NMNAT1 B;L 2 0 0 2 0 443 TRIM13 B;L 1 0 0 1 0 213 NOL9 B;L 2 0 0 2 0 444 TRMT12 B;G 1 0 0 1 0 214 NPHP4 B;L 2 0 0 2 0 445 TSC22D1 B;L 1 0 0 1 0 215 NPPA B;L 2 0 0 2 0 446 VPS13B A;B 1 0 0 1 0 216 NPPB B;L 2 0 0 2 0 447 WDR67 B;G 1 0 0 1 0 217 PARK7 B;L 2 0 0 2 0 448 WDYHV1 B;G 1 0 0 1 0 218 PDPN B;L 2 0 0 2 0 449 WISP1 A;B 1 0 0 1 0 219 PER3 B;L 2 0 0 2 0 450 WWOX B;G 1 0 0 1 0 220 PEX14 B;L 2 0 0 2 0 451 ZFP14 B;L 1 0 0 1 0 221 PGD B;L 2 0 0 2 0 452 ZHX1 B;G 1 0 0 1 0 222 PHF13 B;L 2 0 0 2 0 453 ZHX1-C8ORF76 B;G 1 0 0 1 0 223 PIK3CD B;L 2 0 0 2 0 454 ZHX2 B;G 1 0 0 1 0 224 PLEKHG5 B;L 2 0 0 2 0 455 ZMAT4 B;D 1 0 0 0 1 225 PLEKHM2 B;L 2 0 0 2 0 456 ZNF572 B;G 1 0 0 1 0 226 PLOD1 B;L 2 0 0 2 0 227 PRAMEF1 B;L 2 0 0 2 0 228 PRAMEF10 B;L 2 0 0 2 0 229 PRAMEF11 B;L 2 0 0 2 0 230 PRAMEF12 B;L 2 0 0 2 0 231 PRAMEF13 B;L 2 0 0 2 0
48 Supplementary information - Meyer, Reimand et al.
TUSC1 TEK PLAA MOB3B LRRC19 KLHL9 IZUMO3 IFT74 IFNE IFNA8 IFNA7 IFNA6 IFNA5 IFNA4 IFNA21 event IFNA2 deletion genes IFNA17 IFNA16 IFNA14 IFNA13 IFNA10 IFNA1 ELAVL2 DMRTA1 CAAP1 C9orf11 MTAP CDKN2B CDKN2A C9orf53 472 489 498 primary tumor
Fig. S14: genes with consistent copy number alterations. Genes with consistent copy number alterations in all samples originating from a primary tumor.
49 Supplementary information - Meyer, Reimand et al.
498_uns. 498_7 498_6 498_5 498_4 498_3 498_2 498_1 498_pr. 489_13 489_12 489_10 489_8 489_7 489_6 489_5 489_3 z score 489_1 2 489_uns. 489_pr. 1 482_14 482_7 482_4 0
samples 482_3 482_16 1 482_2 482_1 2 482_13 482_12 482_11 482_9 482_8 482_pr. 472_13 472_18 472_17 472_14 472_9 472_8 472_7 472_4 472_uns. 472_pr. YD7 PEX1 EBPL H6PD PTEN ADC1 CDK6 ENO1 RERE EGFR KRIT1 AT AD1 PHF11 SPSB1 KPNA3 SAMD9 TRIM13 PIK3CA ANKIB1 KCNRG CAB39L G AT AD1 AM133B SETDB2 RCBTB1 SP R FNDC3A SAMD9L CDKN2A CDKN2B C D SLC45A1 F CCDC132 genes Fig S15: clonal copy number alterations observed in TCGA. Genes with copy number alterations that were observed previously in the TCGA GBM study as frequently altered and expression-correlated.
50 Supplementary information - Meyer, Reimand et al.
negative regulation of Kinase protein bindingregulation of Cancer regulation of cyclin-dependent protein binding signaling protein excretion vascular serine/threonine renal sodium process in pathways kinase activity excretion circulatory TRKA signalling protein kinase system protein from the plasma binding serine/threonine regulation of membrane TRKA PI3K-AKT kinase inhibitor excretion regulation of Chronic myeloid activity regulation of blood vessel size Hippo signaling PI3K/AKT Bladder cancer leukemia urine volume regulation of vasodilation pathway signalling renal sodium kinase binding Hippo Non-small cell excretion lung cancer Association of cyclin-dependent regulation of TGF-beta Endometrial Glioma INK4A with protein tube size signaling cancer Cdk4/6 serine/threonine pathway Pathways in Prostate cancer cancer kinase regulator TGFB Thyroid Pancreatic activity Excretion, vascular process hormone PI3K-Akt cancer signaling signaling pathway HIF1 pathway Melanoma p53 signaling pathway Cell cycle oxidoreduction HIF-1 signaling pyridine-containing coenzyme pathway Small cell lung phosphatidylinositol P53 compound metabolic cancer pyridine-containing 3-kinase metabolic process Hepatitis B compound complex mTOR process Cholinergic mTOR signaling biosynthetic synapse pathway process nicotinamide pyridine Metabolism of nucleotide nucleotide vitamins and metabolic biosynthetic cofactors process tumor necrosis apical junction process adherens nicotinamide factor-activated assembly NAD metabolic junction nucleotide Apoptosis receptor activity process Metabolism of pyridine biosynthetic water-soluble cell-cell nucleotide process Adherens vitamins and junction metabolic junction cofactors death receptor assembly tight junction process activity assembly anchoring junction Apoptosis cell-cell junction Metabolism of nucleotides
Cell-cell junction
SCF ubiquitin response to ligase complex fibroblast positive hypoxia response to circadian rhythm proliferation regulation of oxygen levels SCF-dependent fibroblast ubiquitin ligase complex proteasomal proliferation ubiquitin-dependent protein response to catabolic regulation of decreased cullin-RING process fibroblast oxygen levels circadian ubiquitin ligase proliferation behavior complex Fibroblast Response to hypoxia Circadian rhythm Ubiquitin proliferation signaling
negative cellular positive regulation of aspartate family voltage-gated cellular astrocyte response to Steroid regulation of sequence-specific amino acid chloride channel response to development mechanical biosynthesis response to DNA binding biosynthetic activity ketone stimulus DNA damage transcription process stimulus factor activity Fig. S16: pathway analysis of clonal copy number alterations. Functional enrichment map of pathways and processes characteristic to genes with clonal copy number alterations (FDR P<0.05).
51 Supplementary information - Meyer, Reimand et al.
Table S4: pathway analysis of clonal copy number alterations. Enriched pathways and processes characteristic to genes with clonal copy number alterations (FDR p<0.05). GO.ID Description p.Val genes
GO:0007588 excretion 0.00567 EDNRB,UTS2,NPPB,KCNMA1,CLCNKB, CLCNKA
GO:0035812 renal sodium excretion 0.0174 EDNRB,UTS2,NPPB
GO:0044062 regulation of excretion 0.0229 EDNRB,UTS2,NPPB
GO:0035813 regulation of renal 0.0174 EDNRB,UTS2,NPPB sodium excretion
GO:0000079 regulation of cyclin- 0.00158 CDKN2B,CDKN2A,EGFR,CCNY,CCNL2, dependent protein PTEN serine/threonine kinase activity
GO:0014002 astrocyte development 0.0229 EGFR,CDK6,POU3F2
GO:0071260 cellular response to 0.0263 EGFR,ITGB1,TNFRSF8,NPPA,FAS mechanical stimulus
GO:0035809 regulation of urine 0.0129 EDNRB,UTS2,NPPB volume
GO:0009067 aspartate family amino 0.00877 MTAP,PLOD1,MTHFR acid biosynthetic process
GO:1901655 cellular response to 0.00805 EGFR,IRG1,GNB1,CASP9 ketone
GO:2001022 positive regulation of 0.000363 CDKN2A,EGFR,MYC response to DNA damage stimulus
GO:0048144 fibroblast proliferation 0.0274 EGFR,MYC,DACH1,UTS2,CDK6,RNASE H2B
GO:0048145 regulation of fibroblast 0.0222 EGFR,MYC,DACH1,UTS2,CDK6,RNASE proliferation H2B
GO:0048146 positive regulation of 0.0193 EGFR,MYC,UTS2,CDK6,RNASEH2B fibroblast proliferation
GO:0043433 negative regulation of 0.05 CDKN2A,IRG1,COMMD6,PEX14,MAD2L sequence-specific DNA 2,CTNNBIP1,TRIB1 binding transcription factor activity
GO:0003018 vascular process in 0.0185 EGFR,EDNRB,UTS2,NPPB,NPPA,KCNM circulatory system A1,CTNNBIP1,ACTA2,HBB
52 Supplementary information - Meyer, Reimand et al.
GO.ID Description p.Val genes
GO:0035150 regulation of tube size 0.0334 EGFR,EDNRB,UTS2,NPPB,NPPA,KCNM A1,ACTA2,HBB
GO:0050880 regulation of blood 0.0318 EGFR,EDNRB,UTS2,NPPB,NPPA,KCNM vessel size A1,ACTA2,HBB
GO:0042311 vasodilation 0.0133 EGFR,EDNRB,UTS2,NPPB,KCNMA1
GO:0070482 response to oxygen 0.00478 CUL2,GNB1,UTS2,PLOD1,PDPN,NPPA, levels NDRG1,MTOR,MTHFR,KCNMA1,FAS,A GTRAP
GO:0036293 response to decreased 0.0464 CUL2,GNB1,UTS2,PLOD1,NPPA,NDRG1 oxygen levels ,MTOR,MTHFR,KCNMA1,AGTRAP
GO:0001666 response to hypoxia 0.0411 CUL2,GNB1,UTS2,PLOD1,NPPA,NDRG1 ,MTOR,MTHFR,KCNMA1,AGTRAP
GO:0031146 SCF-dependent 0.0246 FBXL3,FBXO6,FBXO2 proteasomal ubiquitin- dependent protein catabolic process
GO:0043393 regulation of protein 0.0131 NRP1,BAMBI,PEX14,PARK7,KRIT1,IFIT2 binding ,CTNNBIP1
GO:0032091 negative regulation of 0.0412 PEX14,PARK7,IFIT2,CTNNBIP1 protein binding
GO:0007623 circadian rhythm 0.0151 EGFR,FBXL3,UTS2,PTEN,PER3,KCNMA 1
GO:0048512 circadian behavior 0.044 UTS2,PTEN,KCNMA1
GO:0006733 oxidoreduction 0.0019 NAMPTL,NADK,UBIAD1,PGD,NMNAT1,H coenzyme metabolic 6PD process
GO:0072524 pyridine-containing 0.0125 NAMPTL,NADK,PGD,NMNAT1,H6PD compound metabolic process
GO:0019362 pyridine nucleotide 0.00707 NAMPTL,NADK,PGD,NMNAT1,H6PD metabolic process
GO:0046496 nicotinamide nucleotide 0.00707 NAMPTL,NADK,PGD,NMNAT1,H6PD metabolic process
GO:0019674 NAD metabolic process 0.0443 NAMPTL,NADK,NMNAT1
GO:0072525 pyridine-containing 0.0247 NAMPTL,NADK,NMNAT1 compound biosynthetic process
