Genomic Approaches to Translocation Sarcomas - · PDF fileGenomic Approaches to Translocation...

Genomic Approaches to Translocation Sarcomas

Marc Ladanyi

Memorial Sloan-Kettering Cancer CenterNew York, NY, USA

Molecular pathology of sarcomas: two major classes

1. Sarcomas with specific reciprocal translocations and relatively simple karyotypes

2. Sarcomas with complex unbalanced karyotypes

and no specific translocations

Less difficultVery difficultMouse models

Looser clusteringRobust clusteringGlobal gene expression profiles

CommonRareIncidence in bilateral retinoblastoma and Li-Fraumeni syndrome

CommonNot observedIncidence in P53-mutant or knockout mice

More frequent, but limited or no prognostic impact

Relatively rare, but strong prognostic impact

P53 pathway alterations

ALT mechanism more common than telomerase

Telomerase common, ALT mechanism rare

Telomere maintenance mechanisms

Non-reciprocal & non-specific, causing gene copy number changes

Reciprocal & specific, producing fusion genes

Translocations

Usually complexUsually simpleKaryotypes

Sarcomas without specific translocations

Sarcomas with specific translocations

Molecular pathology of sarcomas: two major classes

Samuel SingerChris SanderMarc LadanyiCristina AntonescuRobert Maki

MSKCC BROAD Matt MeyersonJordi BarretinaHeidi Greulich Stacey GabrielWendy Winckler

MSKCC & MIT-BROAD

Computational analysis

Targeted functional validation

Samples

RNA and DNA extraction

U133A Affy expression arrays

MicroRNA profiling

250K Affy SNP arrays for copy number and LOH analysis

Sequencing of 225 genes and 450 microRNA genes

Sequenom validation of candidate mutations

Sarcoma Genome Project

Gene selection

Samples run on Affy 250K SNP arrays (as of October 2007)

50complex, 12q rings + giant markers

Dedifferentiated liposarcoma

207TOTAL

27complexLeiomyosarcoma

25simple, t(X;18)Synovial sarcoma

22simple, monosomy 14Gastrointestinal stromal tumor

38complexMFH

24complexPleomorphic liposarcoma

21simple, t(12;16)Myxoid/round cell liposarcoma

TotalKaryotypeSubtype

… + matched normals (205)

Paired Affy U133A expression data for 141 samples

DedifferentiatedLiposarcoma

PleomorphicLiposarcoma

MFH

Round cellLiposarcoma

SynovialSarcoma

GIST

Affy 250K SNP array data, MSKCC analysis

Sarcoma Genome Project: Global view of copy number data

Many genomic copy number changes →strong effect on gene expression profiles

Few genomic copy number changes →weak effect on gene expression profiles

Genomic Approaches to Sarcomas

• Complex karyotype sarcomas will require integration of genomic copy number data with expression profiling data– Expression profiles contain many “passenger genes”

from regions of gains or losses• Translocation sarcomas: expression profiling data

alone more likely to be informative– Reflect lineage + effect of aberrant transcription factor– Probably less biological “noise” than expression profiles

of complex karyotype sarcomas


Agenda

Expression profiling: mining for diagnostic and therapeutic targets

• ChIP-on-chip: integration with expression profiling data to identify target genes of chimeric transcription factors in translocation sarcomas

• Cancer genomics: new approaches to high throughput unbiased discovery of translocations

EWS-ATF1t(12;22)

EWS-CREB1t(2;22)

ACTB-GLIt(7;12)Pericytoma

FUS-BBF2H7t(7;16)Low grade fibromyxoid sarcoma

EWS-TAF2Nt(9;17)EWS-NR4A3t(9;22)Extraskeletal myxoid

chondrosarcoma

FUS-CHOPt(12;16)Myxoid liposarcoma

JAZF1-JJAZ1t(7;17) Endometrial stromal sarcoma

EWS-CREB1t(2;22)Angiomatoid fibrous histiocytoma

ASPL-TFE3t(X;17)Alveolar soft part sarcoma

SYT-SSX1,2t(X;18)Synovial sarcoma

EWS-WT1t(11;22)DSRCTPAX7-FKHRt(1;13)PAX3-FKHRt(2;13)Alveolar

rhabdomyosarcoma

EWS-ATF1t(12;22)Clear cell sarcomaEWS-ERGt(21;22)EWS-FLI1t(11;22)Ewing sarcomaFusion gene Translocation Sarcoma type

Most fusion genes resulting from sarcoma translocations encode aberrant transcription factors

