1

A human B lymphocyte interactome for A human B lymphocyte interactome for the dissection of dysregulated pathways the dissection of dysregulated pathways in lymphoid malignancies.in lymphoid malignancies.

Andrea Califano:Andrea Califano:MAGNetMAGNet: Center for the Multiscale Analysis of Genetic and Cellular Networks: Center for the Multiscale Analysis of Genetic and Cellular Networks

C2B2C2B2: Center for Computational Biology and Bioinformatics: Center for Computational Biology and Bioinformatics

HICCCHICCC: Herbert Irving Comprehensive Cancer Center: Herbert Irving Comprehensive Cancer Center

Columbia UniversityColumbia University

W. Han and R.A.Weinberg, Nature ReviewsW. Han and R.A.Weinberg, Nature Reviews

A subway Map of CancerA subway Map of Cancer

The germinal centerThe germinal center

Naïve B Centroblast Centrocyte

Plasma Cell

Memory B

Pre-GCGerminal Center (GC) Post-GC

B-Cell Subpopulations

B-CLL FollicularLymphoma

BurkittLymphoma

DiffuseLargeCellLymphoma

HodgkinDisease

MultipleMyeloma

B-Cell Derived Malignancies

Mantle CellLymphoma

unmutated mutated Ig V region

(CD5+)(CD5+)

Differential Expression AnalysisDifferential Expression Analysis

Cancer Research ApproachesCancer Research Approaches

• Long List of GenesLong List of Genes

• Unstable Gene SelectionUnstable Gene Selection• Sample SizeSample Size• Experiment SelectionExperiment Selection

• Causal Genes?Causal Genes?

• Dysregulation AbsentDysregulation Absent

InteractomeInteractome

Interactome Definition:Interactome Definition:• Represents > 100 genes or proteinsRepresents > 100 genes or proteins• Visually pleasingVisually pleasing• Void of any information content (if possible)Void of any information content (if possible)• Scale FreeScale Free• Published in Nature(s) or SciencePublished in Nature(s) or Science

Using the Interactome as a MapUsing the Interactome as a Map

TFTF

TFTF

MM

TFTF

MM

MM

Drug-basedDrug-basedPerturbationPerturbation

CausalCausalLesionLesion

Information TheoryInformation Theory

JaneJane

MYCMYC

JoeJoeMaryMary

TERTTERTNotch1Notch1

;I Joe Jane ;I Mary Joe

1; min{ [ 1; ], [ ; ]}I Notch BYSL I Notch MYC I MYC BYSL

; min{ [ ; ], [ ; ]}I Mary Jane I Mary Joe I Joe Jane

;I Mary Jane

ARACNEARACNE

Graphical representation:Graphical representation:• all 56 1all 56 1stst neighbors neighbors• the top ranking 444 2the top ranking 444 2ndnd neighbors neighbors

Previously reported c-MYC targetsPreviously reported c-MYC targets

Neighbor Total # Total Validated by ChIP

1st 5656 22 (39.3%)22 (39.3%) 1111

2nd 20072007 390 (19.4%)390 (19.4%) 251251

ChIP ValidationChIP Validation

BYSLBYSL

5’5’ 3’3’AA

(+158/+348)(+158/+348)BB

no A

bno

Ab

IgG

IgG

-M

YC

-MY

C

no D

NA

no D

NA

inpu

t DN

Ain

put D

NA

AA(+158/+348)(+158/+348)

BB

b)b)

MRPL12MRPL12

EIF3S9EIF3S9

EBNA1BP2EBNA1BP2

BOP1BOP1

ATICATIC

BYSLBYSL

no

Ab

no

Ab

IgG

IgG

-M

YC

-MY

C

no

DN

An

o D

NA

inp

ut

DN

Ain

pu

t D

NA

ZRF1ZRF1

c4orf9c4orf9

a)a)

39418_at39418_at

NOL5ANOL5A

PRMT3PRMT3

TCP1TCP1

c-MYC targetsc-MYC targets

Neighbor Total # Total Validated by ChIP

1st 5656 29 (51.8%)29 (51.8%) 22 (39.3%)22 (39.3%)

2nd 20072007 390 (19.4%)390 (19.4%) 251 (12.5%)251 (12.5%)

