* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Differential gene expression in ES/PNET with type 1 vs type 2 EWS
X-inactivation wikipedia , lookup
Gene desert wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Community fingerprinting wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Molecular evolution wikipedia , lookup
Transcription factor wikipedia , lookup
Genome evolution wikipedia , lookup
Expression vector wikipedia , lookup
Gene expression wikipedia , lookup
Secreted frizzled-related protein 1 wikipedia , lookup
Genomic imprinting wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Ridge (biology) wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Gene regulatory network wikipedia , lookup
Sarcomas with Aberrant Transcription Factors: Biology and Expression Profiling Marc Ladanyi Memorial Sloan-Kettering Cancer Center New York, NY, USA Translocation-associated sarcomas 1. General biological features and comparison to sarcomas with non-specific cytogenetic alterations 2. Insights from microarray-based expression profiling of translocation-associated sarcomas Pathologic genetic rearrangements in human cancers: a family tree Translocations Balanced Promoter Substitution Unbalanced Gene Fusion Chimeric Transcription Factor Deregulated gene expression Loss of Tumor Suppressor Chimeric Tyrosine Kinase Deregulated growth signaling Gain of Oncogene Molecular pathology of sarcomas: two major classes 1. Sarcomas with specific reciprocal translocations and relatively simple karyotypes • • • • approx. 1/3 of all sarcomas 15 different sarcoma types with over 25 different translocations fusion genes: aberrant chimeric transcription factors (most) or aberrant kinases (some) biology: transcriptional deregulation or aberrant signaling 2. Sarcomas with complex unbalanced karyotypes and no specific translocations • • approx. 2/3 of all sarcomas biology: genetic gains & losses, chromosomal instability, telomere dysfunction Molecular pathology of sarcomas: two major classes Sarcomas with specific translocations Sarcomas with non-specific genetic alterations Usually complex Karyotypes Usually simple Translocations Reciprocal & specific, producing fusion genes Telomere maintenance mechanisms Telomerase common, ALT mechanism rare Relatively rare, but strong More frequent, but limited Evidence of “alternative prognostic impact* or no prognostic impact lengthening Not observed Commonof telomeres” P53 pathway alterations Incidence in P53-mutant or knockout mice Non-reciprocal & nonspecific, causing gene copy number changes ALT mechanism more common than telomerase (ALT) telomere maintenance Common mechanism: Incidence in bilateral retinoblastoma and Li-Fraumeni syndrome Rare Global gene expression profiles Robust clustering Ewing sarcoma (0/30) vs osteosarcoma (38/60) Looser clustering (P<0.0001)* * MSKCC study: Genes Chromos Cancer 2004 Molecular pathology of sarcomas: two major classes Sarcomas with specific translocations Sarcomas with non-specific genetic alterations Usually complex Karyotypes Usually simple Translocations Reciprocal & specific, producing fusion genes Telomere maintenance mechanisms Telomerase common, ALT mechanism rare Relatively rare, but strong More frequent, but limited Evidence of “alternative prognostic impact* or no prognostic impact lengthening Not observed Commonof telomeres” P53 pathway alterations Incidence in P53-mutant or knockout mice Incidence in bilateral retinoblastoma and Li-Fraumeni syndrome Rare Global gene expression profiles Robust clustering Non-reciprocal & nonspecific, causing gene copy number changes ALT mechanism more common than telomerase (ALT) telomere maintenance