Expression profiling dataset on sarcomas with chimeric transcription factors

* all significant at Bonferroni p<0.01 relative to 4 other sarcoma types

294215728ES/PNET

531159012ASPS

554316328DSRCT

282151823ARMS

638681646SS

Subset with≥ 2 fold over-expression*

Genes significantly

over- or under-expressed*

Samples

11 ASPL-TFE3 type 13 ASPL-TFE3 type 2

14Alveolar Soft Part Sarcoma (ASPS)

22 EWS-FLI1 type 111 EWS-FLI1 type 2

5 EWS-ERG

38Ewing Sarcoma/PNET (ES)

25 SYT-SSX121 SYT-SSX2

46Synovial Sarcoma (SS)

32 EWS-WT132Desmoplastic Small Round Cell Tumor (DSRCT)

16 PAX3-FKHR7 PAX7-FKHR

23Alveolar Rhabdo-myosarcoma (ARMS)

• Chip: Affymetrix U133A• Data processing: RMA method• Clustering: Pearson correlation• Data used: all 22215 probe sets• n=137 tumors from MSKCC and U.Penn. (F. Barr)• + 4 xenografts + 12 cell lines = 153 total samples

ASPS ES SS DSRCT ARMS

M. Laé, T. Saito, et al., Ladanyi Lab

Mining expression profiles of translocation sarcomas for diagnostic markers

• ApoD in DFSP (van de Rijn group, 2004)• TLE1 in synovial sarcoma (Terry et al., 2007)

ARMS >> ERMS genes: overlap between 3 microarray studies

Mining expression profiles of translocation sarcomas for diagnostic markers

• ApoD in DFSP (van de Rijn group)• TLE1 in synovial sarcoma • TFAP2-β in ARMS vs ERMS

OLIG2 KCNN3

CNR1ELA1ASSPGBD5 ABAT

Wachtel et al. study MSKCC study

Davicioni et al. study

NELL1NHLH1ADRA2CALKNRN1KIAA0746 C16orf45

GARNL4 JAKMIP2 TSC22D2

TNRC9 DCX SYN2

PIPOX TFAP2BPODXL FOXF1KIAA0523DKFZp547P042DKFZp762M127

ELA1

Wachtel et al. JCO 2006

Mining expression profiles of translocation sarcomas for therapeutic targets

• Kinase genes– Family of genes involved in signaling; rich in drug targets

• Approach: – Identified 739 probe sets on U133A chip corresponding to 432

genes with kinase domains based on searches of the Affymetrix annotation database, published data and genome databases

• 83% of the 518 known protein kinase genes– Performed unsupervised clustering and supervised comparisons

focused on these 739 probe sets

Unsupervised clustering based on kinase genesTranslocation sarcomas express distinctive repertoires of kinases

→ 739 probe sets on U133A chip representing 432 kinase genes

ES ARMS DSRCT SS ASPS

Baird et al., 2005 (Meltzer group)

3.0E-103.5EPHA4

1.0E-174.2VRK1

1.0E-134.9JAK1

7.6E-105.4KIT

5.5E-105.9DAPK1

2.1E-1940.3PRKCB1

T test p-value

Fold Kinase Gene

Ewing Sarcoma/PNETDifferentially overexpressed

kinases

7.5E-243.9FGFR1

3.2E-084.7NTRK2

3.0E-268.3FGFR3

6.9E-3614.5PDGFRA

T test p-value

Fold Kinase Gene

Synovial SarcomaDifferentially overexpressed

kinases

1.0E-082.6DAPK1

1.6E-094.2ERBB3

3.4E-116.0MET

4.9E-128.0FGFR4

5.0E-3055.9TTN

T test p-value

Fold Kinase Gene

Alveolar Rhabdomyosarcoma

Differentially overexpressed kinases

Supervised comparisons based on kinase genesTranslocation sarcomas express distinctive repertoires of kinases

Differentially expressed kinases in translocation sarcomas

• Basis: – Lineage-associated (e.g. like KIT in GIST)– Oncogenic role (e.g. like KIT in GIST; like EGFR in

EGFR-mutant lung adenoCA)– Transcriptional targets of chimeric transcription factor

• Pathogenic activation: – Aberrant overexpression due to transcriptional

upregulation– Amplification (unlikely) – Mutation

3.0E-103.5EPHA4

1.0E-174.2VRK1

1.0E-134.9JAK1

7.6E-105.4KIT

5.5E-105.9DAPK1

2.1E-1940.3PRKCB1

T test p-value

Fold Kinase Gene

Ewing Sarcoma/PNETDifferentially overexpressed

kinases

7.5E-243.9FGFR1

3.2E-084.7NTRK2

3.0E-268.3FGFR3

6.9E-3614.5PDGFRA

T test p-value

Fold Kinase Gene

Synovial SarcomaDifferentially overexpressed

kinases

1.0E-082.6DAPK1

1.6E-094.2ERBB3

3.4E-116.0MET

4.9E-128.0FGFR4

5.0E-3055.9TTN

T test p-value

Fold Kinase Gene

Alveolar Rhabdomyosarcoma

Differentially overexpressed kinases

Sequencing of kinases showing relative overexpression in specific translocation sarcomas