53 Supplementary information - Meyer, Reimand et al.
GO.ID Description p.Val genes
GO:0019363 pyridine nucleotide 0.0138 NAMPTL,NADK,NMNAT1 biosynthetic process
GO:0019359 nicotinamide nucleotide 0.0138 NAMPTL,NADK,NMNAT1 biosynthetic process
GO:0007043 cell-cell junction 0.000696 INADL,PARD3,MPP7,ITGB1 assembly
GO:0043297 apical junction assembly 0.000138 INADL,PARD3,MPP7,ITGB1
GO:0070830 tight junction assembly 7.86E-05 INADL,PARD3,MPP7,ITGB1
GO:0000151 ubiquitin ligase complex 0.00445 KLHL9,CUL2,MIB2,LMO7,FBXL3,UBE4B, KLHL21,FBXO6,FBXO44,FBXO2,FBXL4
GO:0031461 cullin-RING ubiquitin 0.0027 KLHL9,CUL2,FBXL3,KLHL21,FBXO6,FB ligase complex XO44,FBXO2
GO:0019005 SCF ubiquitin ligase 0.0053 FBXL3,FBXO6,FBXO44,FBXO2 complex
GO:0005911 cell-cell junction 0.00148 INADL,PARD3,MPP7,KIAA1462,ITGB1,G JD4,LMO7,PLEKHG5,NPHP4,NDRG1,KR IT1,KAZN
GO:0070161 anchoring junction 0.015 PARD3,MPP7,KIAA1462,ITGB1,LMO7,N DRG1,KAZN,FBLIM1,EPHA2,AJAP1
GO:0005912 adherens junction 0.0326 PARD3,MPP7,KIAA1462,ITGB1,LMO7,N DRG1,FBLIM1,EPHA2,AJAP1
GO:0005942 phosphatidylinositol 3- 0.0361 PIK3CD,MTOR,PIK3CA kinase complex
GO:0005247 voltage-gated chloride 0.0196 CLCNKB,CLCNKA,CLCN6 channel activity
GO:0016538 cyclin-dependent protein 0.000544 CDKN2B,CDKN2A,CCNY serine/threonine kinase regulator activity
GO:0030291 protein serine/threonine 0.00174 CDKN2B,CDKN2A,SPRY2 kinase inhibitor activity
GO:0005035 death receptor activity 0.00146 TNFRSF8,TNFRSF25,TNFRSF1B
GO:0005031 tumor necrosis factor- 0.00085 TNFRSF8,TNFRSF25,TNFRSF1B activated receptor activity
GO:0019900 kinase binding 0.0398 CDKN2B,CDKN2A,EGFR,PARD3,ITGB1, CCNY,SPRY2,CCNL2,BORA,PTEN,MAD 2L2,FAS,ERRFI1,CASP9,TRIB1,JAK2,EE F1A1
54 Supplementary information - Meyer, Reimand et al.
GO.ID Description p.Val genes
GO:0019901 protein kinase binding 0.0366 CDKN2B,CDKN2A,EGFR,PARD3,ITGB1, CCNY,SPRY2,CCNL2,BORA,PTEN,MAD 2L2,ERRFI1,CASP9,TRIB1,JAK2,EEF1A 1
KEGG:05161 Hepatitis B 0.00783 IFNA1,MYC,PTEN,PIK3CD,FAS,CDK6,C ASP9,PIK3CA
KEGG:05219 Bladder cancer 8.02E-05 CDKN2A,EGFR,MYC
KEGG:04210 Apoptosis 0.05 PIK3CD,FAS,DFFA,CASP9,PIK3CA
KEGG:04350 TGF-beta signaling 0.00804 CDKN2B,MYC,BAMBI pathway
KEGG:05222 Small cell lung cancer 0.000237 CDKN2B,MYC,ITGB1,PTEN,PIK3CD,CD K6,CASP9,PIK3CA
KEGG:04066 HIF-1 signaling pathway 0.00521 EGFR,CUL2,PIK3CD,NPPA,MTOR,ENO1 ,PIK3CA
KEGG:04110 Cell cycle 0.0241 CDKN2B,CDKN2A,MYC,ZBTB17,MAD2L 2,CDK6
KEGG:05214 Glioma 0.000252 CDKN2A,EGFR,PTEN,PIK3CD,MTOR,C DK6,PIK3CA
KEGG:04151 PI3K-Akt signaling 0.0139 IFNA1,EGFR,MYC,ITGB1,GNB1,PTEN,PI pathway K3CD,MTOR,EPHA2,CDK6,CASP9,PIK3 CA,JAK2
KEGG:05215 Prostate cancer 0.0105 EGFR,PTEN,PIK3CD,MTOR,CASP9,PIK 3CA
KEGG:04520 Adherens junction 0.0157 EGFR,PARD3,LMO7
KEGG:05223 Non-small cell lung 0.000953 CDKN2A,EGFR,PIK3CD,CDK6,CASP9,PI cancer K3CA
KEGG:05212 Pancreatic cancer 0.00233 CDKN2A,EGFR,PIK3CD,CDK6,CASP9,PI K3CA
KEGG:04115 p53 signaling pathway 0.00887 CDKN2A,PTEN,FAS,CDK6,CASP9
KEGG:05218 Melanoma 0.0032 CDKN2A,EGFR,PTEN,PIK3CD,CDK6,PI K3CA
KEGG:04150 mTOR signaling pathway 0.0202 PTEN,PIK3CD,MTOR,PIK3CA,CAB39L
KEGG:04725 Cholinergic synapse 0.0102 GNB1,PIK3CD,KCNQ3,ADCY8,PIK3CA,K CNQ5,JAK2
KEGG:05220 Chronic myeloid 0.0253 CDKN2A,MYC,PIK3CD,CDK6,PIK3CA leukemia
KEGG:04390 Hippo signaling pathway 0.00227 INADL,MYC,PARD3,FZD8
55 Supplementary information - Meyer, Reimand et al.
GO.ID Description p.Val genes
KEGG:05200 Pathways in cancer 0.00153 CDKN2B,CDKN2A,EGFR,MYC,ITGB1,FZ D8,CUL2,PTEN,PIK3CD,MTOR,FAS,CDK 6,CASP9,PIK3CA
KEGG:04919 Thyroid hormone 0.0461 MYC,TBC1D4,PIK3CD,MTOR,CASP9,PI signaling pathway K3CA
KEGG:00100 Steroid biosynthesis 0.014 LIPA,CYP51A1,SQLE
KEGG:05213 Endometrial cancer 0.000632 EGFR,MYC,PTEN,PIK3CD,CASP9,PIK3 CA
REAC:187037 TRKA signalling from the 0.0288 PTEN,MTOR,CASP9,ADCY8,PIK3CA plasma membrane
REAC:198203 PI3K/AKT signalling 0.00302 PTEN,MTOR,CASP9,PIK3CA
REAC:182594 Association of INK4A 7.03E-05 CDKN2B,CDKN2A,CDK6 with Cdk4/6
REAC:196854 Metabolism of vitamins 0.012 NADK,NMNAT1,MTHFR and cofactors
REAC:196849 Metabolism of water- 0.012 NADK,NMNAT1,MTHFR soluble vitamins and cofactors
56 Supplementary information - Meyer, Reimand et al.
Subtype prediction of 472 clones Subtype prediction of 489 clones CL MES PN CL MES PN
● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.3 ● ● ● ● ● ● ● ● 0.3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.2 ● ● ● 0.2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● score
score ● ● ● ● ● ● ● ● ● ● ● 0.1 ● 0.1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.0 ● ● ● ● ● ● ● ● ● 0.0 ● ● ● ● ● ● ● 489_1 489_3 489_5 489_6 489_7 489_8 489_1 489_3 489_5 489_6 489_7 489_8 489_1 489_3 489_5 489_6 489_7 489_8 472_4 472_7 472_8 472_4 472_7 472_8 472_4 472_7 472_8 489_10 489_12 489_13 489_10 489_12 489_13 489_10 489_12 489_13 472_14 472_17 472_18 472_14 472_17 472_18 472_14 472_17 472_18 472_uns. 472_uns. 472_uns. clone 489_uns. 489_uns. clone 489_uns.
Subtype prediction of 498 clones CL MES PN TMZ response
● ● ● ● ● 0.3 ● ● sensitive
● ● resistant ●
● ●
● ● ● ● ● ● ● ● 0.2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●
score ● ● 0.1 ● ●
● ● ● ●
●
● ● ● ● 0.0 ● 498_1 498_2 498_3 498_6 498_7 498_1 498_2 498_3 498_6 498_7 498_1 498_2 498_3 498_6 498_7 clone
Fig S17: Subtype scoring of clones from tumor GBM-472, GBM-489, and GBM-498. Subtypes were scored according to the TCAG classifier gene set (20).