Expected background ~10% (1,300/12,600)Expected background ~10% (1,300/12,600)

Copyright ©2006 by the National Academy of Sciences

Palomero et al. (2006)

Activated NOTCH1 and c-MYC regulatory network in T-ALL. (a) A metagene based on the gene expression signature associated with levels of activated NOTCH1 protein in T-ALL cell lines was integrated in an ARACNe global regulatory network constructed with microarray expression data from T-ALL samples. Neighbors of the NOTCH1 metagene identified as NOTCH1 direct target genes by ChIP-on-chip are shaded in pink, neighbors regulated in T-ALL cells treated with a GSI are shown shaded in blue, and neighbor genes showing both significant promoter occupancy by ChIP-on-chip and regulation upon GSI treatment are shaded in purple. (b) Detailed representation of overlap between NOTCH1 neighbor genes and ChIP-on-chip data. The intensity of each neighboring node represents the significance level of promoter occupancy by NOTCH1. (c) Detailed representation of NOTCH1 neighbor genes regulated in T-ALL cells treated with a GSI. The intensity of each neighboring node, corresponding to the scale panel on the right, represents the significance level of gene regulation by GSI treatment. (d) Representation of direct neighbor genes associated with the NOTCH1 metagene and with c-MYC.

Activated NOTCH1 and c-MYC network in T-ALLActivated NOTCH1 and c-MYC network in T-ALL

A combination of 6 transcription factors combinatorially A combination of 6 transcription factors combinatorially controls a mesenchymal gene expression signature in controls a mesenchymal gene expression signature in glioblastoma, which is associated with poor prognosisglioblastoma, which is associated with poor prognosis

RUNX1

FOSL2 (1)

BHLHB2(1)

To

tal

(2.5

%)

An

ti C

EB

P/b

eta

IgG

No

DN

A

STAT3(1)

STAT3(2)

STAT3(3)

CEBPB

FOSL2 (2)

ZNF238

BHLHB2(2)strong1) taaattgaGCAAtaa (470-84)

2) gaggttctGCAAgtt (496-510)

3) gaagttctGAAAggg (3403-17

BHLHB2

absentgactttggGAAAgcg (801-815)ZNF238

medium1) agaattggGTAAact (560-574)

2) gaggttaaGGAAttc (747-761)FOSL2

strongaaggttgaGCAAtgc (425-439)CEBPB

strong1) ctattatGAAAggc (972-86)

2) attcttatGCAAtaa (2660-74)

3) ggaatttaGAAAgag (2708-22)

4) ggtatttgGAAAagg (2858-72)

STAT3

weakttacttttGAAAtaa (1421-35)RUNX1

ChIP bindingCEBPB-binding siteTF

strong1) taaattgaGCAAtaa (470-84)

2) gaggttctGCAAgtt (496-510)

3) gaagttctGAAAggg (3403-17

BHLHB2

absentgactttggGAAAgcg (801-815)ZNF238

medium1) agaattggGTAAact (560-574)

2) gaggttaaGGAAttc (747-761)FOSL2

strongaaggttgaGCAAtgc (425-439)CEBPB

strong1) ctattatGAAAggc (972-86)

2) attcttatGCAAtaa (2660-74)

3) ggaatttaGAAAgag (2708-22)

4) ggtatttgGAAAagg (2858-72)

STAT3

weakttacttttGAAAtaa (1421-35)RUNX1

ChIP bindingCEBPB-binding siteTF

STAT3STAT3

FOSL2FOSL2

CEBPBCEBPB

ZNF238ZNF238

BHLHB2BHLHB2

RUNX1RUNX1

Iavarone – Califano Collaboration

Mesenchymal Signature Regulation in GlioblastomaMesenchymal Signature Regulation in Glioblastoma

Validation ResultsValidation ResultsFOSL2 (68%) ChIP

EMP3 strongTNC strong

EHD2 strongS100A11 strong

ITGA5 strongBACE2 strongPLAU mediumC1R medium

ACTN1 weakSOCS3 weakFLNA weak

SLC16A3 absentTAGLN absentTHBD absent

MAP2K3 absentACTA2 absent

STAT3 (63%) ChIPRRBP1 strong

SERPINH1 strongTIMP1 medium

COL4A1 mediumACTN1 mediumCHI3L1 mediumCOL4A2 medium/weakCD151 medium/weakICAM1 mediumITGA7 medium/weak

