Supporting Information
Pratilas et al. 10.1073/pnas.0900780106 SI Text HER2) xenografts. In each of these analyses, all noncontrol Determination of Sensitivity to MEK Inhibition. Drug sensitivity probes (n ϭ 22,215) were rank-ordered according to the mag- assays were performed by using a 96-well plate and the Alamar nitude of their difference in expression after MEK inhibition Blue reagent (Invitrogen) as reported (1). The cells were treated compared with DMSO-treated controls, as measured by SAM- with PD0325901 (Pfizer) in a range of concentrations from 0.1 derived d-scores. The positions of the output genes in this signed to 500 nM (Fig. S1). ranking were then scored in a manner similar to that described by the Connectivity Map (2). This empirical and nonparametric Enrichment Analysis Over a Time Course of MEK Inhibition. Af- Kolmogorov-Smirnov-based statistic reflects an enrichment fymetrix U133A 2.0 array analysis of V600EBRAF, SkMel-28 cells, score that ranges from ϩ1toϪ1, where a score of 0 is a lack of at 0, 2, 8, and 24 h after MEK inhibition was completed as enrichment in either direction of expression, or graphically, a described (see Experimental Procedures). Robust multiarray av- random distribution of the output profile genes. Those with erage (RMA) was used to estimate the expression of probe sets. enrichment scores nearer to 1 indicated increasing correlation Each time point from2hto24wascompared with 0 h, and in between the output profile and the rank-ordered gene set from each comparison, we considered only probe sets with an expres- the experimental comparison. sion in log2 space greater than the first quartile of expression on each array. Thus, either time 0 or the time point under study in Oncomine Concepts Map. We performed Oncomine Concepts each comparison had to contribute an expression value exceed- analysis (OCM; www.oncomine.org/ocm) (3, 4) on the 36 probe ing this threshold before a fold change difference was consid- sets not identified as elements of consensus ERK signaling ered. We then generated a fold change difference in expression pathways (as shown in Fig. 2), from the signature of MEK between the 2 experimental time points: 0 and 2, 0 and 8, 0 and inhibition in mutant BRAF cells. Affymetrix probe set identi- 24 h. Genes were annotated and exported if the absolute value fiers for each of the 36 genes were converted to HUGO gene of its fold change was Ͼ2. symbols and batch-loaded to OCM for analysis. Gene concepts Down-regulated, or underexpressed, probe sets (n ϭ 63, 173, sharing significant overlap with this 36-gene set were identified and 187 at time points 2, 8, and 24 h respectively) were grouped and included concepts defined by drug sensitivity, gene expres- as belonging to 1 of 9 monitored classes of gene function as sion, and annotation concepts (Fig. S6). annotated by Gene Ontology (GO) (Table S1). They were selected to reflect the direction of expression of whole classes of MIAME Checklist. Type of experiment. This study used microarray genes relative to the phenomenological activity expected upon expression analysis to identify global changes in transcript al- MEK inhibition. We included both 1 positive control and 2 teration in response to MEK inhibition. Genes under ERK negative control annotations. The former was the homogenous control were identified in a panel of V600EBRAF and RTK- MAP kinase phosphatase activity annotation, containing only activated tumor cells and xenografts, using short-term inhibition members of the DUSP family of phosphatases. The latter two, of ERK activity by the MEK inhibitor PD0325901 (Pfizer). DNA replication and ribosome biogenesis, represent secondary Experimental factors. Cell lines growing in culture (n ϭ 12) and and tertiary function considerably downstream of the primary murine xenografts (n ϭ 2) were treated with the MEK inhibitor expected signaling effect of MEK inhibition. Each gene function PD0325901 or vehicle alone as control. Cell lines were treated annotation was tested for statistically significant enrichment with 50 nM PD0325901 or 0.1% DMSO for 8 h. Mice were (one-sided Fisher exact test) against this down-regulated gene treated with a single oral dose of PD0325901 (25 mg/kg) or set derived at each time point compared with0h(Fig. S2). vehicle alone and killed 8 h later. We found that the positive control annotation of MAPK Number of hybridizations performed in the experiment. A total of 36 phosphatase activity and genes involved in the regulation of hybridizations on oligonucleotide arrays (Affymetrix signal transduction were most significantly enriched at2hwith HGU133A 2.0) were performed. the latter peaking at 8 h. Those genes with functional roles in Hybridization design. The Affymetrix single-color system was used. transcription factor activity, cell proliferation, and cell cycle Quality-control steps taken. Standard Affymetrix control recom- progression were maximally enriched at the 8-h time point, mendations were used. Quality assessment of cRNA was per- whereas genes responsible for cell-to-cell signaling and regula- formed on the RNA 6000 NanoAssay by using a Bioanalyzer tion of transcription from the polymerase II promoter were 2100 (Agilent). maximally enriched at 2 h. We found that genes involved in 2 Origin of the biological sample and its characteristics. Human cell lines processes related to the proliferation of all cells, DNA replica- (SkMel-1, SkMel-5, SkMel-19, SkMel-28) were provided by A. tion and ribosome biogenesis, were maximally enriched at 24 h. Houghton; MALME3M, Colo205, HT29, BT474, SkBr3, MDA- We therefore decided that this latter time point would likely MB-468, A431, and NCI-H1650 were obtained from ATCC. reflect largely secondary effects and not those directly affected Murine xenografts were established in athymic mice by using by suppression of ERK signaling. These data formed the justi- 5–10 ϫ 106 cells per mouse prepared in a 1:1 mixture of fication for investigating a panel of cell lines after an 8-h cells/Matrigel basement membrane and injected s.c. exposure to MEK inhibitor. Protocol for preparing the hybridization extract. Total cellular RNA was extracted from harvested cells or excised xenografts by using Rank Order Analysis. To further assess the significance of the 52 the Qiagen RNeasy extraction kit and methods provided by the genes identified as comprising the ERK output profile in mutant manufacturer. BRAF tumor cell lines, we determined their position in a list of Labeling protocols. Standard Affymetrix protocols (Affymetrix all genes rank-ordered by magnitude of change in expression GeneChip Expression Analysis) were used. after MEK inhibition, in 3 other systems treated with the MEK Protocol and conditions used during hybridization. Standard Af- inhibitor. These included the RTK cell lines (n ϭ 5) and fymetrix protocols (Affymetrix GeneChip Expression Analysis) SkMel-28 (V600EBRAF) and BT474 (WTBRAF, amplified were used.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 1of11 Type of scanning hardware and software used. Microarrays were cell line and xenograft data in both experimental conditions and scanned by using a high-numerical aperture and flying objective subsequently log base 2 transformed. The time-course analysis (FOL) lens in the GS300 scanner (Affymetrix) and quantified by using data were quantified and normalized with Robust Multiarray GeneChip Operating Software version 1.4 (GCOS; Affymetrix). Average (RMA). All statistical analyses were performed in R Type of image analysis software used. GeneChip Operating Software software. version 1.4 (GCOS; Affymetrix) was used. Array design. Platform type was Affymetrix oligonucleotide array. The complete output of the image analysis before data selection and Surface and coating specifications were glass. The arrays used in transformation (spot quantitation matrices). Original Affymetrix out- put files (.CEL files) were used. this study, Affymetrix HG U133Av2 are commercially available Data selection and transformation procedures. GeneChip Operating (Affymetrix). For features and reporters on the array see Software version 1.4 was used for signal quantification of both www.affymetrix.com.
1. Solit DB, et al. (2006) BRAF mutation predicts sensitivity to MEK inhibition. Nature 4. Rhodes DR, et al. (2007) Molecular concepts analysis links tumors, pathways, mecha- 439:358–362. nisms, and drugs. Neoplasia 9:443–454. 2.LambJ,etal. (2006) The Connectivity map: Using gene-expression signatures to 5. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and connect small molecules, genes, and disease. Science 313:1929–1935. powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300. 3. Tomlins SA, et al. (2007) Integrative molecular concept modeling of prostate cancer progression. Nat Genet 39:41–51.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 2of11 Fig. S1. Sensitivity of the panel of cell lines to MEK inhibition. The IC50 of the MEK inhibitor PD0325901 is shown for each of the cell lines used in the panel, segregated by BRAF mutation status: V600E (gray) or WT (blue).