57 Supplementary information - Meyer, Reimand et al.
Table S5: genes with differential expression in TMZ sensitivity. Transcriptional profile of TMZ-sensitive clone 489_9 in GBM-482, ordered by statistical significance (FDR p<0.05). No. Probeset Z-score p-value FDR LogFC Gene No. Probeset Z-score p-value FDR LogFC Gene 1 8051573 -2.259 0.024 0.858 -0.255 CDC42EP3 388 7923442 2.044 0.041 0.858 0.134 SYT2 2 8089082 -2.258 0.024 0.858 -0.173 DCBLD2 389 8033809 2.045 0.041 0.858 0.203 ZNF846 3 8057803 -2.258 0.024 0.858 -0.929 TMEFF2 390 8093494 2.045 0.041 0.858 0.108 CRIPAK 4 8120239 -2.243 0.025 0.858 -0.192 TMEM14A 391 7971218 2.046 0.041 0.858 0.152 KBTBD7 5 8134030 -2.242 0.025 0.858 -0.873 STEAP1 392 8016311 2.046 0.041 0.858 0.072 WNT3 6 7950578 -2.234 0.025 0.858 -0.733 PAK1 393 7895749 2.048 0.041 0.858 0.842 KAT7 7 8121825 -2.231 0.026 0.858 -0.841 RNF217 394 8063187 2.049 0.041 0.858 0.140 EYA2 8 7954985 -2.231 0.026 0.858 -0.633 TMEM117 395 7918300 2.049 0.040 0.858 0.148 PSRC1 9 7909332 -2.226 0.026 0.858 -0.568 CD55 396 7967544 2.049 0.040 0.858 0.298 SCARB1 10 7985213 -2.224 0.026 0.858 -0.319 CHRNA5 397 7952884 2.049 0.040 0.858 0.343 B3GAT1 11 8055294 -2.224 0.026 0.858 -0.252 AJ239322.1 398 8115012 2.050 0.040 0.858 0.064 RN7SKP145 12 8179238 -2.223 0.026 0.858 -0.514 MICA 399 8091910 2.050 0.040 0.858 0.069 SERPINI2 13 8127854 -2.223 0.026 0.858 -0.953 ME1 400 7996290 2.051 0.040 0.858 0.100 CMTM1 14 8135601 -2.220 0.026 0.858 -0.873 MET 401 8176986 2.051 0.040 0.858 0.098 PPP2R3B 15 8114920 -2.219 0.026 0.858 -0.211 DPYSL3 402 8006865 2.052 0.040 0.858 0.175 PPP1R1B 16 8107909 -2.217 0.027 0.858 -0.266 SLC22A4 403 8003503 2.052 0.040 0.858 0.145 FANCA 17 8168749 -2.216 0.027 0.858 -0.427 SRPX2 404 8067361 2.052 0.040 0.858 0.093 TAF4 18 7958253 -2.212 0.027 0.858 -0.415 C12ORF75 405 7957536 2.052 0.040 0.858 0.159 NUDT4 19 8126121 -2.212 0.027 0.858 -0.097 BTBD9 406 8108706 2.053 0.040 0.858 0.131 PCDHB17 20 8057578 -2.206 0.027 0.858 -1.275 CALCRL 407 8158418 2.053 0.040 0.858 0.052 ENDOG 21 8021301 -2.206 0.027 0.858 -0.708 RAB27B 408 7928126 2.053 0.040 0.858 0.220 PALD1 22 8120402 -2.204 0.028 0.858 -0.287 BAG2 409 8053231 2.053 0.040 0.858 0.107 LOXL3 23 7924910 -2.202 0.028 0.858 -0.037 ACTA1 410 8167334 2.054 0.040 0.858 0.122 WAS 24 7948667 -2.202 0.028 0.858 -0.283 AHNAK 411 8066683 2.055 0.040 0.858 0.074 CDH22 25 7910022 -2.198 0.028 0.858 -0.202 CNIH3 412 8091255 2.055 0.040 0.858 0.258 PAQR9 26 8136662 -2.194 0.028 0.858 -0.168 MGAM 413 7946354 2.055 0.040 0.858 0.161 LMO1 27 8173755 -2.194 0.028 0.858 -0.114 ITM2A 414 8169808 2.055 0.040 0.858 0.293 CXORF64 28 8113709 -2.193 0.028 0.858 -0.688 LOX 415 7903028 2.055 0.040 0.858 0.115 RN7SKP123 29 7957260 -2.193 0.028 0.858 -0.178 GLIPR1 416 7958051 2.055 0.040 0.858 0.777 ASCL1 30 8048257 -2.193 0.028 0.858 -0.092 PNKD 417 7894562 2.056 0.040 0.858 0.511 ATP6V0B 31 8146198 -2.191 0.028 0.858 -0.182 POLB 418 7913864 2.056 0.040 0.858 0.062 STMN1 32 8096733 -2.188 0.029 0.858 -0.599 SGMS2 419 7955376 2.056 0.040 0.858 0.207 DIP2B 33 8078479 -2.186 0.029 0.858 -0.070 PDCD6IP 420 8141466 2.057 0.040 0.858 0.093 GPC2 34 8052956 -2.185 0.029 0.858 -0.180 EXOC6B 421 7905258 2.058 0.040 0.858 0.068 SETDB1 35 8171392 -2.184 0.029 0.858 -0.260 ASB9 422 8141757 2.058 0.040 0.858 0.080 ALKBH4 36 7989335 -2.184 0.029 0.858 -0.081 ANXA2 423 8065710 2.058 0.040 0.858 0.111 E2F1 37 8050215 -2.183 0.029 0.858 -0.042 YWHAQ 424 7915408 2.058 0.040 0.858 0.063 FOXJ3 38 7957298 -2.183 0.029 0.858 -0.303 NAV3 425 8143749 2.058 0.040 0.858 0.099 ZNF467 39 8123562 -2.183 0.029 0.858 -0.198 GMDS 426 8020088 2.059 0.040 0.858 0.221 SNOU13 40 8120719 -2.182 0.029 0.858 -0.650 CD109 427 7997839 2.059 0.039 0.858 0.116 CDT1 41 8021169 -2.181 0.029 0.858 -0.707 LIPG 428 8031441 2.059 0.039 0.858 0.180 BRSK1 42 7937150 -2.181 0.029 0.858 -0.096 ADAM8 429 7895765 2.060 0.039 0.858 0.607 KAT7 43 8076704 -2.180 0.029 0.858 -0.104 SMC1B 430 7959354 2.060 0.039 0.858 0.154 BCL7A 44 8137240 -2.176 0.030 0.858 -0.166 GIMAP7 431 8124423 2.061 0.039 0.858 0.177 HIST1H2BG
58 Supplementary information - Meyer, Reimand et al.
45 8135568 -2.172 0.030 0.858 -0.405 MDFIC 432 7980970 2.061 0.039 0.858 0.165 ITPK1 46 8121578 -2.172 0.030 0.858 -0.710 NT5DC1 433 8097753 2.062 0.039 0.858 0.402 DCLK2 47 7963786 -2.171 0.030 0.858 -0.302 ITGA5 434 8173059 2.062 0.039 0.858 0.184 WNK3 48 8057620 -2.170 0.030 0.858 -0.434 COL5A2 435 8160531 2.063 0.039 0.858 0.303 C9ORF72 49 7954245 -2.170 0.030 0.858 -0.476 PLEKHA5 436 7909175 2.064 0.039 0.858 0.171 SRGAP2 50 8105348 -2.167 0.030 0.858 -0.572 GPX8 437 7926189 2.065 0.039 0.858 0.173 SEC61A2 51 8126710 -2.164 0.030 0.858 -0.453 SUPT3H 438 8102982 2.067 0.039 0.858 0.129 AC097658.1 52 8040340 -2.164 0.030 0.858 -0.271 LPIN1 439 7962703 2.067 0.039 0.858 0.111 COL2A1 53 8116418 -2.164 0.030 0.858 -0.502 GFPT2 440 8082368 2.068 0.039 0.858 0.202 PODXL2 54 8151967 -2.163 0.031 0.858 -0.168 STK3 441 8141595 2.068 0.039 0.858 0.088 GIGYF1 55 8146517 -2.162 0.031 0.858 -0.329 CHCHD7 442 8044976 2.069 0.039 0.858 0.195 CNTNAP5 56 8041197 -2.161 0.031 0.858 -0.140 YPEL5 443 7912283 2.069 0.039 0.858 0.163 CTNNBIP1 57 8101992 -2.161 0.031 0.858 -0.572 SLC39A8 444 7900857 2.070 0.038 0.858 0.116 ST3GAL3 58 8120362 -2.159 0.031 0.858 -0.330 BEND6 445 7964499 2.071 0.038 0.858 0.241 AGAP2 59 7902957 -2.159 0.031 0.858 -0.394 EPHX4 446 7942453 2.072 0.038 0.858 0.332 PLEKHB1 60 8151816 -2.159 0.031 0.858 -0.362 GEM 447 7924733 2.072 0.038 0.858 0.108 PARP1 61 7939341 -2.158 0.031 0.858 -0.042 CD44 448 8134827 2.072 0.038 0.858 0.079 NYAP1 62 8089759 -2.158 0.031 0.858 -0.122 TMEM39A 449 7984408 2.073 0.038 0.858 0.144 MAP2K5 63 8120043 -2.158 0.031 0.858 -0.441 RUNX2 450 7966448 2.073 0.038 0.858 0.130 TMEM116 64 8134263 -2.158 0.031 0.858 -0.437 COL1A2 451 8109570 2.073 0.038 0.858 0.140 RNU6-390P 65 8081171 -2.158 0.031 0.858 -0.350 CRYBG3 452 8026830 2.073 0.038 0.858 0.163 FCHO1 66 8066513 -2.156 0.031 0.858 -0.194 SDC4 453 7961198 2.074 0.038 0.858 0.253 KLRAP1 67 7964997 -2.154 0.031 0.858 -0.361 CAPS2 454 7915695 2.074 0.038 0.858 0.069 MUTYH 68 8044263 -2.153 0.031 0.858 -0.125 RANBP2 455 8034806 2.074 0.038 0.858 0.076 DDX39A 69 8041383 -2.151 0.032 0.858 -0.440 LTBP1 456 7955019 2.074 0.038 0.858 0.109 ARID2 70 8091458 -2.150 0.032 0.858 -0.059 SERP1 457 7914214 2.074 0.038 0.858 0.074 SNHG12 71 8090098 -2.150 0.032 0.858 -0.331 MYLK 458 7963263 2.075 0.038 0.858 0.283 Y_RNA 72 7982597 -2.146 0.032 0.858 -0.310 THBS1 459 7939376 2.075 0.038 0.858 0.154 LDLRAD3 73 8051998 -2.146 0.032 0.858 -0.176 MCFD2 460 8064261 2.077 0.038 0.858 0.097 OPRL1 74 8073909 -2.145 0.032 0.858 -0.099 TBC1D22A 461 8085311 2.078 0.038 0.858 0.219 ATP2B2 75 8097148 -2.145 0.032 0.858 -0.086 KIAA1109 462 7943075 2.078 0.038 0.858 0.260 FAT3 76 8122426 -2.144 0.032 0.858 -0.407 PHACTR2 463 8171867 2.079 0.038 0.858 0.141 ARX 77 8129880 -2.142 0.032 0.858 -0.304 PERP 464 7987048 2.080 0.037 0.858 0.063 MTMR10 78 8129418 -2.141 0.032 0.858 -0.183 PTPRK 465 7900540 2.080 0.037 0.858 0.151 RIMKLA 79 8108217 -2.140 0.032 0.858 -0.734 TGFBI 466 7997542 2.081 0.037 0.858 0.143 NECAB2 80 8076734 -2.140 0.032 0.858 -0.560 WNT7B 467 8090803 2.081 0.037 0.858 0.201 RAB6B 81 8058127 -2.137 0.033 0.858 -0.125 CLK1 468 8041582 2.081 0.037 0.858 0.203 PKDCC 82 8041422 -2.136 0.033 0.858 -0.716 RASGRP3 469 8174543 2.081 0.037 0.858 0.517 DCX 83 7917503 -2.135 0.033 0.858 -0.706 GBP3 470 8067985 2.083 0.037 0.858 0.437 NCAM2 84 8154981 -2.133 0.033 0.858 -0.273 UNC13B 471 8111922 2.083 0.037 0.858 0.109 CTD-2201E18.3 85 8113214 -2.133 0.033 0.858 -0.525 GLRX 472 8028030 2.084 0.037 0.858 0.212 ARHGAP33 86 8091972 -2.132 0.033 0.858 -0.521 MECOM 473 8160771 2.084 0.037 0.858 0.104 KIF24 87 7895825 -2.131 0.033 0.858 -0.487 HNRNPM 474 8044133 2.084 0.037 0.858 0.147 NCK2 88 8040238 -2.131 0.033 0.858 -0.250 HPCAL1 475 7914282 2.084 0.037 0.858 0.278 SDC3 89 8046906 -2.130 0.033 0.858 -0.658 GULP1 476 8020226 2.086 0.037 0.858 0.064 SLMO1 90 8129953 -2.130 0.033 0.858 -0.465 HIVEP2 477 8174556 2.086 0.037 0.858 0.211 TRPC5 91 7900546 -2.127 0.033 0.858 -0.322 PPCS 478 7994362 2.087 0.037 0.858 0.139 CCDC101 92 7957665 -2.125 0.034 0.858 -0.231 ELK3 479 8026051 2.088 0.037 0.858 0.137 RNASEH2A 93 8047161 -2.125 0.034 0.858 -0.734 NABP1 480 8071086 2.088 0.037 0.858 0.193 CECR2