SLC16A3 absentPTRF absentOSMR absent

KIAA0963 absentNRP2 absentNLCN absent

BHLHB2 (58%) ChIPSERPINE1 mediumSLC16A3 medium

PPDN mediumBCL3 strongFLNA mediumIFITM3 weak

FC4R2C mediumSLC39A8 absent

LIF absentSOCS3 absentITGA5 absent

EFEMP2 absent

CEPBP (67%) ChIPPTRF strongSHC1 strongMVP strong

SERPINE1 strongTIMP1 weak

SERPINA1 weakS100A11 absentSLC16A3 absent

MYL9 absent

Method works well even on very heterogeneous dataMethod works well even on very heterogeneous dataValidation Rate > ~60%Validation Rate > ~60%

Alcohol and Cocain Addiction in RatsAlcohol and Cocain Addiction in Rats

SLC12A2

EWSR1

APP

CUGBP2EST-1392288_at

HNTEST-1381682_at

KHSRP

PLCB1

GRID1

DAAM1

SARS1

MAPK10

SNRPB

RGD1310066

PBX1

EST-1396530_at

EST-1396952_at

LUC7L2

Inp

ut

DN

A

An

ti P

BX

1

IgG

GRID1

APP

SARS1

MAPK10

PLCB1

SNRPB

EST-1396952_at

EWSR1

EST-1392288_at

EST-1396530_at

DAAM1

UBE2B2

ChIP Validation of PBX1 ChIP Validation of PBX1 Predicted TargetsPredicted Targets

MPF cNAc sNAcCeABLA dlBNST vBNST VTA

ARACNe Predictions: TF control of addiction signatureARACNe Predictions: TF control of addiction signature

Rat Brain Microarray Expression ProfilesRat Brain Microarray Expression Profiles

Regulatory Control in Eukaryotic Regulatory Control in Eukaryotic CellsCells

• The ability of a transcription factor (TF) to control its The ability of a transcription factor (TF) to control its target genes is regulated at multiple levels:target genes is regulated at multiple levels:

– Post-transcriptionally (On TF protein)Post-transcriptionally (On TF protein)• PhosphorylationPhosphorylation

• AcetylationAcetylation

• StabilityStability

• OthersOthers

– Epigenetically (On Target Genes)Epigenetically (On Target Genes)• DNA Methylation DNA Methylation

• Histone methylation and acetylationHistone methylation and acetylation

• Formation of transcription complex with co-factorsFormation of transcription complex with co-factors

• TF Competition or SynergyTF Competition or Synergy

Thus, the function of a TF is dependent upon the presence of other molecules (modulators)Thus, the function of a TF is dependent upon the presence of other molecules (modulators)

Modulator AnalysisModulator Analysis

JoeJoeMaryMary

; |I Mary Joe Tony

TonyTony

MYCMYC TERTTERT

GSK3GSK3

DegradationDegradation

SignalSignal

; |TF t mI g g g

MYCMYC

TERTTERT

GSK3GSK3

MINDY (Modulator Inference by Network Dynamics)MINDY (Modulator Inference by Network Dynamics)

Single Modulator, Multiple TargetsSingle Modulator, Multiple Targets

Repressed TargetsRepressed Targets

Activated TargetsActivated Targets

High/Low Modulator ExpressionHigh/Low Modulator ExpressionLow/High Modulator ExpressionLow/High Modulator Expression

MYC Modulation in Human B LymphocytesMYC Modulation in Human B Lymphocytes

• Phenotypic variabilityPhenotypic variability– 254 microarray gene expression 254 microarray gene expression

profilesprofiles– 27 distinct natural occurring and 27 distinct natural occurring and

tumor derived B cell phenotypestumor derived B cell phenotypes

• MYC proto-oncogeneMYC proto-oncogene– Important genetic hub of B cell Important genetic hub of B cell

physiologyphysiology– Transcription factor known to be Transcription factor known to be

widely differentially regulatedwidely differentially regulated

4% 1%

3%

2%

2%

2%

1%

2%

2%

2%

4%

2%

1%

6%

7%

3%

10%2%2%

9%

3%

8%

6%

6%

3%

4%5%

BL-line

BL-line type III

DLCL-line

HD-line

LCL-line

MM-line

undefined line

Cord blood

N

M

CB

CC

GC

B-CLL unmut

B-CLL mut

AIDS-BL

BL

AIDS-DLCL-CB

AIDS-DLBCL-IB

DLCL-CB

DLCL-IB

DLCL

FL

HCL

MCL

PEL

SLVL

Kinases, Phosphatases & Kinases, Phosphatases & Acetyltransferases Acetyltransferases