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 3of11 Fig. S2. Enrichment of gene function over a time course of MEK inhibition of a V600EBRAF cell line. (A) Nine classes of genes are plotted by functional annotation related to downstream pathway activities. The gene content of each is derived from only those genes whose expression is down-regulated at the plotted time point (x axis) relative to 0 h with a fold change Ͼ2. Plotted on the y axis is the Ϫlog10 of the P value for the enrichment of each class at each time point (one-sided Fisher exact test). (B) For the selected time point (8 h), the aggregate expression of those genes in A are plotted in each functional annotation. Aggregate expression is the mean Z-score of expression.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 4of11 Fig. S3. Microarray analysis of RTK-activated tumor cells in response to MEK inhibition. (A) RTK activated tumor cells (BT474 and SKBr3, HER2 overexpressing; H1650, EGFR mutant; A431 and MDA468, EGFR overexpressing) growing in culture were treated with 50 nM PD0325901 or DMSO as control for 8 h. Immunoblots of cell lysates are shown, probed with antibodies against phosphorylated and total ERK 1/2. The samples used for these immunoblots were generated in duplicate with those used for microarray analysis. (B) Heat map representation of the 60-probe set (52-gene) profile derived from MEK inhibition of BRAF mutant tumor cells. The 60 probe sets are significantly altered (56 down, 4 up) in response to MEK inhibition in V600E BRAF tumor cells and xenografts (Left). In the RTK-activated tumor cells and xenografts, the basal expression of the same gene profile is lower, and their response to MEK inhibition is not significant (Right).
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 5of11 Fig. S4. (A) Inhibition of ERK activity in xenograft tumors treated with PD0325901. Ten mice bearing SkMel-28 (V600EBRAF) xenografts and 8 mice bearing BT474 (HER2 overexpressing) xenografts were randomized to receive 25 mg/kg PD0325901 or vehicle as a single oral dose. Excised tumors were analyzed by immunoblots, and the inhibition of phospho-ERK in MEK-inhibited tumors is shown. Tumor sections from SkMel-28 mice 2 and 5 (control) and 7 and 10 (treated) and from BT474 mice 1 and 4 (control) and 5 and 7 (treated) were used for RNA extraction and microarray analysis. (B) Immunoblot of protein products of selected output genes (SPRY, ETV, and cyclin D1) following oral exposure of MEK inhibitor in mice bearing SkMel-28 xenograft tumors.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 6of11 Fig. S5. Basal expression levels of genes in the MEK/ERK output profile between RTK-activated and V600EBRAF tumor cells. (A and B) Genes in the profile fall into 2 classes: those overexpressed in tumors with V600EBRAF (A) and those expressed at similar levels in V600EBRAF and RTK cells (but MEK-dependent only in the former) (B). Boxplots of microarray expression are shown for representative genes in each class, where the y axis represents normalized expression values. Significance of difference in expression between RTK and V600EBRAF cells was determined by using a two-tailed Student’s t test and subsequent multiple hypothesis correction with the Benjamini–Hochberg FDR procedure (5). Genes were considered differentially expressed if the FDR was Ͻ10%. Examples of genes in each class are listed. (C) The aggregate expression of the MEK/ERK output profile was calculated as described in Experimental Procedures as a single value for each cell line and xenograft sample and plotted on the x axis. V600EBRAF tumors are denoted in red, RTK tumors are in blue. SkMel-28 and BT474 xenografts are denoted by the x1 and x2 superscripts to indicate values for 2 separate tumors of each type.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 7of11 Fig. S6. Oncomine concept map. A network view of selected molecular concepts enriched for genes differentially expressed upon MEK inhibition of mutant BRAF cells. The 36 genes not otherwise implicated in ERK signaling (see Fig. 2) are used as the reference gene set and are represented by the central black node. Nodes included in the network are signatures of gene concepts sharing overlap with the reference gene set. The size of each concept (number of genes) is proportional to the diameter of the node, and node colors reflect the source of the concept as annotated in the legend. Edges (gray) reflect statistically significant enrichment between any 2 connected nodes (concepts or signatures), and edge weight increases with the increasing significance of enrichment (P Ͻ 0.01).