59 Supplementary information - Meyer, Reimand et al.
94 7981161 -2.124 0.034 0.858 -0.189 SYNE3 481 7905406 2.089 0.037 0.858 0.094 CGN 95 8117447 -2.124 0.034 0.858 -0.191 BTN2A2 482 8072979 2.089 0.037 0.858 0.160 POLR2F 96 7986010 -2.123 0.034 0.858 -0.108 IQGAP1 483 8098344 2.091 0.036 0.858 0.309 HAND2-AS1 97 8136347 -2.123 0.034 0.858 -0.120 CALD1 484 8077815 2.092 0.036 0.858 0.199 LINC00852 98 7943398 -2.121 0.034 0.858 -0.168 YAP1 485 8159616 2.094 0.036 0.858 0.072 NDOR1 99 8082869 -2.119 0.034 0.858 -0.465 PPP2R3A 486 7946853 2.094 0.036 0.858 0.111 KCNJ11 100 8078405 -2.119 0.034 0.858 -0.397 CMTM7 487 8068231 2.094 0.036 0.858 0.416 OLIG2 101 8164217 -2.119 0.034 0.858 -0.198 FAM129B 488 8158823 2.094 0.036 0.858 0.284 RNU6-881P 102 8037374 -2.117 0.034 0.858 -0.309 PLAUR 489 7921752 2.095 0.036 0.858 0.069 ARHGAP30 103 8122099 -2.117 0.034 0.858 -0.808 ENPP1 490 7992131 2.096 0.036 0.858 0.192 SOX8 104 8105181 -2.117 0.034 0.858 -0.078 MRPS30 491 7991735 2.096 0.036 0.858 0.060 SNRNP25 105 8035896 -2.115 0.034 0.858 -0.633 TSHZ3 492 8137091 2.096 0.036 0.858 0.113 ZNF398 106 8021418 -2.115 0.034 0.858 -0.411 MALT1 493 7994506 2.097 0.036 0.858 0.069 NFATC2IP 107 8175492 -2.114 0.034 0.858 -0.265 ATP11C 494 8135436 2.097 0.036 0.858 0.216 SLC26A4 108 7951614 -2.114 0.035 0.858 -0.141 PPP2R1B 495 8109179 2.098 0.036 0.858 0.079 PPARGC1B 109 8042993 -2.113 0.035 0.858 -0.090 CTNNA2 496 7975926 2.098 0.036 0.858 0.147 CIPC 110 7902074 -2.112 0.035 0.858 -0.125 LEPR 497 8109585 2.099 0.036 0.858 0.092 LSM11 111 8176174 -2.112 0.035 0.858 -0.273 MPP1 498 8007607 2.099 0.036 0.858 0.142 RUNDC3A 112 7945245 -2.111 0.035 0.858 -0.215 NTM 499 8068235 2.099 0.036 0.858 0.291 OLIG1 113 8116859 -2.110 0.035 0.858 -0.055 TMEM14C 500 8030448 2.099 0.036 0.858 0.253 CPT1C 114 8021470 -2.110 0.035 0.858 -0.417 PMAIP1 501 7915245 2.099 0.036 0.858 0.118 HPCAL4 115 8160559 -2.110 0.035 0.858 -0.292 DDX58 502 8065444 2.100 0.036 0.858 0.216 ACSS1 116 7982366 -2.109 0.035 0.858 -0.253 SCG5 503 7996081 2.100 0.036 0.858 0.205 GPR56 117 7938035 -2.108 0.035 0.858 -0.144 TRIM22 504 7945045 2.101 0.036 0.858 0.320 DDX25 118 8138442 -2.107 0.035 0.858 -0.183 TWIST1 505 7975876 2.101 0.036 0.858 0.150 ESRRB 119 8105741 -2.106 0.035 0.858 -0.347 MAST4 506 8103235 2.101 0.036 0.858 0.122 TIGD4 120 8024111 -2.101 0.036 0.858 -0.207 CNN2 507 8092520 2.101 0.036 0.858 0.368 C3ORF70 121 7962516 -2.100 0.036 0.858 -0.588 SLC38A1 508 8066451 2.102 0.036 0.858 0.040 RIMS4 122 8058604 -2.099 0.036 0.858 -0.540 MYL1 509 8004804 2.102 0.036 0.858 0.107 PFAS 123 8134036 -2.097 0.036 0.858 -0.450 STEAP2 510 7997135 2.103 0.035 0.858 0.126 FLJ00418 124 7961546 -2.096 0.036 0.858 -0.246 EPS8 511 7917792 2.104 0.035 0.858 0.244 MTND4P11 125 8154836 -2.096 0.036 0.858 -0.119 ANXA2P2 512 8137906 2.104 0.035 0.858 0.074 RADIL 126 8141052 -2.095 0.036 0.858 -0.135 PON1 513 7981490 2.104 0.035 0.858 0.125 TMEM179 127 7952557 -2.094 0.036 0.858 -0.079 SRPR 514 8006834 2.105 0.035 0.858 0.108 LASP1 128 7923141 -2.094 0.036 0.858 -0.543 DENND1B 515 7993946 2.105 0.035 0.858 0.170 EEF2K 129 8151281 -2.094 0.036 0.858 -0.108 TRAM1 516 7927915 2.105 0.035 0.858 0.295 STOX1 130 8121838 -2.094 0.036 0.858 -0.755 TPD52L1 517 8022356 2.105 0.035 0.858 0.130 SPIRE1 131 7948997 -2.094 0.036 0.858 -0.084 ATL3 518 8015554 2.105 0.035 0.858 0.153 KCNH4 132 8117071 -2.094 0.036 0.858 -0.087 FAM8A1 519 7915955 2.106 0.035 0.858 0.282 SPATA6 133 8099340 -2.093 0.036 0.858 -0.081 WDR1 520 7979888 2.106 0.035 0.858 0.106 SLC8A3 134 8081235 -2.093 0.036 0.858 -0.447 COL8A1 521 8134460 2.106 0.035 0.858 0.153 RP4-607J23.2 135 8106393 -2.092 0.036 0.858 -0.227 F2R 522 8176286 2.106 0.035 0.858 0.090 PLCXD1 136 8072170 -2.091 0.037 0.858 -0.207 KREMEN1 523 8179011 2.107 0.035 0.858 0.370 MOG 137 8176578 -2.091 0.037 0.858 -0.158 USP9Y 524 7998002 2.108 0.035 0.858 0.151 ZNF276 138 8041519 -2.090 0.037 0.858 -0.294 RMDN2 525 7970691 2.109 0.035 0.858 0.194 GPR12 139 8053975 -2.090 0.037 0.858 -0.082 LMAN2L 526 7908694 2.110 0.035 0.858 0.154 NAV1 140 7908940 -2.089 0.037 0.858 -0.286 ATP2B4 527 8026638 2.111 0.035 0.858 0.094 MYO9B 141 8096845 -2.089 0.037 0.858 -0.206 EGF 528 7985786 2.111 0.035 0.858 0.254 ACAN 142 8160637 -2.089 0.037 0.858 -0.229 B4GALT1 529 8090343 2.112 0.035 0.858 0.334 KLF15