Common mechanism: Translocation sarcomas (0/9) vs other sarcomas Looser clustering (7/9) (P=0.002)* * Hopkins study: AJP 2004 Molecular pathology of sarcomas: two major classes Sarcomas with specific translocations Usually simple Translocations Reciprocal & specific, producing fusion genes Telomere maintenance mechanisms Telomerase common, ALT mechanism rare Relatively rare, but strong prognostic impact Not observed Rare Global gene expression profiles Robust clustering Common No P53 mutation (n=52) 0.6 Incidence in bilateral retinoblastoma and Li-Fraumeni syndrome 0.8 Incidence in P53-mutant or knockout mice More frequent, but limited or no prognostic impact Common P53 in Ewing sarcoma* Looser clustering p<0.0001 * MSKCC study: JCO in press 0.0 0.2 0.4 P53 pathway alterations Non-reciprocal & nonspecific, causing gene copy number changes ALT mechanism more common than telomerase 1.0 Karyotypes Sarcomas with non-specific genetic alterations Usually complex 0 P53 mutated (n=8) 20 40 60 80 100 120 Molecular pathology of sarcomas: two major classes Sarcomas with specific translocations Karyotypes Usually simple Translocations Reciprocal & specific, producing fusion genes Telomere maintenance mechanisms Telomerase common, ALT mechanism rare Relatively rare, but strong prognostic impact Not observed P53 pathway alterations Incidence in P53-mutant or knockout mice Sarcomas with non-specific genetic alterations Usually complex Non-reciprocal & nonspecific, causing gene copy number changes ALT mechanism more common than telomerase More frequent, but limited or no prognostic impact Common Incidence in bilateral retinoblastoma and Li-Fraumeni syndrome Rare Common Global gene expression profiles Robust clustering Looser clustering Major fusion genes in sarcomas: biological overview Sarcoma type Translocation Fusion gene Transcriptional Deregulation Aberrant Signaling Ewing sarcoma t(11;22) EWS-FLI1 X t(21;22) EWS-ERG X Clear cell sarc. t(12;22) EWS-ATF1 X Myxoid LPS t(12;16) TLS-CHOP X Alveolar rhabdomyosarcoma t(2;13) PAX3-FKHR X t(1;13) PAX7-FKHR X DSRCT t(11;22) EWS-WT1 X Extr. myx. CS t(9;22) EWS-CHN X Synovial sarc. t(X;18) SYT-SSX1,2 X DFSP t(17;22) COL1A1-PDGFB X Cong. FS t(12;15) ETV6-NTRK3 X IMT t(2p23) ALK fusions X End. str. sarc. t(7;17) JAZF1-JJAZ1 X ASPS t(X;17) ASPL-TFE3 X Low grade MFS t(7;16) FUS-BBF2H7 X Pericytoma t(7;12) ACTB-GLI X Target Genes of Chimeric Transcription Factors in Sarcomas • Slow progress in identifying genuine biologically critical target genes of chimeric transcription factors – e.g. well-established targets: • EWS-FLI1 <15 • EWS-WT1, PAX3-FKHR <5 each – Low throughput gene-by-gene studies – False leads generated by assays using exogenous target promoters and/or heterologous cells – Most representative cellular background for inducible systems still subject of active investigation – Need for more alternative higher throughput approaches Target Genes of Chimeric Transcription Factors in Selected Sarcomas Tumor Fusion protein Type of DNA BD Direct or indirect targets Ewing sarcoma / EWS-FLI1 PNET EWS-ERG ETS TGFBR2 (), p57KIP2 (), MYC, PDGF-C, ID2, CCND1, UBE2C, IGFBP3() DSRCT EWS-WT1 Zn finger PDGF-A, IL-2/15R, TALLA1, BAIAP3 Alveolar RMS PAX3-FKHR PAX7-FKHR Paired box MET, CXCR4 Synovial Sarcoma SYT-SSX1 SYT-SSX2 (none) XRCC4, TLE1 Alveolar Soft Part Sarcoma ASPL-TFE3 bHLH-LZ CYP17A1, UPP1 Sarcomas with Aberrant Transcription Factors 1. General biological features and comparison to sarcomas with non-specific cytogenetic alterations 2. Insights from microarray-based expression profiling of translocation-associated sarcomas Expression Profiling of Sarcomas with Chimeric Transcription Factors Why is it interesting? 