ModulatorModulator M#M# M+M+ M-M- ModeMode DescriptionDescription EvidenceEvidence

CSNK2A1CSNK2A1 205205 205205 00 ++ Casein kinase 2, alpha 1Casein kinase 2, alpha 1 HPRDHPRD

PPAP2BPPAP2B 120120 00 120120 -- Phosphatidic acid phosphatase 2BPhosphatidic acid phosphatase 2B Activates GSK3Activates GSK3

HCKHCK 118118 00 118118 -- Hemopoietic cell kinaseHemopoietic cell kinase BCR pathwayBCR pathway

SATSAT 109109 00 109109 -- Spermidine N1-acetyltransferaseSpermidine N1-acetyltransferase   

DUSP2DUSP2 9595 00 9595 -- Dual specificity phosphatase 2Dual specificity phosphatase 2 Dephosphorylates ERK2Dephosphorylates ERK2

MAP4K4MAP4K4 9494 00 9494 -- MAP kinase kinase kinase kinase 4MAP kinase kinase kinase kinase 4 BCR pathwayBCR pathway

PPM1APPM1A 9292 00 9292 -- Protein phosphatase 1AProtein phosphatase 1A   

CSNK1DCSNK1D 9090 00 9090 -- Casein kinase 1, deltaCasein kinase 1, delta   

GCATGCAT 8686 8686 00 ++ Glycine C-acetyltransferaseGlycine C-acetyltransferase   

TRIOTRIO 8484 00 8484 -- Triple functional domainTriple functional domain   

PRKCIPRKCI 6363 6363 00 ++ Protein kinase C, iotaProtein kinase C, iota BCR pathwayBCR pathway

PRKACBPRKACB 5757 00 5757 -- Protein kinase, catalytic, betaProtein kinase, catalytic, beta BCR pathwayBCR pathway

STK38STK38 5656 5656 00 ++ Serine/threonine kinase 38Serine/threonine kinase 38   

MTMR6MTMR6 5555 22 5353 -- Myotubularin related protein 6Myotubularin related protein 6   

NEK9NEK9 5353 5353 00 ++ NIMA-related kinase 9NIMA-related kinase 9   

MYST1MYST1 4747 4747 00 ++ MYST histone acetyltransferase 1MYST histone acetyltransferase 1   

MAPK13MAPK13 4545 4545 00 ++ MAP kinase 13MAP kinase 13 BCR pathwayBCR pathway

OXSR1OXSR1 4545 00 4545 -- Oxidative-stress responsive 1Oxidative-stress responsive 1   

DUSP4DUSP4 4343 00 4343 -- Dual specificity phosphatase 4Dual specificity phosphatase 4   

MAP2K3MAP2K3 4242 00 4242 -- MAP kinase kinase 3MAP kinase kinase 3 BCR pathwayBCR pathway

PPP4R1PPP4R1 3939 00 3939 -- Protein phosphatase 4, R1Protein phosphatase 4, R1   

ERK2ERK2 3737 3737 00 ++ MAP kinase 1MAP kinase 1 BCR pathwayBCR pathway

MAP4K1MAP4K1 3636 00 3636 -- MAP kinase kinase kinase kinase 1MAP kinase kinase kinase kinase 1 BCR pathwayBCR pathway

CSNK1ECSNK1E 3535 3434 11 ++ Casein kinase 1, epsilonCasein kinase 1, epsilon   

FYNFYN 3333 00 3333 -- FYN oncogeneFYN oncogene   

NEK7NEK7 3333 3333 00 ++ NIMA-related kinase 7NIMA-related kinase 7   

CSNK2A2CSNK2A2 3131 3131 00 ++ Casein kinase 2, alphaCasein kinase 2, alpha Related to CSNK2A1Related to CSNK2A1

DUSP5DUSP5 3030 00 3030 -- Dual specificity phosphatase 5Dual specificity phosphatase 5   

……   

PP2APP2A 2828 2828 00 ++ Serine/threonine-protein phosphatase 2A   

HDAC1HDAC1 88 00 88 -- Histone deacetylaseHistone deacetylase   (12/28, 43%)(12/28, 43%)

WB: STK38

Inpu

t [2.