60 Supplementary information - Meyer, Reimand et al.
143 8140534 -2.088 0.037 0.858 -0.364 SEMA3C 530 8049394 2.112 0.035 0.858 0.100 TRPM8 144 7947599 -2.088 0.037 0.858 -0.268 CHST1 531 8013660 2.112 0.035 0.858 0.090 ALDOC 145 8173551 -2.087 0.037 0.858 -0.244 PHKA1 532 8024306 2.113 0.035 0.858 0.217 APC2 146 8081055 -2.087 0.037 0.858 -0.193 CHMP2B 533 8046428 2.114 0.035 0.858 0.269 RAPGEF4 147 7928944 -2.086 0.037 0.858 -0.456 PAPSS2 534 7964300 2.115 0.034 0.858 0.097 ZBTB39 148 8081233 -2.084 0.037 0.858 -0.498 RNU6-1263P 535 7931500 2.115 0.034 0.858 0.117 GPR123 149 8147344 -2.083 0.037 0.858 -0.053 PDP1 536 8105456 2.116 0.034 0.858 0.232 SETD9 150 8168817 -2.083 0.037 0.858 -0.386 DRP2 537 8106573 2.116 0.034 0.858 0.186 THBS4 151 8029006 -2.082 0.037 0.858 -0.269 AXL 538 7907032 2.117 0.034 0.858 0.394 MAEL 152 8121257 -2.082 0.037 0.858 -0.259 PRDM1 539 7932867 2.118 0.034 0.858 0.250 ZNF438 153 7983811 -2.082 0.037 0.858 -0.132 PIGB 540 8083071 2.118 0.034 0.858 0.239 SPSB4 154 8071069 -2.082 0.037 0.858 -0.143 IL17RA 541 8027574 2.119 0.034 0.858 0.270 CHST8 155 7902495 -2.081 0.037 0.858 -0.515 NEXN 542 7957072 2.119 0.034 0.858 0.419 RAB3IP 156 7924207 -2.081 0.037 0.858 -0.138 PTPN14 543 8054872 2.119 0.034 0.858 0.403 TFCP2L1 157 7968915 -2.081 0.037 0.858 -0.275 GTF2F2 544 8148517 2.119 0.034 0.858 0.460 BAI1 158 7946972 -2.080 0.037 0.858 -0.171 SAA3P 545 7966293 2.120 0.034 0.858 0.133 C12ORF76 159 8075728 -2.080 0.038 0.858 -0.198 MYH9 546 8145490 2.120 0.034 0.858 0.135 PTK2B 160 8124040 -2.080 0.038 0.858 -0.123 ATXN1 547 8079037 2.120 0.034 0.858 0.244 TRAK1 161 7914557 -2.079 0.038 0.858 -0.435 SYNC 548 8062766 2.121 0.034 0.858 0.139 MYBL2 162 7944011 -2.078 0.038 0.858 -0.193 REXO2 549 8005978 2.121 0.034 0.858 0.246 TRAF4 163 8001449 -2.078 0.038 0.858 -0.231 IRX3 550 8014679 2.122 0.034 0.858 0.115 SRCIN1 164 8059854 -2.077 0.038 0.858 -0.154 ARL4C 551 8002792 2.122 0.034 0.858 0.177 FA2H 165 8147580 -2.076 0.038 0.858 -0.098 VPS13B 552 8114898 2.122 0.034 0.858 0.603 RNA5SP196 166 8120880 -2.076 0.038 0.858 -0.456 TPBG 553 7894782 2.123 0.034 0.858 0.064 EIF4G2 167 7963280 -2.076 0.038 0.858 -0.190 SMAGP 554 8087530 2.124 0.034 0.858 0.148 CAMKV 168 8102567 -2.075 0.038 0.858 -0.276 PRDM5 555 7998053 2.124 0.034 0.858 0.170 MC1R 169 8096032 -2.075 0.038 0.858 -0.366 PRDM8 556 8017150 2.124 0.034 0.858 0.153 TUBD1 170 8122986 -2.074 0.038 0.858 -0.199 SNX9 557 7995776 2.125 0.034 0.858 0.192 MT3 171 8150881 -2.073 0.038 0.858 -0.562 PLAG1 558 7981013 2.125 0.034 0.858 0.216 PRIMA1 172 8180253 -2.073 0.038 0.858 -0.095 FUNDC2P3 559 8034974 2.127 0.033 0.858 0.205 EPHX3 173 7971077 -2.073 0.038 0.858 -0.630 POSTN 560 8143397 2.128 0.033 0.858 0.172 DENND2A 174 8058914 -2.072 0.038 0.858 -0.137 AAMP 561 7893102 2.128 0.033 0.858 0.485 PRPF8 175 8107005 -2.069 0.039 0.858 -0.192 CAST 562 7910915 2.129 0.033 0.858 0.144 CHRM3 176 8107133 -2.069 0.039 0.858 -0.261 PAM 563 8137670 2.129 0.033 0.858 0.289 PDGFA 177 7936529 -2.069 0.039 0.858 -0.758 KIAA1598 564 7897691 2.130 0.033 0.858 0.240 PTCHD2 178 8130962 -2.068 0.039 0.858 -0.046 PDCD2 565 7991173 2.132 0.033 0.858 0.131 KLHL25 179 8080926 -2.068 0.039 0.858 -0.106 ARL6IP5 566 7970648 2.133 0.033 0.858 0.114 AMER2 180 7962579 -2.067 0.039 0.858 -0.684 AMIGO2 567 7992043 2.133 0.033 0.858 0.101 FAM173A 181 8140579 -2.067 0.039 0.858 -1.002 CACNA2D1 568 8174513 2.133 0.033 0.858 0.719 CHRDL1 182 8048772 -2.065 0.039 0.858 -0.082 RHBDD1 569 8021349 2.133 0.033 0.858 0.181 ST8SIA3 183 7963567 -2.065 0.039 0.858 -0.112 KRT8 570 8097586 2.134 0.033 0.858 0.244 GAB1 184 8146115 -2.064 0.039 0.858 -0.430 C8ORF4 571 7954143 2.135 0.033 0.858 0.245 PTPRO 185 8142886 -2.064 0.039 0.858 -0.106 UBE2H 572 7924987 2.135 0.033 0.858 0.620 AGT 186 8156228 -2.063 0.039 0.858 -0.286 CTSL 573 8132667 2.137 0.033 0.858 0.243 ADCY1 187 8056206 -2.063 0.039 0.858 -0.089 RBMS1 574 8030022 2.140 0.032 0.858 0.056 GRIN2D 188 8131600 -2.062 0.039 0.858 -0.350 TSPAN13 575 7975799 2.140 0.032 0.858 0.247 FLVCR2 189 8054479 -2.061 0.039 0.858 -0.453 MALL 576 7953469 2.141 0.032 0.858 0.245 GNB3 190 8102482 -2.061 0.039 0.858 -0.165 SEC24D 577 8170716 2.141 0.032 0.858 0.203 PLXNB3 191 8116610 -2.060 0.039 0.858 -0.092 NQO2 578 8124510 2.141 0.032 0.858 0.165 HIST1H2BL
61 Supplementary information - Meyer, Reimand et al.
192 8120102 -2.058 0.040 0.858 -0.458 CD2AP 579 8128247 2.142 0.032 0.858 0.197 BACH2 193 8121277 -2.057 0.040 0.858 -0.319 AIM1 580 8021120 2.143 0.032 0.858 0.342 RNF165 194 8135069 -2.056 0.040 0.858 -0.404 SERPINE1 581 7944339 2.143 0.032 0.858 0.121 RN7SL688P 195 7946332 -2.054 0.040 0.858 -0.099 OR10A3 582 8043630 2.145 0.032 0.858 0.128 ARID5A 196 8102912 -2.054 0.040 0.858 -0.484 TBC1D9 583 8155284 2.145 0.032 0.858 0.380 FRMPD1 197 8041212 -2.054 0.040 0.858 -0.201 LCLAT1 584 7977868 2.146 0.032 0.858 0.061 C14ORF93 198 8151999 -2.054 0.040 0.858 -0.269 RGS22 585 8034583 2.146 0.032 0.858 0.106 SYCE2 199 8065868 -2.053 0.040 0.858 -0.060 EIF6 586 8112428 2.146 0.032 0.858 0.380 CD180 200 8008454 -2.052 0.040 0.858 -0.171 ABCC3 587 7910416 2.147 0.032 0.858 0.140 URB2 201 8038407 -2.051 0.040 0.858 -0.211 RRAS 588 7900931 2.147 0.032 0.858 0.037 B4GALT2 202 7906919 -2.049 0.040 0.858 -0.476 RGS4 589 7989313 2.148 0.032 0.858 0.463 Y_RNA 203 7957551 -2.048 0.041 0.858 -0.212 SOCS2 590 8103745 2.148 0.032 0.858 0.227 HAND2 204 8051387 -2.048 0.041 0.858 -0.080 DPY30 591 7937135 2.150 0.032 0.858 0.153 TTC40 205 8174779 -2.046 0.041 0.858 -0.078 LAMP2 592 7969050 2.150 0.032 0.858 0.568 CYSLTR2 206 8097717 -2.046 0.041 0.858 -0.278 ARHGAP10 593 7998749 2.151 0.031 0.858 0.135 BRICD5 207 7906852 -2.046 0.041 0.858 -0.063 UHMK1 594 7995055 2.152 0.031 0.858 0.087 KAT8 208 8170531 -2.045 0.041 0.858 -0.084 MAGEA4 595 7920687 2.152 0.031 0.858 0.200 GBAP1 209 8121095 -2.045 0.041 0.858 -0.087 ANKRD6 596 8137202 2.153 0.031 0.858 0.130 ZNF862 210 7923131 -2.043 0.041 0.858 -0.526 DENND1B 597 8117207 2.153 0.031 0.858 0.193 ALDH5A1 211 8132860 -2.043 0.041 0.858 -0.306 EGFR 598 8056457 2.154 0.031 0.858 0.183 SCN1A 212 8160260 -2.043 0.041 0.858 -0.534 BNC2 599 7975687 2.154 0.031 0.858 0.068 LIN52 213 7978376 -2.042 0.041 0.858 -0.219 STXBP6 600 7933488 2.155 0.031 0.858 0.462 VSTM4 214 7965627 -2.042 0.041 0.858 -0.123 LTA4H 601 8013243 2.156 0.031 0.858 0.207 SHMT1 215 8100298 -2.042 0.041 0.858 -0.473 OCIAD2 602 7977418 2.156 0.031 0.858 0.100 C14ORF80 216 8129677 -2.041 0.041 0.858 -0.219 SGK1 603 8141664 2.156 0.031 0.858 0.157 VGF 217 8046020 -2.041 0.041 0.858 -0.293 SCN2A 604 7917347 2.157 0.031 0.858 0.125 DDAH1 218 8067233 -2.041 0.041 0.858 -0.231 PMEPA1 605 7986195 2.158 0.031 0.858 0.259 SV2B 219 7986214 -2.040 0.041 0.858 -0.257 SLCO3A1 606 7901229 2.158 0.031 0.858 0.095 FAAH 220 8121588 -2.040 0.041 0.858 -0.464 DSE 607 8045075 2.160 0.031 0.858 0.199 GPR17 221 7907092 -2.040 0.041 0.858 -0.109 MPZL1 608 7895497 2.160 0.031 0.858 0.064 IK 222 8085984 -2.038 0.042 0.858 -0.335 OSBPL10 609 7899075 2.160 0.031 0.858 0.156 EXTL1 223 8023598 -2.036 0.042 0.858 -0.261 RNF152 610 7951928 2.160 0.031 0.858 0.174 DSCAML1 224 8061958 -2.035 0.042 0.858 -0.054 CHMP4B 611 7957092 2.160 0.031 0.858 0.426 MYRFL 225 8007169 -2.035 0.042 0.858 -0.120 KLHL10 612 8151496 2.161 0.031 0.858 0.486 ZNF704 226 8062312 -2.035 0.042 0.858 -0.266 MYL9 613 7894240 2.161 0.031 0.858 0.284 PRPF8 227 8092523 -2.034 0.042 0.858 -0.125 EHHADH 614 8061227 2.162 0.031 0.858 0.124 SLC24A3 228 8069301 -2.033 0.042 0.858 -0.269 COL6A2 615 7922018 2.162 0.031 0.858 0.496 ILDR2 229 7948048 -2.033 0.042 0.858 -0.071 TRIM51GP 616 7903632 2.163 0.031 0.858 0.326 CELSR2 230 7977299 -2.033 0.042 0.858 -0.163 CEP170B 617 8009493 2.163 0.031 0.858 0.629 KCNJ16 231 8041447 -2.032 0.042 0.858 -0.152 CRIM1 618 8133983 2.165 0.030 0.858 0.459 ADAM22 232 8149793 -2.032 0.042 0.858 -0.104 ENTPD4 619 8143759 2.170 0.030 0.858 0.193 ATP6V0E2-AS1 233 8063394 -2.031 0.042 0.858 -0.084 PTPN1 620 8005953 2.170 0.030 0.858 0.368 SNORD4A 234 8134981 -2.031 0.042 0.858 -0.127 TRIP6 621 7987405 2.170 0.030 0.858 0.741 RASGRP1 235 8048940 -2.031 0.042 0.858 -0.394 SP100 622 7992775 2.170 0.030 0.858 0.138 PAQR4 236 8051204 -2.031 0.042 0.858 -0.043 SUPT7L 623 7959657 2.171 0.030 0.858 0.061 ATP6V0A2 237 8042468 -2.030 0.042 0.858 -0.311 ANXA4 624 8131143 2.171 0.030 0.858 0.363 LFNG 238 8124650 -2.029 0.042 0.858 -1.462 UBD 625 8072870 2.171 0.030 0.858 0.099 PDXP 239 7944803 -2.028 0.043 0.858 -0.331 VWA5A 626 8038225 2.172 0.030 0.858 0.141 PLEKHA4 240 8143327 -2.028 0.043 0.858 -0.258 PARP12 627 8155250 2.173 0.030 0.858 0.076 GRHPR