• Mutation screens -> essentially negative– PRKCB1: 0/28 ES/PNET– JAK1: 0/31 ES/PNET– EPHA4: 1/68 ES/PNET– PDGFRA: 0/27 SS (1 reported)– FGFR3: 0/30 SS– Etc…

Neal Shukla, M.D.

Differentially expressed kinases in translocation sarcomas

• Basis: – Lineage-associated (e.g. like KIT in GIST)– Oncogenic role (e.g. like KIT in GIST; like EGFR in

EGFR-mut lung CA)– Transcriptional targets of chimeric transcription factor

• Pathogenic activation: – Aberrant overexpression due to transcriptional

upregulation– Amplification (unlikely) – Mutation

Kinase Fold T-test p-valuePIM1 3.1 6.42E-10MAPK3 1.6 1.28E-09MET 4.9 2.91E-09H11 6.4 9.73E-09MAPKAPK2 1.6 1.68E-08PRKAG2 8.7 2.74E-08MKNK2 2.6 3.16E-08GPRK6 8.1 4.31E-08LYN 2.3 5.69E-08PACSIN2 2.1 1.02E-07FLT1 3.9 8.99E-07MAPKAP1 1.8 2.13E-06

MET is a highly differentially overexpressed kinase in ASPS

Comparison of 16 ASPS samples vs 123 samples of 4 other sarcomas

Masumi Tsuda, Ph.D.

Alveolar Soft Part Sarcoma(ASPS)

soft tissue

ASPL-TFE3 fusion gene in Alveolar Soft Part Sarcoma

der(17)t(X;17)(p11;q25)

ASPL-TFE3 fusion arising fromt(X;17)(q25.3; p11.2)

– Rare soft tissue sarcoma– First described in 1952 (here)– F:M = 3:2, age range: 15-35– highly chemotherapy and

radiation therapy resistant– site: within or next to skeletal

muscle– typical presentation: thigh mass

ASPL- 17q25- ubiquitously expressed cytoplasmic

protein involved in processing of glucose transporters

TFE3- Xp11.2- bHLH-LZ family transcription factor

highly related to MITF and TFEB- contains DNA binding domain and NLS- Consensus sequence CANNTG

ASPL-TFE3- replaces the N-terminal portion of TFE3 by the fused ASPL sequences- retains DNA binding domain and NLS, functions as transcription factor- Type 2 retains activating domain as well- more active than TFE3 in model assays

ASPL-TFE3 type1

1

1

311 591

311 626AD

ASPL1 553

311UBX

TFE3 AD bHLH-LZ

1 575

296261

bHLH-LZ

bHLH-LZ

ASPL-TFE3 type2

12NLS

NLS

NLS

ASPL-TFE3 fusion geneChimeric Transcription Factor

ASPL-TFE3: a gene fusion found in both a sarcoma and a carcinoma

Cellular background

Kidney Soft tissue

ASPL-TFE3 Renal Carcinoma Alveolar Soft Part SarcomaOnly existing human cell line with endogenous ASPL-TFE3 translocation is from an ASPL-TFE3 renal carcinoma; there is no ASPS cell line available.

TFE3 gene fusions in human tumors

t(X;17)(p11;q23)14Renal CACLTC-TFE3

inv(X)(p11.2;q12)39Renal CANonO-TFE3

t(6;11)(p21;q13)10-42Renal CAAlpha-TFEB

t(X;1)(p11.2;p34)5-68Renal CAPSF-TFE3

t(X;1)(p11.2;q12)2-70Renal CAPRCC-TFE3

t(X;17)(p11;q25)2-68Renal CAASPL-TFE3

der(17)t(X;17)(p11;q25)5-40ASPSASPL-TFE3

TranslocationAgeTumorFusion

“Translocation carcinomas of the kidney”

02000400060008000

1000012000140001600018000

basic Met-pro

Fold

act

ivat

ion

MOCKTFE3ASPL-TFE3 type1ASPL-TFE3 type2

P<0.01

P<0.01

ASPL-TFE3 activates exogenous MET promoter construct

Masumi Tsuda, Ph.D.