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 8of11 Table S1. Gene Ontology (GO) annotations monitored and enriched during time-course study of SkMel-28 response to MEK inhibition Identifier Type Term U133a2*
GO:0017017 MF MAP kinase phosphatase activity 9 GO:0009966 BP Regulation of signal transduction 11 GO:0007267 BP Cell-cell signaling 273 GO:0006357 BP Regulation of transcription from Pol II promoter 155 GO:0003700 MF Transcription factor activity 676 GO:0008283 BP Cell proliferation 242 GO:0007046 BP Ribosome biogenesis 15 GO:0006260 BP DNA replication 94 GO:0000074 BP Regulation of progression through cell cycle 197
BP, biological process; MF, molecular function. *Count of distinct genes represented by annotated probe sets.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 9of11 Table S2. MEK dependence and differential expression of genes in the V600EBRAF ERK output profile Expression ratio Symbol Gene name MEKi/ control* BRAF V600E/ WT† ProbeSetID DUSP6 dual specificity phosphatase 6 Ϫ124.31 9.59 208891at DUSP6 dual specificity phosphatase 6 Ϫ52.46 10.21 208893sat DUSP6 dual specificity phosphatase 6 Ϫ49.95 54.07 208892sat FOSL1 FOS-like antigen 1 Ϫ18.16 9.17 204420at SPRY2 sprouty homolog 2 (Drosophila) Ϫ15.47 9.96 204011at ETV5 ets variant gene 5 (ets-related molecule) Ϫ11.13 15.93 203349sat FOS v-fos FBJ murine osteosarcoma viral oncogene homolog Ϫ10.38 23.86 209189at IER3 immediate early response 3 Ϫ10.11 1.37 201631sat DUSP4 dual specificity phosphatase 4 Ϫ9.08 3.00 204014at LIF leukemia inhibitory factor (cholinergic differentiation factor) Ϫ9.07 6.74 205266at ETV5 ets variant gene 5 (ets-related molecule) Ϫ8.03 11.13 203348sat MAFF v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (avian) Ϫ6.55 3.92 36711at CCND1 cyclin D1 (PRAD1: parathyroid adenomatosis 1) Ϫ6.54 1.17 208711sat ETV1 ets variant gene 1 Ϫ6.03 11.21 221911at SPRY4 sprouty homolog 4 (Drosophila) Ϫ5.93 4.31 221489sat PYCRL pyrroline-5-carboxylate reductase-like Ϫ5.89 4.24 218944at TNC tenascin C (hexabrachion) Ϫ5.72 3.29 216005at IL8 interleukin 8 Ϫ5.52 8.37 202859xat MYC v-myc myelocytomatosis viral oncogene homolog (avian) Ϫ5.43 1.65 202431sat PLK3 polo-like kinase 3 (Drosophila) Ϫ4.87 2.27 204958at CCND1 cyclin D1 (PRAD1: parathyroid adenomatosis 1) Ϫ4.74 1.26 208712at PHLDA2 pleckstrin homology-like domain, family A, member 2 Ϫ4.54 1.23 209803sat ETV4 ets variant gene 4 (E1A enhancer binding protein, E1AF) Ϫ4.49 8.88 211603sat DUSP4 dual specificity phosphatase 4 Ϫ4.48 2.62 204015sat GPR3 G protein-coupled receptor 3 Ϫ4.19 6.68 214613at EGR1 early growth response 1 Ϫ4.18 1.04 201694sat LNK SH2B adaptor protein 3 Ϫ4.10 9.83 203320at SPRED2 sprouty-related, EVH1 domain containing 2 Ϫ3.72 2.33 212466at SPRED2 sprouty-related, EVH1 domain containing 2 Ϫ3.43 2.90 212458at RRS1 RRS1 ribosome biogenesis regulator homolog (S. cerevisiae) Ϫ3.42 1.74 209567at ETV5 