1. Translocation-associated sarcomas already have an objective molecular classification (detection of specific translocation) that can be used to evaluate unsupervised clustering based on expression profiles 2. Transcriptional deregulation is likely to be central to the pathogenesis of translocationassociated sarcomas with chimeric transcription factors Expression profiling of sarcomas with chimeric transcription factors • 137 tumor samples from MSKCC and U.Penn. (F. Barr) • + 4 xenografts + 12 cell lines = 153 total samples • Classification of all samples confirmed by fusion transcript RT-PCR Alveolar Rhabdomyosarcoma (ARMS) Desmoplastic Small Round Cell Tumor (DSRCT) Synovial Sarcoma (SS) 23 Ewing Sarcoma/PNET (ES) 38 Alveolar Soft Part Sarcoma (ASPS) 14 32 46 16 PAX3-FKHR 7 PAX7-FKHR 32 EWS-WT1 25 SYT-SSX1 21 SYT-SSX2 22 EWS-FLI1 type 1 11 EWS-FLI1 type 2 5 EWS-ERG 11 ASPL-TFE3 type 1 3 ASPL-TFE3 type 2 Gene expression data analysis • Hybridization to Affymetrix U133A GeneChip (22215 probe sets, 18500 transcripts, 14500 genes) • Unsupervised Hierarchical Clustering Raw data processing Clustering Number of genes MAS v5.0 Cluster/Treeview RMA method Pearson correlation Subsets All probe sets (22215) • Selection of differentially expressed genes Two-tailed t-tests; p<0.01 after Bonferroni correction Unsupervised hierarchical clustering n°1 MAS v5.0, Cluster/Treeview, different subsets of most variable probe sets 7200 probe sets ARMS SS ES DSRCT SS ES ASPS 3200 probe sets ASPS DSRCT ARMS ES SS ES SS 2200 probe sets DSRCT ASPS SS ARMS SS ES ARMS Variability of clustering results according to number of probe sets selected Unsupervised Hierarchical Clustering n°2 RMA method, Pearson correlation, all 22215 probe sets n=153 ES ASPS cell lines DSRCT ARMS ES SS Unsupervised hierarchical clustering n°2 RMA method, Pearson correlation, all 22215 probe sets ES/PNET orphan case n=137 tumors only ASPS ES SS DSRCT ARMS Unsupervised hierarchical clustering Multi-dimensional scaling analysis 3 different views of same data Ewing’s sarcoma Alveolar rhabdomyosarcoma Alveolar soft part sarcoma Synovial sarcoma Desmoplastic small round cell tumor n=137 Distribution of tumor types and numbers of differentially expressed genes by sarcoma type Samples Genes significantly over- or underexpressed* Subset with 2 fold overexpression* SS 46 6816 638 ARMS 23 1518 282 DSRCT 28 3163 554 ASPS 12 1590 531 ES/PNET 28 2157 294 * all significant at Bonferroni p<0.01 relative to 4 other sarcoma types Numbers of differentially expressed genes “inflated” by comparison to reference group of remaining four sarcomas, each with strong distinctive expression profile The gene expression profiles of translocation sarcomas contain many previously reported differentially expressed genes “Literature validation” Sarcoma Gene P-value Fold change ARMS MyoD1 10-18 12 FGFR4 10-11 7.7 Myogenin MIC2 10-9 10-18 4.5 2.8 MYC 10-17 5.2 CCND1 10-12 4.5 PRAME 10-39 5.6 TLE1 10-33 8.5 FZD1 10-24 8.4 ES SS • Differentially expressed genes in sarcomas with chimeric transcription factors – Target genes regulated by fusion protein (direct or indirect) – Genes reflecting pre-existing phenotype of host cell – Genes reflecting secondary genetic or epigenetic alterations Use of microarray data to predict classification of translocation sarcomas Multiple-class Prediction using Supervised Methods Support Vector Machine Prediction Rule 90% Training set 10% Validation set Selection of a subset of informative genes for each class Creation of a Prediction Model Predictor/Classifier Assess accuracy on the validation set 10 fold Cross validation Performance