62 Supplementary information - Meyer, Reimand et al.
241 8068593 -2.028 0.043 0.858 -0.435 ETS2 628 8086600 2.173 0.030 0.858 0.296 CCR1 242 8048026 -2.027 0.043 0.858 -0.283 CPS1 629 7909425 2.173 0.030 0.858 0.313 CAMK1G 243 7981142 -2.026 0.043 0.858 -0.414 CLMN 630 7910727 2.174 0.030 0.858 0.439 ACTN2 244 8149774 -2.026 0.043 0.858 -0.315 LOXL2 631 7952249 2.175 0.030 0.858 0.466 USP2 245 8046003 -2.026 0.043 0.858 -0.877 GCA 632 8163109 2.176 0.030 0.858 0.262 FRRS1L 246 7943521 -2.026 0.043 0.858 -0.134 DDI1 633 7909628 2.176 0.030 0.858 0.153 FLVCR1 247 8047403 -2.026 0.043 0.858 -0.155 CASP10 634 7990683 2.176 0.030 0.858 0.121 ACSBG1 248 8060963 -2.025 0.043 0.858 -0.417 SNAP25 635 8061094 2.177 0.029 0.858 0.281 PCSK2 249 8097521 -2.023 0.043 0.858 -0.064 SCOC 636 7940508 2.179 0.029 0.858 0.216 DAGLA 250 8104592 -2.022 0.043 0.858 -0.102 FBXL7 637 8141371 2.179 0.029 0.858 0.316 GJC3 251 8107421 -2.021 0.043 0.858 -0.060 AP3S1 638 8105506 2.180 0.029 0.858 0.114 ZSWIM6 252 8105328 -2.021 0.043 0.858 -0.114 SNX18 639 7959786 2.180 0.029 0.858 0.049 AACS 253 8049635 -2.021 0.043 0.858 -0.193 TRAF3IP1 640 7941214 2.181 0.029 0.858 0.096 POLA2 254 8155946 -2.020 0.043 0.858 -0.405 VPS13A 641 7897522 2.181 0.029 0.858 0.592 RBP7 255 8056060 -2.020 0.043 0.858 -0.113 BAZ2B 642 8169061 2.181 0.029 0.858 0.604 PLP1 256 7903461 -2.020 0.043 0.858 -0.399 NTNG1 643 7964089 2.182 0.029 0.858 0.169 PAN2 257 7969861 -2.020 0.043 0.858 -0.427 ITGBL1 644 7950726 2.182 0.029 0.858 0.226 FAM181B 258 7982060 -2.019 0.044 0.858 -0.144 SNORD115-27 645 7918449 2.183 0.029 0.858 0.813 KCNA2 259 8137089 -2.018 0.044 0.858 -0.140 RNU6-650P 646 7999177 2.184 0.029 0.858 0.077 ANKS3 260 7958414 -2.017 0.044 0.858 -0.064 ISCU 647 7923662 2.185 0.029 0.858 0.464 PIK3C2B 261 7893017 -2.016 0.044 0.858 -0.056 SART3 648 8157650 2.188 0.029 0.858 0.803 PTGS1 262 8031884 -2.015 0.044 0.858 -0.329 ZNF544 649 8133459 2.188 0.029 0.858 0.110 CLIP2 263 8018786 -2.014 0.044 0.858 -0.196 MXRA7 650 8058542 2.189 0.029 0.858 0.982 C2ORF80 264 7967456 -2.013 0.044 0.858 -0.114 RILPL2 651 8016841 2.189 0.029 0.858 0.404 TMEM100 265 8098328 -2.013 0.044 0.858 -0.165 GALNT7 652 7906786 2.190 0.029 0.858 0.275 FCRLA 266 7917850 -2.013 0.044 0.858 -0.605 ARHGAP29 653 7941946 2.191 0.028 0.858 0.040 NDUFV1 267 7957861 -2.013 0.044 0.858 -0.609 ANO4 654 8040419 2.192 0.028 0.858 0.349 MYCN 268 7958000 -2.013 0.044 0.858 -0.089 CHPT1 655 8156373 2.192 0.028 0.858 0.060 FGD3 269 8140971 -2.012 0.044 0.858 -0.326 SAMD9L 656 7984155 2.192 0.028 0.858 0.171 SNX1 270 8174972 -2.012 0.044 0.858 -0.074 DCAF12L1 657 8149500 2.193 0.028 0.858 0.203 MTUS1 271 8129573 -2.012 0.044 0.858 -0.284 MOXD1 658 7963880 2.194 0.028 0.858 0.328 ITGA7 272 8154245 -2.012 0.044 0.858 -0.582 PDCD1LG2 659 7977432 2.196 0.028 0.858 0.142 TMEM121 273 7902965 -2.012 0.044 0.858 -0.268 BTBD8 660 8158783 2.196 0.028 0.858 0.161 NUP214 274 7997346 -2.012 0.044 0.858 -0.115 CLEC3A 661 7938925 2.196 0.028 0.858 0.106 NELL1 275 7902452 -2.011 0.044 0.858 -0.422 AK5 662 7999754 2.196 0.028 0.858 0.438 XYLT1 276 8096919 -2.010 0.044 0.858 -0.384 ALPK1 663 8013022 2.197 0.028 0.858 0.237 FAM211A 277 7938816 -2.010 0.044 0.858 -0.229 ZDHHC13 664 8105436 2.197 0.028 0.858 0.420 MAP3K1 278 8129974 -2.008 0.045 0.858 -0.147 FUCA2 665 7914433 2.197 0.028 0.858 0.264 BAI2 279 8058614 -2.007 0.045 0.858 -0.071 LANCL1 666 7961381 2.197 0.028 0.858 0.422 GPR19 280 7995552 -2.005 0.045 0.858 -0.190 CYLD 667 8012605 2.197 0.028 0.858 0.407 GAS7 281 7996198 -2.005 0.045 0.858 -0.294 CCDC113 668 8101788 2.197 0.028 0.858 0.380 UNC5C 282 8174474 -2.005 0.045 0.858 -0.321 ACSL4 669 7967736 2.198 0.028 0.858 0.164 POLE 283 7917322 -2.005 0.045 0.858 -0.316 SYDE2 670 8172204 2.198 0.028 0.858 0.476 MAOB 284 8020973 -2.004 0.045 0.858 -0.132 FHOD3 671 8064277 2.199 0.028 0.858 0.293 MYT1 285 8057677 -2.003 0.045 0.858 -0.444 SLC40A1 672 8137485 2.200 0.028 0.858 0.633 DPP6 286 8054862 -2.003 0.045 0.858 -0.125 TMEM185B 673 8142770 2.200 0.028 0.858 0.195 RBM28 287 7940191 -2.002 0.045 0.858 -0.311 STX3 674 8098161 2.200 0.028 0.858 0.175 RNU6-284P 288 8008113 -2.002 0.045 0.858 -0.137 CALCOCO2 675 7975787 2.200 0.028 0.858 0.191 JDP2 289 8036460 2.001 0.045 0.858 0.234 DPF1 676 8033956 2.202 0.028 0.858 0.054 S1PR2