ets variant gene 5 (ets-related molecule) Ϫ3.33 3.76 216375sat ARID5A AT rich interactive domain 5A (MRF1-like) Ϫ3.28 1.22 213138at B4GALT6 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 6 Ϫ3.16 5.75 206233at HMGA2 high mobility group AT-hook 2 Ϫ3.11 11.98 208025sat SLC4A7 solute carrier family 4, sodium bicarbonate cotransporter, member 7 Ϫ2.98 1.10 209884sat TNFRSF12A tumor necrosis factor receptor superfamily, member 12A Ϫ2.92 Ϫ2.15 218368sat MAP2K3 mitogen-activated protein kinase kinase 3 Ϫ2.75 2.28 215498sat ELOVL6 ELOVL family member 6, elongation of long chain fatty acids-like 6 Ϫ2.74 Ϫ1.65 204256at CD3EAP CD3E antigen, epsilon polypeptide associated protein Ϫ2.69 1.21 205264at CHSY1 carbohydrate (chondroitin) synthase 1 Ϫ2.52 1.32 203044at HSPC111 hypothetical protein HSPC111 Ϫ2.38 1.18 214011sat GEMIN4 gem (nuclear organelle) associated protein 4 Ϫ2.37 1.52 205527sat YRDC yrdC domain containing (E. coli) Ϫ2.37 1.65 218647sat BYSL bystin-like Ϫ2.35 1.67 203612at SLC1A5 solute carrier family 1 (neutral amino acid transporter), member 5 Ϫ2.35 Ϫ1.02 208916at PPAN peter pan homolog (Drosophila) Ϫ2.33 Ϫ1.09 221649sat POLR3G polymerase (RNA) III (DNA directed) polypeptide G (32kD) Ϫ2.30 3.94 206653at BXDC2 brix domain containing 2 Ϫ2.27 Ϫ1.46 219177at PPAT phosphoribosyl pyrophosphate amidotransferase Ϫ2.15 1.28 209434sat WDR3 WD repeat domain 3 Ϫ2.13 1.34 218882sat DDX21 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 Ϫ2.12 Ϫ1.12 208152sat FLJ10534 TSR1, 20S rRNA accumulation, homolog (S. cerevisiae) Ϫ2.11 1.95 218156sat GTPBP4 GTP binding protein 4 Ϫ2.10 1.06 218238at GNL3 guanine nucleotide binding protein-like 3 (nucleolar) Ϫ2.04 1.01 217850at POLR3G polymerase (RNA) III (DNA directed) polypeptide G (32kD) Ϫ2.00 3.95 206654sat POLR1C polymerase (RNA) I polypeptide C, 30kDa Ϫ1.96 Ϫ1.05 209317at KIR3DL2 killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 2 3.28 Ϫ3.22 207313xat SEMA6A semaphorin 6A 4.32 6.88 215028at HYDIN hydrocephalus inducing 4.38 Ϫ2.30 220098at ALF general transcription factor Iia, 1-like 6.49 Ϫ3.90 213413at
*Mean paired expression ratio between control and MEK-inhibited mutant BRAF cell lines. †Mean unpaired expression ratio by group, between mutant BRAF and WT BRAF/ RTK activated cell lines.
Pratilas et al. www.pnas.org/cgi/content/short/0900780106 10 of 11 Table S3. Selected molecular concepts enriched relative to genes differentially expressed upon MEK inhibition of mutant BRAF cells Concept type Concept Overlap† P value Odds ratio Content Connectivity map* Underexpressed in 505, MCF7 13 1.30E-09 13.29 POLR3G,CD3EAP,CHSY1,RRS1,GTPBP4, treated with wortmannin TNFRSF12A,HSPC111,PPAN,PYCRL, (0.00000001 M) BYSL,PHLDA2,YRDC,DDX21 Underexpressed in 55, PC3 treated 12 1.70E-08 11.66 WDR3,RRS1,GTPBP4,IL8,TNFRSF12A, with LY-294002 (0.00001 M) HSPC111,PPAT,BXDC2,PPAN,BYSL, DDX21,POLR1C Underexpressed in 506, MCF7 11 