5%]

IP: C

-Myc

IP: M

ouse

IgGSTK38 co-precipitates STK38 co-precipitates

with c-Myc in HeLawith c-Myc in HeLa

Transcription Factor AnalysisTranscription Factor AnalysisModulatorModulator M#M# M+M+ M-M- ModeMode DescriptionDescription EvidenceEvidence PPBSBS

AHRAHR 186186 00 186186 -- Aryl hydrocarbon receptorAryl hydrocarbon receptor    --

SMAD3SMAD3 163163 00 163163 -- Mothers against DPP homolog 3Mothers against DPP homolog 3 HPRDHPRD --

CREMCREM 139139 00 139139 -- cAMP responsive element modulatorcAMP responsive element modulator    1×101×10-36-36

DDIT3DDIT3 106106 00 106106 -- DNA-damage-inducible transcript 3DNA-damage-inducible transcript 3    0.040.04

DRAP1DRAP1 101101 100100 11 ++ DR1-associated protein 1DR1-associated protein 1    --

ZKSCAN1ZKSCAN1 100100 100100 00 ++ Zinc finger with KRAB and SCAN 1Zinc finger with KRAB and SCAN 1    --

BHLHB2BHLHB2 8888 00 8888 -- Basic HLH domain containing, B2Basic HLH domain containing, B2    9×109×10-58-58

NR4A1NR4A1 8888 00 8888 -- Nuclear receptor subfamily 4, A1Nuclear receptor subfamily 4, A1    --

ATF3ATF3 8686 00 8686 -- Activating transcription factor 3Activating transcription factor 3    1×101×10-17-17

UBTFUBTF 7979 7979 00 ++ Upstream binding transcription factorUpstream binding transcription factor    --

NR4A2NR4A2 7777 00 7777 -- Nuclear receptor subfamily 4, A2Nuclear receptor subfamily 4, A2    --

SOX5SOX5 7474 00 7474 -- SRY-box 5SRY-box 5    0.660.66HOXB7HOXB7 6969 6969 00 ++ Homeo box B7Homeo box B7    --

BACH1BACH1 6868 00 6868 -- bZIP transcription factor 1bZIP transcription factor 1    5×105×10-9-9

ARNTARNT 6161 6060 11 ++ AHR nuclear translocatorAHR nuclear translocator    --

ETV5ETV5 6161 00 6161 -- ETS variant gene 5 ETS variant gene 5    --IRF1IRF1 6060 00 6060 -- Interferon regulatory factor 1Interferon regulatory factor 1    6×106×10-8-8

TCF12TCF12 6060 6060 00 ++ Transcription factor 12Transcription factor 12    2×102×10-3-3

GTF2IGTF2I 5555 5555 00 ++ General transcription factor II, i General transcription factor II, i HPRDHPRD --NFKB2NFKB2 5555 00 5555 -- NFkB 2 (p49/p100)NFkB 2 (p49/p100) BCR pathwayBCR pathway 0.700.70

FOSFOS 4444 00 4444 -- v-Fos oncogene homologv-Fos oncogene homolog    2×102×10-3-3

JUNJUN 4242 00 4242 -- v-Jun oncogene homologv-Jun oncogene homolog    1×101×10-7-7

ZNF354AZNF354A 4141 4141 00 ++ Zinc finger protein 354AZinc finger protein 354A    --

CUTL1CUTL1 4040 33 3737 -- Cut-like 1 proteinCut-like 1 protein    --

SMARCB1SMARCB1 3939 3939 00 ++ Regulator of chromatin, B1Regulator of chromatin, B1 HPRDHPRD --

CBFA2T3CBFA2T3 3737 3737 00 ++ Core-binding factor, alpha 2T3Core-binding factor, alpha 2T3    --