Reporter co-transfection and transactivation assay

nega

. con

.

posi

. con

.

inpu

t

IgG

anti-

myc

inpu

t

IgG

anti-

myc

inpu

t

IgG

anti-

myc

MOCKASPL-TFE3

type1ASPL-TFE3

type2

100 bp

100 bp

Tet 0 h

Tet 46 h

ASPL-TFE3 is present at the MET promoter

・Chromatin Immunoprecipitation assay (293 cells with inducible ASPL-TFE3)

-2000 bp -200bp-1340bp

-1310 ↓

Transcription Start Site

CANNTG(consensus binding

sequences for native TFE3 and ASPL-TFE3)

Time after induction (hrs)

0.51

1.5

2

2.5

33.5

4

0 12 24 36 48

MOCKASPS1ASPS2

Normalized MET expression

IB: anti-myc

64 Ktype2type1

0 48Tet 2412 0 482412 0 48 (h)2412

MOCK293 cells with

ASPL-TFE3 type 1

ASPL-TFE3 activates endogenous MET promoter

(ASPL-TFE3 inducible 293 cells with T-Rex system)

293 cells with ASPL-TFE3 type 2

Membrane

ASPL-TFE3 Met

Transcription cell growthadhesionmotilityinvasion

ASPS and renal carcinomas with TFE3 fusions

AutophosphorylationCytoplasm

Nucleus

p

MET

HGF

Gab1CrkRASc-Raf

MEK1/2

MAPK/Erk1/2

p

pp

p

p p

MYCp p

PD98059U0126

p

MET

MetMet

genes

p

p pStat3

PI3-k p

Aktp

LY294002PHA665752 (Pfizer)

Oncogenic TFE3 fusion proteins activate MET signaling by transcriptional upregulation

p?

Tsuda M, Davis IJ, Argani P, Shukla N, McGill GG, Nagai M, Saito T, Laé M, Fisher DE, Ladanyi M. Cancer Res 67:919-929, 2007.

•ASPL-TFE3 stimulates MET kinase activity and downstream signaling

FU-U

R1RH3

0A6

73HS-

SY-II

Fuji

IB: Met

IB: actin

00.20.40.60.8

11.2

0 0.05 0.1 0.2 0.5 1 10 20 50

% v

iabi

lity

(µM)

PHA665752

PHA665752

FU-UR1RH30A673HS-SY-IIFuji

Effects of MET inhibitor PHA665752 on cell viability

0

0.2

0.4

0.6

0.8

1

1.2

0 10 20 50 100

PD98059 (MEK inhib.)

(µM)LY294002 (PI3K inhib)

00

0.2

0.4

0.6

0.8

1

1.2

10 20 50 100(µM)

Other sarcomas ASPL-TFE3 RCC line (no ASPS line available)

#1 #2 #2-2

#9 #12

#15

#18

#18-

2

ASPS

#14

#17

#20

#31

#31-

2

ASPL-TFE3

Met

#21

#28

#28

#29

PRCC-TFE3

IP: Met, IB: p-Met(Y1234/1235)

140 K

140 K

HGF69 K

Renal tumors

ASPS ASPL-TFE3 RCC

MET

Frequent co-expression of MET and HGF

and expression of phospho-MET in tumors with TFE3 fusions

HGF

Oncogenic activation of MET signaling• Kinase domain missense mutations (activating)

– Familial and sporadic (15%) papillary renal cell carcinomas– rare in other cancers

• MET fusion protein (constitutively activated)– TPR-MET (only in carcinogen-treated cell line MNNG-HOS)

• Autocrine signaling – MET and HGF co-expression

• MET amplification – Gastric cancer (10-20%)– EGFR-mut lung adenocarcinomas resistant to EGFR TKIs

• Aberrant transcriptional upregulation of MET– by amplified MITF in some melanomas (MET is a direct target of MITF)– by TFE3 fusion proteins in ASPS and renal carcinomas with TFE3

fusions

• Translocation sarcomas are usually highly dependent on their specific CTF• Deregulation of gene expression by these CTFs is a major part of the

biology of these sarcomas • Transcription factors = poor therapeutic targets

– Need to identify critical downstream transcriptional targets for which drugs exist or are in development

• Slow progress in identifying genuine biologically critical CTF target genes• Approach: obtain a more comprehensive view of genuine in vivo direct

targets of CTFs through the integration of ChIP-on-chip data with:– expression profiling data from tumors– expression profiling data from homologous or heterologous cell lines in which

CTF levels are modulated

Target Genes of Chimeric Transcription Factors (CTFs) in Sarcomas


Agenda

• Expression profiling: mining for diagnostic and therapeutic targetsChIP-on-chip: integration with expression profiling data to identify target genes of chimeric transcription factors in translocation sarcomas