63 Supplementary information - Meyer, Reimand et al.
290 7904751 2.001 0.045 0.858 0.202 RBM8A 677 8000310 2.203 0.028 0.858 0.084 EARS2 291 8047963 2.002 0.045 0.858 0.094 SNAI1P1 678 8096025 2.203 0.028 0.858 0.089 RN7SL127P 292 7989922 2.002 0.045 0.858 0.196 SCARNA14 679 8170319 2.204 0.028 0.858 0.504 TMEM257 293 8002969 2.003 0.045 0.858 0.249 MAF 680 8158771 2.207 0.027 0.858 0.641 AIF1L 294 7977373 2.003 0.045 0.858 0.097 MTA1 681 7937667 2.207 0.027 0.858 0.237 BRSK2 295 7992639 2.003 0.045 0.858 0.102 TBC1D24 682 8029219 2.208 0.027 0.858 0.299 TMEM145 296 8163116 2.005 0.045 0.858 0.340 EPB41L4B 683 8112182 2.208 0.027 0.858 0.136 MIER3 297 8160935 2.006 0.045 0.858 0.114 FANCG 684 8115122 2.208 0.027 0.858 0.527 CAMK2A 298 8097449 2.006 0.045 0.858 0.578 PCDH10 685 8172573 2.209 0.027 0.858 0.203 SYP 299 7953723 2.006 0.045 0.858 0.070 CLEC4A 686 8122279 2.209 0.027 0.858 0.469 KIAA1244 300 8143534 2.006 0.045 0.858 0.110 KEL 687 8100507 2.209 0.027 0.858 0.434 HOPX 301 8135931 2.007 0.045 0.858 0.110 CICP18 688 8171653 2.209 0.027 0.858 0.310 MAP3K15 302 8133845 2.007 0.045 0.858 0.400 Y_RNA 689 8133167 2.210 0.027 0.858 0.071 KCTD7 303 7904254 2.008 0.045 0.858 0.065 ATP1A1 690 8140319 2.210 0.027 0.858 0.319 HIP1 304 7934945 2.009 0.045 0.858 0.132 PANK1 691 8036862 2.211 0.027 0.858 0.160 CTC-425O23.2 305 8000467 2.009 0.044 0.858 0.134 GSG1L 692 8033025 2.211 0.027 0.858 0.086 DUS3L 306 7963911 2.010 0.044 0.858 0.063 CD63 693 8073929 2.211 0.027 0.858 0.158 FAM19A5 307 8168303 2.010 0.044 0.858 0.084 TAF1 694 7990643 2.212 0.027 0.858 0.327 LINGO1 308 7915252 2.010 0.044 0.858 0.408 BMP8B 695 7975390 2.212 0.027 0.858 0.658 SMOC1 309 7946757 2.010 0.044 0.858 0.281 SOX6 696 8105596 2.212 0.027 0.858 0.642 RGS7BP 310 7969017 2.010 0.044 0.858 0.044 RB1 697 7897561 2.212 0.027 0.858 0.226 KIF1B 311 8079753 2.011 0.044 0.858 0.138 DAG1 698 8161224 2.213 0.027 0.858 0.104 ZBTB5 312 7970162 2.013 0.044 0.858 0.188 ATP11A 699 8009326 2.213 0.027 0.858 0.341 CACNG5 313 7904000 2.013 0.044 0.858 0.081 DDX20 700 8034700 2.213 0.027 0.858 0.181 C19ORF57 314 8108847 2.014 0.044 0.858 0.032 RNF14 701 8067969 2.213 0.027 0.858 0.613 CHODL 315 8021131 2.014 0.044 0.858 0.174 KATNAL2 702 8132151 2.213 0.027 0.858 0.723 ADCYAP1R1 316 7992973 2.015 0.044 0.858 0.041 DNAJA3 703 8126035 2.214 0.027 0.858 0.313 CPNE5 317 8173106 2.015 0.044 0.858 0.143 ITIH6 704 8135262 2.215 0.027 0.858 0.864 LHFPL3 318 8080562 2.015 0.044 0.858 0.318 IL17RB 705 7920027 2.215 0.027 0.858 0.350 CELF3 319 8156476 2.016 0.044 0.858 0.115 PHF2 706 7920877 2.216 0.027 0.858 0.185 ARHGEF2 320 8137208 2.016 0.044 0.858 0.187 ATP6V0E2 707 7920928 2.217 0.027 0.858 0.305 PAQR6 321 7990714 2.017 0.044 0.858 0.219 CHRNA3 708 7950764 2.219 0.026 0.858 0.502 DLG2 322 7930341 2.017 0.044 0.858 0.195 SORCS3 709 7952813 2.220 0.026 0.858 0.429 IGSF9B 323 7918558 2.017 0.044 0.858 0.618 KCND3 710 8028719 2.221 0.026 0.858 0.643 DLL3 324 7993035 2.017 0.044 0.858 0.116 UBN1 711 8000930 2.222 0.026 0.858 0.373 RNU6-1043P 325 7899192 2.018 0.044 0.858 0.240 RPS6KA1 712 8067546 2.222 0.026 0.858 0.385 RP11-93B14.5 326 8014057 2.018 0.044 0.858 0.265 OMG 713 8007828 2.223 0.026 0.858 0.408 MAPT 327 8023377 2.018 0.044 0.858 0.079 MEX3C 714 8013833 2.224 0.026 0.858 0.658 SEZ6 328 8060205 2.019 0.043 0.858 0.120 PASK 715 8046746 2.224 0.026 0.858 0.581 PPP1R1C 329 7973820 2.019 0.043 0.858 0.193 RNU6-541P 716 7948912 2.224 0.026 0.858 0.236 CHRM1 330 7992092 2.020 0.043 0.858 0.100 CHTF18 717 7955348 2.225 0.026 0.858 0.274 GPD1 331 8026877 2.020 0.043 0.858 0.043 SLC5A5 718 7975889 2.226 0.026 0.858 0.417 VASH1 332 8131709 2.020 0.043 0.858 0.162 SP4 719 8114767 2.226 0.026 0.858 0.374 PCDH1 333 8152119 2.020 0.043 0.858 0.323 NCALD 720 8160823 2.227 0.026 0.858 0.634 CNTFR 334 7953835 2.021 0.043 0.858 0.191 KLRG1 721 7980344 2.228 0.026 0.858 0.692 ZDHHC22 335 7986836 2.021 0.043 0.858 0.080 RNA5SP391 722 7921012 2.228 0.026 0.858 0.579 C1ORF61 336 8070182 2.021 0.043 0.858 0.104 RCAN1 723 8022085 2.229 0.026 0.858 0.144 DLGAP1 337 8008768 2.021 0.043 0.858 0.352 PPM1E 724 7911458 2.229 0.026 0.858 0.311 ACAP3 338 7927694 2.022 0.043 0.858 0.761 PHYHIPL 725 7995739 2.229 0.026 0.858 0.354 GNAO1
64 Supplementary information - Meyer, Reimand et al.
339 8015706 2.022 0.043 0.858 0.231 RAMP2-AS1 726 7992219 2.230 0.026 0.858 0.298 BAIAP3 340 7906757 2.022 0.043 0.858 0.571 FCGR2A 727 8066757 2.230 0.026 0.858 0.395 SLC13A3 341 8180242 2.023 0.043 0.858 0.075 RP11-798L4.1 728 7921533 2.231 0.026 0.858 0.794 KCNJ10 342 7960261 2.023 0.043 0.858 0.057 RAD52 729 7940851 2.231 0.026 0.858 0.376 FLRT1 343 7969736 2.024 0.043 0.858 0.126 FARP1 730 7935401 2.233 0.026 0.858 0.454 ARHGAP19-SLIT1 344 8067626 2.024 0.043 0.858 0.216 KCNQ2 731 8036055 2.233 0.026 0.858 0.377 LGI4 345 7977615 2.026 0.043 0.858 0.125 RNASE1 732 7958305 2.233 0.026 0.858 0.744 RFX4 346 8056611 2.026 0.043 0.858 0.085 LRP2 733 7992147 2.234 0.026 0.858 0.201 CACNA1H 347 8135488 2.026 0.043 0.858 0.191 LRRN3 734 7968260 2.234 0.025 0.858 0.193 GSX1 348 8058869 2.026 0.043 0.858 0.157 TNS1 735 7951717 2.235 0.025 0.858 0.337 TMPRSS5 349 7995477 2.028 0.043 0.858 0.179 RNY4P3 736 8024808 2.235 0.025 0.858 0.330 SHD 350 8092661 2.028 0.043 0.858 0.288 MASP1 737 7927803 2.235 0.025 0.858 0.624 LRRTM3 351 8087145 2.028 0.043 0.858 0.175 CELSR3 738 8071559 2.237 0.025 0.858 0.086 SDF2L1 352 7906177 2.028 0.043 0.858 0.087 TTC24 739 8073623 2.237 0.025 0.858 0.364 MPPED1 353 7993680 2.028 0.043 0.858 0.042 C16ORF62 740 8027152 2.238 0.025 0.858 0.202 NCAN 354 7895536 2.028 0.043 0.858 0.111 IK 741 7905664 2.238 0.025 0.858 0.375 SLC27A3 355 8158028 2.029 0.043 0.858 0.142 LRSAM1 742 8161964 2.239 0.025 0.858 0.468 FRMD3 356 7999008 2.029 0.042 0.858 0.111 SLX4 743 7991246 2.239 0.025 0.858 0.476 RLBP1 357 8030578 2.029 0.042 0.858 0.135 CTD-2126E3.1 744 8086799 2.240 0.025 0.858 0.476 CSPG5 358 7959777 2.029 0.042 0.858 0.149 BRI3BP 745 8033487 2.244 0.025 0.858 0.269 FBN3 359 8118669 2.031 0.042 0.858 0.285 KIFC1 746 7914974 2.245 0.025 0.858 0.756 GRIK3 360 8176655 2.031 0.042 0.858 0.115 NLGN4Y 747 7921003 2.247 0.025 0.858 0.894 C1ORF61 361 7908841 2.031 0.042 0.858 0.205 PPP1R12B 748 7952475 2.247 0.025 0.858 0.573 HEPACAM 362 8143702 2.032 0.042 0.858 0.125 ZNF425 749 7905606 2.248 0.025 0.858 0.360 NPR1 363 8168274 2.032 0.042 0.858 0.088 GJB1 750 7946860 2.248 0.025 0.858 0.254 ABCC8 364 8021783 2.032 0.042 0.858 0.065 ZNF236 751 8153322 2.249 0.025 0.858 0.336 ARC 365 8103535 2.032 0.042 0.858 0.082 GK3P 752 7949080 2.251 0.024 0.858 0.347 NRXN2 366 8162533 2.032 0.042 0.858 0.218 PTCH1 753 8067602 2.252 0.024 0.858 0.481 NKAIN4 367 8131496 2.032 0.042 0.858 0.217 GLCCI1 754 7908812 2.252 0.024 0.858 0.515 GPR37L1 368 7924977 2.033 0.042 0.858 0.256 PGBD5 755 8139330 2.253 0.024 0.858 0.390 CAMK2B 369 8154953 2.033 0.042 0.858 0.105 KIAA1045 756 8064059 2.253 0.024 0.858 0.503 COL20A1 370 8021565 2.033 0.042 0.858 0.297 PHLPP1 757 7956120 2.253 0.024 0.858 0.571 ERBB3 371 8140356 2.035 0.042 0.858 0.289 HIP1 758 7922482 2.253 0.024 0.858 0.654 TNR 372 8093852 2.035 0.042 0.858 0.084 MSX1 759 8022666 2.254 0.024 0.858 0.876 CHST9 373 8093013 2.036 0.042 0.858 0.085 TNK2 760 7935930 2.254 0.024 0.858 0.496 KCNIP2 374 7980358 2.037 0.042 0.858 0.151 POMT2 761 7906205 2.254 0.024 0.858 0.757 BCAN 375 8024605 2.037 0.042 0.858 0.173 CELF5 762 7973403 2.255 0.024 0.858 0.503 CMTM5 376 8001547 2.037 0.042 0.858 0.330 PLLP 763 8022655 2.256 0.024 0.858 0.699 AQP4 377 8154012 2.038 0.042 0.858 0.180 KANK1 764 7984174 2.258 0.024 0.858 0.444 SNX22 378 8000799 2.039 0.041 0.858 0.175 GDPD3 765 8121152 2.258 0.024 0.858 0.615 FUT9 379 7938738 2.039 0.041 0.858 0.239 KCNC1 766 8099685 2.259 0.024 0.858 0.582 LGI2 380 8049075 2.039 0.041 0.858 0.110 B3GNT7 767 8170307 2.259 0.024 0.858 0.783 SLITRK2 381 8032509 2.041 0.041 0.858 0.388 GNG7 768 8098021 2.259 0.024 0.858 0.914 GRIA2 382 8147715 2.042 0.041 0.858 0.066 RNU6ATAC8P 769 7908508 2.259 0.024 0.858 0.352 CRB1 383 8108579 2.042 0.041 0.858 0.088 TMCO6 770 8114780 2.260 0.024 0.858 0.388 PCDH12 384 8065612 2.043 0.041 0.858 0.137 C20ORF112 771 7913385 2.261 0.024 0.858 0.369 RAP1GAP 385 8079058 2.043 0.041 0.858 0.188 RNU4-78P 772 8077833 2.262 0.024 0.858 0.483 SLC6A1 386 8085360 2.043 0.041 0.858 0.529 TIMP4 773 8099218 2.263 0.024 0.858 0.651 PPP2R2C 387 8129231 2.044 0.041 0.858 0.130 FAM184A 774 8014812 2.263 0.024 0.858 0.669 STAC2