DBPDBP 3737 3737 00 ++ Albumin D-box binding proteinAlbumin D-box binding protein    6×106×10-3-3

MAFMAF 3636 00 3636 -- v-Maf oncogene homologv-Maf oncogene homolog    --

RELBRELB 3535 00 3535 -- v-Rel oncogene homolog Bv-Rel oncogene homolog B    --ZNF217ZNF217 3434 3434 00 ++ Zinc finger protein 217Zinc finger protein 217    --

ESR1ESR1 3333 3333 00 ++ Estrogen receptor 1Estrogen receptor 1    0.040.04

NFATC4NFATC4 3131 3131 00 ++ Nuclear factor of activated T-cells 4Nuclear factor of activated T-cells 4 BCR pathwayBCR pathway 0.920.92

TFECTFEC 3131 00 3131 -- Transcription factor ECTranscription factor EC    --

CITED2CITED2 3030 00 3030 -- Cbp/p300-interacting transactivator 2Cbp/p300-interacting transactivator 2    --

NFYBNFYB 3030 3030 00 ++ NFkB binding proteinNFkB binding protein   HPRDHPRD 2×102×10-3-3

……   

MEF2BMEF2B 1414 1111 33 ++ MADS box transc. enhancer factor 2, polypeptide B --

(18/35, 51%)(18/35, 51%)

Genome-wide Kinome-TransfactomeGenome-wide Kinome-Transfactome

   DUSP10DUSP10 CDC25BCDC25B STK17BSTK17B PPAP2BPPAP2B PRKACBPRKACB TRIB2TRIB2 CCL2CCL2 DUSP7DUSP7 HCKHCK MAP3K5MAP3K5 ...... ABL1ABL1 FESFES AURKCAURKC EPHB2EPHB2 ROR2ROR2 JARID1BJARID1B AKT1AKT1 AAK1AAK1 PTPN3PTPN3 RAF1RAF1

ETS1ETS1 00 00 00 00 00 22 5656 1010 1717 00...... 00 11 00 00 00 00 00 00 11 00

MAFFMAFF 00 11 00 00 00 00 00 1515 00 00...... 22 00 00 00 00 00 00 00 00 00

SMAD3SMAD3 33 1212 1313 00 66 11 00 44 00 22...... 00 00 00 00 00 00 00 00 11 00

ATF3ATF3 11 00 11 00 11 2121 00 77 00 1212...... 00 00 00 00 00 11 00 00 00 00

ZNF85ZNF85 1212 88 1414 00 4747 22 00 88 00 1111...... 00 00 00 00 00 00 00 00 00 00

SOX5SOX5 22 33 00 00 11 00 00 2222 11 00...... 00 00 00 22 00 00 00 00 00 00

CREMCREM 00 00 00 00 11 11 33 1010 00 11...... 00 00 00 00 00 00 00 00 00 00

CHD4CHD4 3434 1515 99 00 11 00 66 88 00 00...... 00 00 00 00 00 00 00 00 00 00

E2F5E2F5 33 00 77 00 1212 1515 00 00 22 4949...... 00 00 00 00 00 00 00 00 00 00

GTF3AGTF3A 44 77 00 22 11 00 55 11 00 33...... 00 00 00 00 00 00 00 00 00 00

...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ......

SS18L1SS18L1 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

HOXC4HOXC4 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

FOXD1FOXD1 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

KLF5KLF5 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

WT1WT1 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

HOXC5HOXC5 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

MYT1MYT1 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

CRXCRX 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

DGCR6LDGCR6L 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

DUX2DUX2 00 00 00 00 00 00 00 00 00 00...... 00 00 00 00 00 00 00 00 00 00

Kinome (846)

Transfactom

e (889)

PPIPPITotalTotal

++ --

MINDYMINDY ++ 86648664 2508825088 3375233752-- 6810868108 262305262305 330413330413

TotalTotal 7677276772 287393287393 364165364165 = 889x846= 889x846

Genome Wide Pathway Genome Wide Pathway ValidationValidation

2 = 470.6, p < 1x10-16

Consider a set of ‘gold standard’ Consider a set of ‘gold standard’ modulators of a TF to include:modulators of a TF to include:

• Direct modulators: signaling Direct modulators: signaling proteins with known PPIs with proteins with known PPIs with the TFthe TF