• Cancer genomics: new approaches to high throughput unbiased discovery of translocations

LM-PCR

Cells expressing transcription

factor of interest

Antibody to transcription

factor of interest

Cy3 label Cy5 label

Crosslink, Sonicate

Hybridize to Microarray

ChiP on CHIP

Ligation-mediated PCR

Chromatin immunoprecipitation combined with DNA promoter chip hybridization for genome-wide analysis of transcription factor localization

Size of DNA fragments after LM-PCR: 200 - 700bp

Enrichment of bound DNA vs. total DNA by chromatin IP

Agilent Human Promoter Microarray

• 2 x 244K arrays– Promoters of ~19,143 genes– 25 probes/promoter– Probes ~ 60nt oligomers

• Spaced every ~ 200 bp– Location of Probes within Promoter Region

• 5500 bp upstream from transcriptional start site• 2500 bp downstream from transcriptional start site

– Genome version: UCSC May 2004 Build 34

Validation of cell lines and antibodies used for ChIP-on-chip

• EWS-FLI1– Cell line: TC-71 (Ewing’s sarcoma with EWS-FLI1)

• No native FLI1 by western– Antibody: FLI1 C-terminal– Positive control ChIP targets: TGFBR2, ID2

• ASPL-TFE3– Cell line: FU-UR-1 (renal carcinoma with ASPL-TFE3)

• No native TFE3 by western and RT-PCR– Antibody: TFE3 C-terminal– Positive control ChIP targets: MET, CYP17A1

Zhen Lu, Ph.D.

Rachel Kobos, M.D.

ChIP on CHIP resultsfor ASPL-TFE3 in FU-UR-1 cells

Enriched by IP

Significantly enriched by IP

Triplicate ChIPTriplicate array hybridizations

Data analyzed by TileMap- Significant genes = Tstat >3.0

2193 genes (11.5%) significant for ASPL-TFE3

ChIP on CHIP resultsfor EWS-FLI1 in TC71 cells

Enriched by IP

Significantly enriched by IP

Triplicate ChIPTriplicate array hybridizations

Data analyzed by TileMap- Significant genes = Tstat >3.0

2617 genes (13.7%) significant for EWS-FLI1

Reproducibility of ChIP-Chip Results:Binding of MET promoter by ASPL-TFE3

TGFBR2

Previously known target of EWS-FLI1

gaactcctgagtggtgtgggagggcggtgaggggcagctgaaagtcggccaaagctctcggaggggctggtctaggaaacatgattggcagctacgagagagctaggggctggacgtcgaGGAGAGGGAGAAGGCTCTCGGGCGGAGAGAGGTCCTGCCCAGCTGTTGGCGAGGAGTTTCCTGTTTCCCCCGCAGCGCTGAGTTGAAGTTGAGTGAGTCACTCGCGCGCACGGAGCGACGACACCCCCGCGCGTGCACCCGCTCGGGACAGGAGCCGGACTCCTGTGCAGCTTCCCTCGGCCGCCGGGGGCCTCCCCGCGCCTCGCCGGCCTCCAGGCCCCCTCCTGGCTGGCGAGCGGGCGCCACATCTGGCCCGCACATCTGCGCTGCCGGCCCGGCGCGGGGTCCGGAGAGGGCGCG

Significant Probes

ChIP primers used in Fukuma et al 2002.

ChIP primers used in Siligan et al 2005.

TGFBR2

Previously known target of EWS-FLI1

Previously known target of ASPL-TFE3MET

gggggccgatttccctctgggtggtgccagtccccacctcagcggtcctcggaacccgcggactaggggacggacagcacgcgaggcagacagacacgtgctggggcgggcaggcgagcgcctcagtctggtcgcctggcggtgcctccggccccaacgcgcccgggccgccgcgggccgcgcgcgccgatgcccggctgagtcactggcagggcagcgcgcgtgtgggaaggggcggagggagtgcggccggcgggcgggcggggcgctgggctcagcccggccgcaggtgacccggagGCCCTCGCCGCCCGCGGCGCCCCGAGCGCTTTGTGAGCAGATGCGGAGCCGAGTGGAGGGCGCGAGCCAGATGCGGGGCGACAGCTGACTTGCTGAGAGGAGGCGGGGAGGCGCGGAGCGCGCGTGTGGTCCTTGCGCCGCTGACTTCTCCACTGGTTCCTGGGCACCGAAAGgtaaaattgcagcccctttcagatccagtacccaatcCctcgcctcaggggttctgctttctttgttcccctaagagacctgactgctgttccagggggcaaaaccAcgtaggtgggctagagtttaggggcttcggaaactgaagagacgtggccacggcgaggacgaaactagaatggggcttgtctttttagggggttgcttctgatggccacctgtatgacttaggagggagaggggcgctgggacagtgggtgatgtgtgactgttacggcccagcaagttttaaagctgggatctgactcagcccttacaaaagggatccggtcatcctcgtcccaccgtgatgcagctggcaa

ChIP primers used in Tsuda et al 2007.