65 Supplementary information - Meyer, Reimand et al.
775 8133904 2.265 0.024 0.858 0.830 GRM3
776 8027748 2.266 0.023 0.858 0.553 FXYD3
777 7964852 2.266 0.023 0.858 0.795 BEST3
66 Supplementary information - Meyer, Reimand et al. Table S6: pathway analysis of TMZ expression profile. Significantly enriched pathways and processes characteristic to the transcriptional profile of TMZ sensitivity in clone 482_9 of GBM-482 (FDR p<0.05). GO.ID Description FDR p-value Expression Genes
GO:0030205 dermatan sulfate metabolic process 0.0396 Increased BCAN,NCAN,CSPG5
GO:0030208 dermatan sulfate biosynthetic process 0.0306 Increased BCAN,NCAN,CSPG5
GO:0050654 chondroitin sulfate proteoglycan metabolic process 0.0136 Increased BCAN,XYLT1,CHST9,NCAN, CSPG5
GO:0030166 proteoglycan biosynthetic process 0.00615 Increased BCAN,XYLT1,CHST9,NCAN, CSPG5
GO:0030204 chondroitin sulfate metabolic process 0.0115 Increased BCAN,XYLT1,CHST9,NCAN, CSPG5
GO:0050650 chondroitin sulfate proteoglycan biosynthetic process 0.000253 Increased BCAN,XYLT1,CHST9,NCAN, CSPG5
GO:0030206 chondroitin sulfate biosynthetic process 0.000139 Increased BCAN,XYLT1,CHST9,NCAN, CSPG5
GO:0007270 neuron-neuron synaptic transmission 0.0185 Increased SLC6A1,GRIA2,TNR,GRIK3
GO:0050804 regulation of synaptic transmission 0.00666 Increased SLC6A1,GRM3,TNR,CAMK2 A,GRIK3,KCNJ10,SYP,CSP G5
GO:0050805 negative regulation of synaptic transmission 0.0223 Increased SLC6A1,TNR,GRIK3
GO:1990351 transporter complex 0.00735 Increased FXYD3,BEST3,GRIA2,ABCC 8,GRIK3,KCNIP2,CACNA1H ,TRPC5
GO:1902495 transmembrane transporter complex 0.00626 Increased FXYD3,BEST3,GRIA2,ABCC 8,GRIK3,KCNIP2,CACNA1H ,TRPC5
GO:0034702 ion channel complex 0.00298 Increased FXYD3,BEST3,GRIA2,ABCC 8,GRIK3,KCNIP2,CACNA1H ,TRPC5
GO:0008066 glutamate receptor activity 0.00821 Increased GRM3,GRIA2,GRIK3
GO:0022803 passive transmembrane transporter activity 0.0134 Increased FXYD3,AQP4,BEST3,GRIA2 ,GRIK3,KCNIP2,CACNA1H, KCNJ10,TRPC5
GO:0015267 channel activity 0.0134 Increased FXYD3,AQP4,BEST3,GRIA2 ,GRIK3,KCNIP2,CACNA1H, KCNJ10,TRPC5
GO:0022838 substrate-specific channel activity 0.00871 Increased FXYD3,AQP4,BEST3,GRIA2 ,GRIK3,KCNIP2,CACNA1H, KCNJ10,TRPC5
KEGG:04728 Dopaminergic synapse 0.00814 Increased PPP2R2C,GRIA2,CAMK2A, MAOB,GNAO1
KEGG:04724 Glutamatergic synapse 0.0118 Increased GRM3,GRIA2,GRIK3,GNAO 1,ADCY1
KEGG:05030 Cocaine addiction 0.0452 Increased GRM3,GRIA2,MAOB
KEGG:04713 Circadian entrainment 0.00524 Increased GRIA2,CAMK2A,CACNA1H, GNAO1,ADCY1
67 Supplementary information - Meyer, Reimand et al.
KEGG:04010 MAPK signaling pathway 0.0215 Decreased RASGRP3,PAK1,EGFR
KEGG:04014 Ras signaling pathway 0.00949 Decreased RASGRP3,PAK1,EGFR,MET
KEGG:05120 Epithelial cell signaling in Helicobacter pylori 0.00462 Decreased PAK1,EGFR,MET infection
68 Supplementary information - Meyer, Reimand et al.
Bibliography 1. Pollard SM, et al. (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4(6):568-580.
2. Sun Y, et al. (2008) Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Mol Cell Neurosci 38(2):245-258.
3. Esteller M, et al. (2000) Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343(19):1350-1354.
4. Suzuki T, et al. (2011) The correlation between promoter methylation status and the expression level of O6-methylguanine-DNA methyltransferase in recurrent glioma. Jpn J Clin Oncol 41(2):190-196.
5. Bayani J & Squire JA (2004) Preparation of cytogenetic specimens from tissue samples. Curr Protoc Cell Biol Chapter 22:Unit 22 22.
6. Singh SK, et al. (2004) Identification of human brain tumour initiating cells. Nature 432(7015):396-401.
7. Bengtsson H, Wirapati P, & Speed TP (2009) A single-array preprocessing method for estimating full-resolution raw copy numbers from all Affymetrix genotyping arrays including GenomeWideSNP 5 & 6. Bioinformatics 25(17):2149-2156.
8. Silversides CK, et al. (2012) Rare copy number variations in adults with tetralogy of Fallot implicate novel risk gene pathways. PLoS Genet 8(8):e1002843.
9. Olshen AB, Venkatraman ES, Lucito R, & Wigler M (2004) Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics 5(4):557-572.
10. Iafrate AJ, et al. (2004) Detection of large-scale variation in the human genome. Nat Genet 36(9):949-951.
11. Anonymous (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455(7216):1061-1068.
12. Krzywinski M, et al. (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19(9):1639-1645.
13. Ronquist F & Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12):1572-1574.
14. Smyth GK, Michaud J, & Scott HS (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21(9): 2067-2075.
69 Supplementary information - Meyer, Reimand et al.
15. Reimand J, Arak T, & Vilo J (2011) g:Profiler--a web server for functional interpretation of gene lists (2011 update). Nucleic acids research 39(Web Server issue):W307-315.
16. Mitelman F (2000) Recurrent chromosome aberrations in cancer. Mutation research 462(2-3):247-253.
17. Hahn WC & Weinberg RA (2002) Modelling the molecular circuitry of cancer. Nature reviews. Cancer 2(5):331-341.
18. Futreal PA, et al. (2004) A census of human cancer genes. Nature reviews. Cancer 4(3):177-183.
19. Vogelstein B & Kinzler KW (2004) Cancer genes and the pathways they control. Nature medicine 10(8):789-799.
20. Verhaak RG, et al. (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer cell 17(1):98-110.
21. Patel AP, et al. (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344(6190):1396-1401.
22. Ashburner M, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature genetics 25(1):25-29.
23. Croft D, et al. (2011) Reactome: a database of reactions, pathways and biological processes. Nucleic acids research 39(Database issue):D691-697.
24. Kanehisa M, Goto S, Furumichi M, Tanabe M, & Hirakawa M (2010) KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic acids research 38(Database issue):D355-360.
25. Ruepp A, et al. (2010) CORUM: the comprehensive resource of mammalian protein complexes--2009. Nucleic acids research 38(Database issue):D497-501.
26. Merico D, Isserlin R, Stueker O, Emili A, & Bader GD (2010) Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PloS one 5(11):e13984.
27. Cline MS, et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nature protocols 2(10):2366-2382.
70