• Pathway modulators: Pathway modulators: signaling proteins on any signaling proteins on any pathway that includes the TFpathway that includes the TF

Kegg PathwaysKegg Pathways

The B Cell InteractomeThe B Cell Interactome

An integrated Cellular Network for

the Dissection of Lymphoid Malignancies

Celine LefebvreCeline LefebvreKai WangKai WangWei Keat LimWei Keat Lim

Katia BassoKatia BassoRiccardo Dalla FaveraRiccardo Dalla Favera

Protein-Protein interactions

Orthologous interactions in mouse

ARACNE(254 B cell expression profiles)

TFBS (MATCH)

Target gene co-expression

TFBScoexpmousearacnePDI LRLRLRLRLR

Protein-DNA interactions

HTorthcoexpGWGOPPI LRLRLRLRLRLR

Y2H, MassSpec

Orthologous interactions

Biological Processannotations

RGS4 block RASD1

CKS1A interact SKP2

CD4 bind TFAP2A

GPNMB contain PPFIBP1

TACR1 require PARP1

GeneWaysGene co-expression (254 B cell expression

profiles)

3Lefebvre et. al. A context-specific network of protein-DNA and protein-protein interactions reveals new regulatory motifs in human B cells. Submitted for Recomb Satellite on Systems Biology, Dec. 1-2, 2006

B Cell InteractomeB Cell Interactome• Mixed interaction network (Bayesian Evidence Integration)

– 40,000 Protein-Protein Interactions predicted from:40,000 Protein-Protein Interactions predicted from:

• Orthologous interactions (fly, mouse, worm, yeast)

• Yeast 2-hybrid human datasets

• Gene co-expression (B cells - mutual information)

• Gene Ontology biological process annotations

• GeneWays (literature mining algorithm)

• Structural Clues (to be added)

– 10,000 Protein-DNA Interactions from:10,000 Protein-DNA Interactions from:

• Orthologous interactions in mouse (Transfac, BIND)

• Transcription factor binding sites (MATCH)

• GeneWays

• ARACNE

– 120,000 Post-transcriptional Modulator Interactions from:120,000 Post-transcriptional Modulator Interactions from:

• MINDY

• Structural Clues (to be added)

– 8,500 genes (900 TFs)8,500 genes (900 TFs)

Using the Network Using the Network for Cancer Researchfor Cancer Research

Network Disregulation in Cancer

Kartik ManiKartik ManiCeline LefebvreCeline LefebvreKai WangKai WangWei Keat LimWei Keat LimAdam MargolinAdam Margolin

Katia BassoKatia BassoRiccardo Dalla FaveraRiccardo Dalla Favera

Algorithm OverviewAlgorithm Overview

1. Network ModelCreation

2. Dysregulated Edge Analysis

3. Root CauseIntegrative Analysis

TF

TF

TF

T1

T2

T3 T4

X

X

X

Entrez Name Score p-value

5090 PBX3 Inf 0

10745 PHTF1 400.932 3.03E-172

4799 NFX1 389.755 2.11E-167

7639 ZNF85 357.854 1.38E-153

10589 DRAP1 314.070 1.26E-134

Edge Phenotype MapEdge Phenotype Map

LOF

GOF

Edge Phenotype Map - B-Cell MAS 5.0

Phenotypes

Mu

tua

l In

form

ati

on

B-CLL (mut) (unmut) BL DLCL (CB) (IB) FL GC (CB) (CC) HCL MCL PEL SLVL non-GC (N) (M) (Cord)

0.1

0.2

0.3

0.40.50.60.7

Network AnalysisNetwork Analysis

Dysregulated P-PDysregulated P-PNormal P-PNormal P-P ModulationModulationNormal P-DNormal P-D Dysregulated P-DDysregulated P-D

Analyze theAnalyze theB Cell Knowledge BaseB Cell Knowledge Base

Given a Phenotype Selection (E.g., Follicular Lymphoma)Given a Phenotype Selection (E.g., Follicular Lymphoma)

Score = - Log Score = - Log pp((ggii in in SS) - Log ) - Log pp((ggii modulates modulates SS))

Probability computed by Fisher Exact Test (FET)Probability computed by Fisher Exact Test (FET)