1.90E-07 10.19 POLR3G,RRS1,GTPBP4,TNFRSF12A, treated with LY-294002 HSPC111,PPAN,BYSL,PHLDA2,DDX21, (0.00001 M) B4GALT6,SLC4A7 Underexpressed in 506, MCF7 10 1.90E-06 8.85 WDR3,RRS1,GTPBP4,GNL3,HSPC111, treated with sirolimus PPAT,PPAN,PYCRL,PHLDA2, YRDC (0.0000001 M) Underexpressed in 35, HL60 8 1.20E-04 6.49 WDR3,GTPBP4,IL8,KIR3DL2,HSPC111, treated with LY-294002 BXDC2,PPAN,BYSL (0.00001 M) Underexpressed in 71, SKMEL5 7 1.20E-04 6.49 POLR3G,RRS1,LIF,TNFRSF12A,HSPC111, treated with LY-294002 BYSL,PHLDA2 (0.00001 M) GO biological process Ribosome biogenesis 3 1.50E-05 80.59 RRS1,GTPBP4,BXDC2 HPRD interaction sets FN1 3 0.003 12.19 GTPBP4,TNC,DDX21 KEGG pathway Purine metabolism 3 0.008 9.98 POLR3G,PPAT,POLR1C Literature-defined concepts Down-regulated in response to 10 7.70E-10 21.25 POLR3G,CD3EAP,RRS1,GTPBP4,HSPC111, hypoxia and to HIF-1 expression PPAT,SLC1A5,BYSL,ELOVL6,DDX21 Up-regulated genes in human 6 1.40E-09 73.04 WDR3,RRS1,GTPBP4,GNL3,HSPC111, B-cell line P493–6 expressing DDX21 c-Myc Up-regulated genes in human B 6 3.40E-09 62.12 WDR3,RRS1,GTPBP4,GNL3,HSPC111, cells upon Myc gene expression DDX22 Down-regulated in SKMEL-2 3 0.004 11.18 RRS1,SEMA6A,POLR1C melanoma cells overexpressing transcription factor E2F-1 Promoter binding site (Transfac) E2F-1 9 0.002 3.74 CD3EAP,CHSY1,GNL3,KIR3DL2,GEMIN4, PPAT,SLC1A5,BYSL, B4GALT6 Oncomine gene expression Oncogene transfected - top 5% 14 3.60E-09 11.02 WDR3,GTPBP4,GNL3,TNC,IL8,LIF, signatures underexpressed in E2F3‡ TNFRSF12A,HSPC111,SLC1A5,BYSL, YRDC,DDX21,SLC4A7,POLR1C Oncogene transfected - top 5% 13 4.40E-07 9.62 CD3EAP,GTPBP4,MAFF,GNL3,TNC,IL8, overexpressed in activated LIF,TNFRSF12A,PHLDA2,YRDC,HMGA2, H-Ras‡ DDX21,SLC4A7 Oncogene transfected - top 5% 11 1.90E-06 7.9 POLR3G,WDR3,GTPBP4,GNL3,HSPC111, overexpressed in c-Myc‡ PPAT,BXDC2,BYSL,YRDC,DDX21,POLR1C Prostate ETS status - top 20% 15 3.10E-04 3.73 POLR3G,WDR3,ALF,RRS1,GTPBP4,GNL3, overexpressed in ERG, ETV1, TNC,LIF,PPAT,BXDC2,PHLDA2,ELOVL6, ETV4 YRDC,DDX21,SLC4A7 Prostate cancer, ETS 7 0.005 4.05 WDR3,ALF,GTPBP4,HSPC111,PHLDA2, overexpression - top 10% ELOVL6,DDX21 overexpressed in ERG overexpression, ETV1 overexpression, ETV4 overexpression Melanoma culture - top 20% 13 0.005 2.78 POLR3G,CHSY1,RRS1,GTPBP4,GNL3, overexpressed in cutaneous LIF,TNFRSF12A,HSPC111,PPAT,BXDC2, melanoma, metastasic SLC1A5,PYCRL,POLR1C melanoma Melanoma type - top 1% 3 0.005 9.46 GNL3,PPAT,SLC4A7 overexpressed in lymph node metastasis, metastatic growth phase melanoma, metastatic melanoma culture, vertical growth phase melanoma Metastatic melanoma, BRAF 6 0.007 4.45 CD3EAP,ALF,GEMIN4,HSPC111,PHLDA2, mutation - top 10% B4GALT6 overexpressed in V599E
*Unless otherwise noted, top 5% of genes underexpressed at 6 h. †Count of 36 non-ERK-implicated genes enriched in each concept. ‡Human primary mammary epithelial cells.
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