of microarray-based predictor 136/137 sarcomas were accurately predicted Model Prediction ARMS 23 0 0 0 0 Histological ASPS and Molecular DSRCT Diagnosis SS 0 12 0 0 0 0 0 28 0 0 0 0 0 46 0 ES 1 0 0 0 27 Similar results with other predictions methods Single misclassified case = orphan case in unsupervised clustering ES with EWS-FLI1 type 1 fusion + p16 deletion and no PAX-FKHR Expression Profiling of Sarcomas with Chimeric Transcription Factors – Global analyses / Microarray-based classifier • Translocation sarcomas are associated with very distinctive gene expression profiles – can be used to classify these sarcomas as accurately as translocation detection • Parameters for raw data processing and clustering can have strong effects on unsupervised analyses – Contribution of chimeric transcription factor target genes to specific expression profiles Contribution of chimeric transcription factor target genes to specific expression profiles of translocation sarcomas Approaches 1. Differential expression of known target genes 2. Cross-referencing of profiles from microarray experiments using inducible cell lines 3. Identification of candidates for target gene analyses based on expression profiling data from primary tumors The expression profiles of translocation sarcomas contain known target genes of their chimeric transcription factors ES: EWS-FLI1 targets Differential expression in ES/PNET (n=38 vs 115) P-value Fold change Proposed Target Gene Reported Effect of EWS-FLI1 MYC 10-17 5.2 ID2 10-9 3.2 CCND1 10-12 4.5 UBE2C 10-8 2.7 PDGFC 10-9 3.5 The expression profiles of translocation sarcomas contain known target genes of their chimeric transcription factors DSRCT: EWS-WT1 targets Proposed Target Gene Reported Effect of EWS-WT1 Differential expression in DSRCT (n=32 vs 121) P-value Fold change TALLA-1 10-15 5.5 PDGFA 10-14 2.7 IL2RB 10-13 4.6 BAIAP3 10-9 4 EWS-WT1 target genes defined in a heterologous cell line are over-represented among genes differentially expressed in DSRCTs • • • Induction of EWS-WT1 protein expression in U2OS human osteosarcoma cells with tetracycline-inducible EWS-WT1 construct Hybridized to U133A chips W. Gerald Lab, MSKCC • 102 genes demonstrated at least a 3 fold alteration in expression level at 24h following induction of EWS-WT1 – 22 down-regulated, 80 upregulated EWS-WT1 target genes defined in a heterologous cell line are over-represented among genes differentially expressed in DSRCTs U2OS cell experiment 80 genes upregulated at least 3 fold Expression profiles of DSRCT tumors 35 genes in common 553 genes that were 2 fold overexpressed in DSRCT relative to other translocation sarcomas • 17-fold enrichment for EWS-WT1 target genes among genes in the DSRCT expression profile (Chi-square p<0.0001) • include several previously validated EWS-WT1 targets: – BAIAP3, PDGFA, TALLA1, IL2RB EWS-WT1 target genes defined in a heterologous cell line are over-represented among genes differentially expressed in DSRCTs • 44% of genes upregulated by induction of EWS-WT1 in the U2OS human osteosarcoma cell line were also significantly overexpressed in DSRCTs • 6% of the DSRCT expression profile corresponds to genes induced by EWS-WT1 in the model system EWS-WT1 target genes defined in a heterologous cell line are over-represented among genes differentially expressed in DSRCTs • 44% of genes upregulated by induction of EWS-WT1 in the U2OS human osteosarcoma cell line were also significantly overexpressed in DSRCTs • 6% of the DSRCT expression profile corresponds to genes induced by EWS-WT1 in the model system • Comparison with similar data in Ewing’s sarcoma • Lessnick SL, Dacwag CS, Golub TR. Cancer