SS = Set of dysregulated interactions and associated genes = Set of dysregulated interactions and associated genes

TFTF

TFTF

MM

TFTF

MM

MM

Phenotype-Based Phenotype-Based Dysregulation InteractionsDysregulation Interactions

TFTF

TFTF

MM

TFTF

MM

MM

Analyze anyAnalyze anyDysregulated ModuleDysregulated Module

TFTF

TFTF

MM

MM

Gene ScoringGene Scoring

TF1TF1

T3T3T2T2T1T1

T5T5T4T4

TF2TF2

T3T3

T2T2

T1T1

• There are a total of 5 There are a total of 5 dysregulated interactionsdysregulated interactions

• 3 out of 5 are connected to 3 out of 5 are connected to TF1 (out of 6 total)TF1 (out of 6 total)

• 2 out of 5 are modulated by 2 out of 5 are modulated by TF1TF1

• We calculate probability of We calculate probability of observing this pattern by observing this pattern by chance (Fisher Exact Test)chance (Fisher Exact Test)

Normal Interaction

Dysregulated Interaction

Score(TF1) = sig.(Direct Effect) + sig.(Modulator Effect)

Benchmarking (cont’d)Benchmarking (cont’d)

Phenotype Causal Gene Description Rank T-Test

Germinal Center (GC) BCL6 BCL6 necessary for GC formation 12 238

Follicular Lymphoma (FL) BCL2 t(14;18) 11 696

Burkitt Lymphoma (BL)MYC

(MTA1 2nd)

t(8;14), t(8;2), t(8;22)

MTA1 KO mice do not

develop BL with MYC transl.

5 39

Mantle Cell Lymphoma (MCL)

Cyclin D1/BCL1 t(11;14) 16 8

ConclusionsConclusions

• Computational algorithms and integrative methods are starting to achieve high precision in human cells, equivalent to experimental assays (>80%)

• Cellular Context Specificity is Critical

• Higher-order interactions cannot be discarded a priori

• Networks are useful to dissect cellular phenotypes

• Tools availability:– ARACNe and the B Cell Interactome are available:

http://wiki.c2b2.columbia.edu/califanolab/index.php/Software

– MINDY will be available by Summer ‘07

AcknowledgmentsAcknowledgments• Califano Lab ComputationalCalifano Lab Computational

– Adam MargolinAdam Margolin– Kai WangKai Wang– Ilya Nemenman (LANL)Ilya Nemenman (LANL)– Nilanjana Banerjee (Philips)Nilanjana Banerjee (Philips)– Celine LefebvreCeline Lefebvre– Wei Keat LimWei Keat Lim– Kartik ManiKartik Mani– In Sock JangIn Sock Jang– Alberto Ambesi-ImparatoAlberto Ambesi-Imparato– Manjunath KustagiManjunath Kustagi– Sean ZhouSean Zhou– Kaushal KumarKaushal Kumar– Quian FengQuian Feng– Achint SethiAchint Sethi

• Califano Lab ExperimentalCalifano Lab Experimental– Rachel CoxRachel Cox– Mariano AlvarezMariano Alvarez– Brygida BisikirskaBrygida Bisikirska– Presha RajbhandariPresha Rajbhandari

• Institute of Cancer GeneticsInstitute of Cancer Genetics– Riccardo Dalla FaveraRiccardo Dalla Favera– Katia BassoKatia Basso– Mas SaitoMas Saito– Ulf KleinUlf Klein

• Rzhetsky Lab (GeneWays)Rzhetsky Lab (GeneWays)

• Honig LabHonig Lab– Alona SosinskiAlona Sosinski

• IBM CBCIBM CBC– Gustavo StolovitzkyGustavo Stolovitzky– Yuhai TuYuhai Tu

• geWorkbench TeamgeWorkbench Team– John WatkinsonJohn Watkinson– Beerooz BadiiBeerooz Badii– Ken SmithKen Smith– Matt HallMatt Hall– Xiaoqing ZhangXiaoqing Zhang– Eileen DalyEileen Daly– Kiran KeshavKiran Keshav– Pavel MorozovPavel Morozov– Mary Van GinholvenMary Van Ginholven

Funding provided by NCI, NIAID, and the NIH RoadmapFunding provided by NCI, NIAID, and the NIH Roadmap