Significant Probes

METPreviously known target of ASPL-TFE3

Example of Gene of Interest for ASPL-TFE3

PIM1 kinase

Kinase Fold T-test p-valuePIM1 3.1 6.42E-10MAPK3 1.6 1.28E-09MET 4.9 2.91E-09H11 6.4 9.73E-09MAPKAPK2 1.6 1.68E-08PRKAG2 8.7 2.74E-08MKNK2 2.6 3.16E-08GPRK6 8.1 4.31E-08LYN 2.3 5.69E-08PACSIN2 2.1 1.02E-07FLT1 3.9 8.99E-07MAPKAP1 1.8 2.13E-06

PIM1 is a highly differentially overexpressed kinase in ASPS

Comparison of 16 ASPS samples vs 123 samples of 4 other sarcomas

- PIM1 inhibitors exist- Implicated in other cancers

Example of Gene of Interest for EWS-FLI1TERT

- EWS-FLI1 previously reported to upregulate TERT (Takahashi, CR 2003)- Ulaner et al, 2004: TERT+/TRAP+ in

- 21/30 Ewing sarcomas- 9/10 Ewing cell lines

Relationship of ChIP-on-chip bound genes to differentially expressed genes in tumor expression profiles

• Unsupervised clustering of 137 sarcoma samples based on genes bound in the ChIP-chip analysis

Unsupervised clustering of 137 sarcoma samples based on 2193 genes bound by ASPL-TFE3 in theChIP-chip analysis

Unsupervised clustering of 137 sarcoma samples based on 2617 genes bound by EWS-FLI1 in the

ChIP-chip analysis


• Unsupervised clustering of 137 sarcoma samples based on genes bound in the ChIP-chip analysis

• Comparison of ChIP-chip bound genes to genes significantly differentially expressed in tumor expression profiles

– Subset comparisons (stringently defined subsets of over- or under-expressed genes)

Expression profiling dataset on sarcomas with chimeric transcription factors

* all significant at Bonferroni p<0.01 relative to 4 other sarcoma types

294215728ES/PNET

531159012ASPS

554316328DSRCT

282151823ARMS

638681646SS

Subset with≥ 2 fold over-expression*

Genes significantly

over- or under-expressed*

Samples

11 ASPL-TFE3 type 13 ASPL-TFE3 type 2

14Alveolar Soft Part Sarcoma (ASPS)

22 EWS-FLI1 type 111 EWS-FLI1 type 2

5 EWS-ERG

38Ewing Sarcoma/PNET (ES)

25 SYT-SSX121 SYT-SSX2

46Synovial Sarcoma (SS)

32 EWS-WT132Desmoplastic Small Round Cell Tumor (DSRCT)

16 PAX3-FKHR7 PAX7-FKHR

23Alveolar Rhabdo-myosarcoma (ARMS)

• Chip: Affymetrix U133A• Data processing: RMA method• Clustering: Pearson correlation• Data used: all 22215 probe sets• n=137 tumors from MSKCC and U.Penn. (F. Barr)• + 4 xenografts + 12 cell lines = 153 total samples

ASPS ES SS DSRCT ARMS

M. Laé, T. Saito, et al., Ladanyi Lab


• ASPS / ASPL-TFE3– ≥ 2X overexpressed genes

• Ewing’s / EWS-FLI1– ≥ 2X overexpressed genes

12713286NotBound

1372129Bound

Not over-expressed

Over-expressedP<10-31

12558195NotBound

171433Bound

Not over-expressed

Over-expressedP=0.25


• ASPS / ASPL-TFE3– ≥ 2X overexpressed genes

• Ewing’s / EWS-FLI1– ≥ 2X overexpressed genes

12713286NotBound

1372129Bound

Not over-expressed

Over-expressedP<10-31

12558195NotBound

171433Bound

Not over-expressed

Over-expressedP=0.25

Genes of interest: MET, CYP17A1, UPP1, PIM1, IGF2R, MAFF

Genes of interest: NR0B1, NKX2.2, ID2, CCND1, GAS1


• ASPS / ASPL-TFE3– Top 100 ≥ 1.5X underexpressed genes,

Sorted by p-value

• Ewing’s / EWS-FLI1– Top 100 ≥ 1.5X underexpressed genes,

sorted by p-value

1290693NotBound

14947Bound

Not under-expressed

Under-expressed

P=0.32

1267776NotBound

172324Bound

Not under-expressed

Under-expressedP<10-3

Genes of interest: FZD10, SIX2, FOXF1, NCAM1

12880119NotBound

147229Bound

Not down-regulated

Down-regulated

Relationship of EWS-FLI1 ChIP-on-chip bound genes to genes whose expression is significantly altered upon knockdown of EWS-FLI1 in a Ewing sarcoma cell line