Cell 1:393-401, 2002 • 46% of the EWS-FLI1-upregulated genes appeared in the ES/PNET expression profile obtained from primary tumors • 8% of the ES/PNET expression profile corresponded to genes induced by EWS-FLI1 in the model system Alveolar Soft Part Sarcoma Top 20 significantly overexpressed genes Fold change P-value Gene 121 10-11 CYP17A1 86 10-24 INHBE 75 10-9 MIBP 62 10-37 GPNMB 52 10-28 GOS2 51 10-34 Hs.57548 49 10-9 DEFB1 47 10-11 SULT1C1 47 10-10 UPP1 43 10-11 SULT1C1 43 10-15 SV2B 42 10-9 GPR56 41 10-7 NTSR2 40 10-19 Hs.37189 36 10-7 AGXT2L1 30 10-14 CLI 29 10-16 CLI 27 10-2 3 PTDGS 25 10-29 PTDGS 24 10-39 PTDGS Can we use this list to identify new target genes of sarcoma fusion proteins? Ranked by fold-change Makoto Nagai, MD PhD Alveolar Soft Part Sarcoma Top 20 significantly overexpressed genes Fold change P-value Gene 121 10-11 CYP17A1 86 10-24 INHBE 75 10-9 MIBP 62 10-37 GPNMB 52 10-28 GOS2 51 10-34 Hs.57548 45000 49 10-9 DEFB1 47 10-11 SULT1C1 40000 35000 47 10-10 UPP1 43 10-11 SULT1C1 20000 15000 43 10-15 SV2B 42 10-9 GPR56 10000 5000 41 10-7 NTSR2 40 10-19 Hs.37189 36 10-7 AGXT2L1 30 10-14 CLI 29 10-16 CLI 27 10-2 3 PTDGS 25 10-29 PTDGS 24 10-39 PTDGS Cytochrome P450 subfamily 17A1 (CYP17A1) 30000 25000 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 Ranked by fold-change Identification of CYP17A1 as a direct target of ASPL-TFE3 based on its strong differential overexpression in the expression profile of ASPS • Promoter region of CYP17A1 contains 3 potential TFE3 sites • CYP17A1 promoter strongly activated by ASPL-TFE3, but not native TFE3 • Strong physical binding of ASPLTFE3 to sites #1 and #3 by EMSA • In vivo presence of ASPL-TFE3 at CYP17A1 promoter in model cell line (293) by chromatin IP • Induction of ASPL-TFE3 in model cell line (293) results in upregulation of endogenous CYP17A1 by real-time Q-RT-PCR Alveolar Soft Part Sarcoma Top 20 significantly overexpressed genes Fold change P-value Gene 121 10-11 CYP17A1 86 10-24 INHBE 75 10-9 MIBP 62 10-37 GPNMB 52 10-28 GOS2 51 10-34 Hs.57548 49 10-9 DEFB1 47 10-11 SULT1C1 47 10-10 UPP1 43 10-11 SULT1C1 43 10-15 SV2B 42 10-9 GPR56 41 10-7 NTSR2 40 10-19 Hs.37189 36 10-7 AGXT2L1 30 10-14 CLI 29 10-16 CLI 27 10-2 3 PTDGS 25 10-29 PTDGS 24 10-39 PTDGS Can we do this with another gene from the list? Ranked by fold-change Alveolar Soft Part Sarcoma Top 20 significantly overexpressed genes Fold change P-value Gene 121 10-11 CYP17A1 86 10-24 INHBE 75 10-9 MIBP 62 10-37 GPNMB 52 10-28 GOS2 51 10-34 Hs.57548 49 10-9 DEFB1 47 10-11 SULT1C1 47 10-10 UPP1 43 10-11 SULT1C1 43 10-15 SV2B 42 10-9 GPR56 41 10-7 NTSR2 40 10-19 Hs.37189 36 10-7 AGXT2L1 30 10-14 CLI 29 10-16 CLI 27 10-2 3 PTDGS 25 10-29 PTDGS 24 10-39 PTDGS 203234_at gb:NM_003364.1 Uridine Uridine Phosphorylase (UPP1) phosphorylase (UP) 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 Ranked by fold-change Identification of UPP1 as a direct target of ASPL-TFE3 based on its strong differential overexpression in the expression profile of ASPS • UPP1 promoter more strongly activated by ASPL-TFE3 than by native TFE3 • Induction of ASPL-TFE3 in model cell line (293) results in upregulation of endogenous UPP1, as measured by real-time quantitative RT-PCR Identification of UPP1 as a direct target of ASPL-TFE3 based on its strong differential overexpression in the expression profile of ASPS • UPP1 promoter more strongly activated by ASPL-TFE3 than by native TFE3 • Induction of ASPL-TFE3 in model cell line (293) results in upregulation of endogenous UPP1, as measured by real-time quantitative RT-PCR Identification of UPP1 as a direct target of ASPLTFE3 based on its strong differential overexpression