• Published expression profile: – Steve Lessnick et al., Expression profiling of EWS/FLI identifies NKX2.2 as a critical target

gene in Ewing's sarcoma. Cancer Cell 2006 • Ewing’s sarcoma cell line A673 with EWS-FLI1

– shRNA knockdown, U133A arrays

– 32 genes upregulated by EWS-FLI1 148 genes downregulated by EWS-FLI1

1297227NotBound

14965Bound

Not Upregulated

UpregulatedP=0.37 P<10-3

12880119NotBound

147229Bound

Not down-regulated

Down-regulated

Relationship of EWS-FLI1 ChIP-on-chip bound genes to genes whose expression is significantly altered upon knockdown of EWS-FLI1 in a Ewing sarcoma cell line

• Published expression profile: – Steve Lessnick et al., Expression profiling of EWS/FLI identifies NKX2.2 as a critical target

gene in Ewing's sarcoma. Cancer Cell 2006 • Ewing’s sarcoma cell line A673 with EWS-FLI1

– shRNA knockdown, U133A arrays

– 32 genes upregulated by EWS-FLI1 148 genes downregulated by EWS-FLI1

1297227NotBound

14965Bound

Not Upregulated

UpregulatedP=0.37 P<10-3

Genes of interest: NR0B1, NKX2.2, GAS1, RET

Genes of interest: IGFBP3, IGFBP7, KLF6

Cross-reference genes bound by EWS-FLI1 with 7 sources of expression profiles of EWS-FLI1-upregulated genes

238 genesSarcoma Profiling

227 genesLadanyi

Unpublished

Transfected EWS-FLI1 in

Human Fibroblasts92 genes

Lessnick SL et al,2002

SRBCT Profiling33 genes

Staege MS et al, 2004.

Transfected EWS-FLI1intoERMS cells87 genes

Hu-Lieskovan Set al 2005.

Sarcoma Profiling261 genes

Baird K et al, 2005.

EWS-FLI1 KD in A673 cells

32 genesSmith R et al,

2006.

EWS-FLI1 KD in TC71, EWS502

1312 genesKinsey M et al,

2006.

ChIP – chipdata

2617 genes bound by EWS-FLI1

ZDHHC3 PPFIA1 ID2 EGR2

UAP1 PFKP GAS1EFNB1

SRPK2 NR0B1FVT1 DAPK1

SQLE MYO10 FNBP1 CCND1

RUNX3 SH2B3 FANCACARHSP1

RDX LEPROTL1 FADS1 ATP2A2

RAP1GAP IMPA2 EPB41 ATP1A1

28 EWS-FLI1 bound genes found to be “up” in >1 expression profile

Cross-reference genes bound by EWS-FLI1 with 7 sources of expression profiles of EWS-FLI1-upregulated genes

238 genesSarcoma Profiling

227 genesLadanyi

Unpublished

Transfected EWS-FLI1 in

Human Fibroblasts92 genes

Lessnick SL et al,2002

SRBCT Profiling33 genes

Staege MS et al, 2004.

Transfected EWS-FLI1intoERMS cells87 genes

Hu-Lieskovan Set al 2005.

Sarcoma Profiling261 genes

Baird K et al, 2005.

EWS-FLI1 KD in A673 cells

32 genesSmith R et al,

2006.

EWS-FLI1 KD in TC71, EWS502

1312 genesKinsey M et al,

2006.

ChIP – chipdata

2617 genes bound by EWS-FLI1

Future Experiments

• Systematic knockdown of genes upregulated by EWS-FLI1 using siRNA or shRNA libraries

• Chemical inhibition of selected targets (RTKs)


Agenda

• Expression profiling: mining for diagnostic and therapeutic targets

• ChIP-on-chip: integration with expression profiling data to identify target genes of chimeric transcription factors in translocation sarcomasCancer genomics: new approaches to high throughput unbiased discovery of translocations

>75% of genes somatically altered in human tumors are translocatedHeightened interest following discovery of gene fusions in common

carcinomas (TMPRSS2-ERG in prostate CA; EML4-ALK in lung CA)High throughput unbiased screening methods becoming available

Translocations in human cancers

New approaches to high throughput unbiased discovery of translocations and

intragenic rearrangements

• Genomic DNA– BAC end-sequence profiling– Massively parallel single-molecule sequencing of paired end

tags

• mRNA-based– Search for chimeric transcripts in EST libraries – Massively parallel single-molecule cDNA sequencing

• Microarray-based

Translocations can be associated with genomic copy number changes

1 2 3 4 5 6 7 8

…..…..