in the expression profile of ASPS • UPP1 promoter more strongly activated by ASPL-TFE3 than by native TFE3 • Induction of ASPL-TFE3 in model cell line (293) results in upregulation of endogenous UPP1, as measured by real-time quantitative RT-PCR • Potential therapeutic interest of uridine phosphorylase – converts the pyrimidine analogue, 5'-deoxy5'fluorouridine, to 5-FU – allows administration of 5'deoxy-5'fluorouridine as a prodrug with low toxicity to non-neoplastic cells expressing only basal levels of uridine phosphorylase Identification of potential target genes of SYT-SSX based on strong differential overexpression in the expression profile of synovial sarcoma Top 5 ranked by p-value Probe set Fold T test p-value Gene Symbol 203221_at 10.9 2.68E-43 TLE1 205031_at 6.1 4.54E-40 EFNB3 206315_at 14.0 8.98E-38 CRLF1 204086_at 11.2 1.82E-37 PRAME 58780_s_at 3.2 6.66E-36 FLJ10357 203131_at 14.5 6.87E-36 PDGFRA 221004_s_at 5.1 1.17E-35 ITM2C 213479_at 28.2 1.61E-35 NPTX2 202575_at 7.4 1.27E-33 CRABP2 220326_s_at 2.6 1.72E-33 FLJ10357 204724_s_at 18.4 1.38E-32 COL9A3 209806_at 4.7 3.71E-32 HIST1H2BK 218502_s_at 15.3 1.97E-31 TRPS1 203222_s_at 8.2 3.56E-31 TLE1 210497_x_at 5.2 5.40E-31 SSX2 204735_at 3.8 5.56E-31 PDE4A 217957_at 2.4 1.06E-30 GTL3 214761_at 4.2 1.61E-30 OAZ 212599_at 6.8 2.27E-30 AUTS2 221447_s_at 6.1 2.67E-30 LOC83468 213194_at 5.4 2.95E-30 FLJ10539 Tsuyoshi Saito, 218397_at 2.7 3.23E-30 PHF9M.D. Ph.D. 213698_at 3.1 3.66E-30 MGC14276 200975_at 2.2 4.52E-30 PPT1 203180_at 10.7 1.45E-29 ALDH1A3 See Pathology Poster # 5 (not in printed program) TLE1 (transducin-like enhancer of split 1) a transcriptional repressor of Wnt/-catenin signaling Expression Profiling of Sarcomas with Chimeric Transcription Factors – Global analyses / Microarray-based classifier – Contribution of chimeric transcription factor target genes to specific expression profiles • Significant subsets of genes in specific expression profiles may be chimeric transcription factor target genes • Specific expression profiles can be used to identify new candidates for target gene analyses Kinases as therapeutic targets in sarcomas with chimeric transcription factors • kinases or signaling pathways directly activated by the specific aberrant transcription factor Tumor Fusion protein Signaling proteins confirmed or proposed to be upregulated Ewing sarcoma / PNET EWS-FLI1 EWS-ERG PIM3 Alveolar RMS PAX3-FKHR PAX7-FKHR MET Desmoplastic small round cell tumor EWS-WT1 PDGF-A, FGFR4 Kinases as therapeutic targets in sarcomas with chimeric transcription factors • kinases or signaling pathways directly activated by the specific aberrant transcription factor • kinases or signaling pathways overexpressed in specific sarcomas apparently unrelated to direct action of aberrant transcription factor • ERBB2/Her2/neu in synovial sarcoma • EGFR in synovial sarcoma • KIT in Ewing’s sarcoma • kinases activated by mutations as secondary or cooperating events in sarcomas with aberrant transcription factors (like FLT3 mutations in leukemias) • none identified so far • chimeric transcription factors regulated by phosphorylation • EWS-WT1 Expression profiling of sarcomas with aberrant transcription factors and related studies MSKCC • Ladanyi Lab – – – – – – • Tsuyoshi Saito Makoto Nagai Marick Laé Violetta Barbashina Man Yee Lui Zhiquan Zhao W. Gerald Lab – William Gerald – Lishi Chen • – Pete Argani • U. Pennsylvania – Fred Barr Biostatistics – Adam Olshen – Shannon Chuai • • Johns Hopkins • U. Michigan – Larry Baker – Dafydd Thomas Other MSKCC collaborators – Cristina Antonescu – John Healey, Murray Brennan, Sam Singer – Paul Meyers, Len Wexler, Robert Maki