A

B

Examples: gain of Ph chromosome in blastic CML, PAX7-FKHR gains in ARMS

1 2 3

…..

A

Array-based strategies for unbiased detection of gene fusions

• aCGH approach (part of MSKCC TCGA contribution)– dense cancer gene coverage optimally suited for the

detection of gene fusions associated with genomic gains or losses (Cameron Brennan, MSKCC)

– design: exon-level coverage of gene classes involved in gene fusions: transcription factors, kinases, etc…

– detects: • gene fusions occurring through interstitial deletions • non-reciprocal translocations• reciprocal translocations followed by loss or duplication of

one of the derivatives

Array-based strategies for unbiased detection of gene fusions

• aCGH approach (part of MSKCC TCGA contribution):– dense cancer gene coverage optimally suited for the detection of

gene fusions associated with genomic gains or losses

• Exon expression array approach – based on intragenic change points in expression levels due to 3’

exons driven by promoter of 5’ fusion partner gene

Human Exon 1.0 ST ArrayHuman Genome U133 Plus 2.0 Array

exon

target of array probe

Fusion gene A-B+

Fusion gene B-A

Breakpoint

Exon

arr

ayR

elat

ive

Expr

essi

on le

vel

1 2 3 4 5 6 7Gene B

Normalized to samples without translocation

Tumor with fusion gene A-B

0

Change pointin expression level

Reciprocal fusion transcriptFusion gene B-A

Gene A

Exon array probes

PromoterA

mRNAcDNA

1 2 3 4 5

Gene BPromoter

B

Exon array probescDNA

mRNA

1 2 3 4 5 6 7

Fusion gene A-B

Promoter A

mRNA

cDNA

Breakpoint

Exon array probes

1 2 4 5 6 7

Weak promoter

Low

High

Strong promoter

Transcript levels

Fusion GeneA-B

Fusion GeneB-A

9-9TMPRSS2-ERGProstate cancer1-1PML-RARaAML

1-1EWS-FLI1 10-61-1EWS-FLI1 10-5

31

1111223232

1333

TissueNumber of samples

6

-----1--11

-1-2

Cell line

1BCR-ABL p210

1EWS-FLI1 type24EWS-FLI1 type1

3EWS-ERG

5SYT-SSX1

1ETV6-NTRK3Infantile fibrosarcoma

1BCR-ABL p190CML

2EWS-NR4A3Extraskeletal myxoid chondrosarcoma3EWS-ATF1Clear cell sarcoma

1TLS-CHOPMyxoid liposarcoma

Ewing’s sarcoma

3SYT-SSX2Synovial sarcoma

TotalFusion geneTumor

4EWS-WT1Desmoplastic small round cell sarcoma

46Total

3ASPL-TFE3 Type1Alveolar soft part sarcoma2PAX3-FKHRAlveolar rhabdomyosarcoma

Test set for detection of fusion transcripts using Affy Exon array data

Toru Motoi, M.D.,Ph.D.

Use of Affymetrix Exon Arrays to detect gene fusions

5’ 3’

Discontinuity of expression levels of WT1 exons in JN-DSRCT1 cell

with EWS-WT1fusion


5’ 3’

Discontinuity of expression levels of SSX1 exons in HS-SY-II cell line

with SYT-SSX1fusion


in primary tumor samples

Discontinuity of expression levels of WT1 exons

in a clinical DSRCT sample

Discontinuity of expression levels ofSSX1 exons in clinical synovial

sarcoma sample with SYT-SSX1

Development of a computational algorithm to score genes with discontinuity of expression levels between 5’ and 3’ exons

SSX1 in Syn Sarccell line

WT1 in DSRCTcell line

Fusion Score

SSX1 in Syn Sarc clinical sample

Nick Socci & Adam Olshen, MSKCC


Agenda

• Expression profiling: mining for diagnostic and therapeutic targets

• ChIP-on-chip: integration with expression profiling data to identify target genes of chimeric transcription factors in translocation sarcomas

• Cancer genomics: new approaches to high throughput unbiased discovery of translocations and intragenic rearrangements

Genomic Approaches to Translocation Sarcomas - · PDF fileGenomic Approaches to Translocation...

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