NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth

Palomero et al. 10.1073/pnas.0606108103.

Supporting Information

Files in this Data Supplement:

Supporting Figure 7
Supporting Figure 8
Supporting Table 1
Supporting Table 2
Supporting Figure 9
Supporting Figure 10
Supporting Table 3
Supporting Figure 11
Supporting Methods




Fig. 7. ICN1 expression antagonizes the effects of GSIs on the expression of NOTCH1 target genes. Control DND41 T-ALL cells expressing GFP and DND41 cells infected with retroviruses driving expression of ICN1 and GFP were treated with GSI (CompE 100 nM 48 h). Expression of NOTCH1 target genes c-MYC, DELTEX1, IFRD2 and CD3D was up-regulated in ICN1 expressing cells compared to controls. GAPDH mRNA levels were used as normalization control. Data shows mean and SD of triplicate measurements.





Fig. 8. Inhibition of NOTCH1 signaling with GSIs impairs cell cycle progression in T-ALL cells. CUTLL1 and DND41 cells were treated with GSI (CompE 100 nM) or vehicle only (DMSO) for 72 h. Cell cycle was analyzed by flow cytometry analysis of DNA content after PI staining. A reduction in proliferation with accumulation of cells in G1 was observed. Histograms show representative results of triplicate experiments. Similar results were obtained in HPB-ALL, ALL-SIL, KOPTK1, and TALL1 cells.





Fig. 9. Venn diagrams representing the overlap between NOTCH1 direct target genes identified by ChIP-on-chip and genes regulated by GSI treatment in T-ALL cell lines. a. Overlap with ChIP-on-chip targets with error model P-value cutoff <0.05. (b) Overlap with ChIP-on-chip targets with error model P-value cutoff <0.001. The significance of the overlap was assessed using Fisher's exact test.





Fig. 10. Gene expression changes induced by GSI in genes identified as NOTCH1 targets by ChIP-on-chip. Genes with error model binding P values <0.05 and expression t test P values <0.001 are shown ranked by signal-to-noise ratio. Heat map represents color coded expression levels for each sample with respect to mock treatment controls.





Fig. 11. Structure of the proposed feed-forward loop regulatory motif controlling cell growth genes downstream of NOTCH1 and MYC in T-ALL. Input in the circuit is provided by activation of NOTCH1 signaling (by interaction of the NOTCH1 receptor with DSL ligands or by ligand independent activation induced by NOTCH1 mutations) and by signaling pathways up-regulating the expression or the activity of the MYC oncoprotein. This model support s that the intensity of input signals that enter this circuitry upstream of MYC may tune the cell growth response to NOTCH1 and may potentially enhance or attenuate the antitumor effect of drugs targeting NOTCH1 signaling.





Table 1. Top NOTCH1 direct target genes identified by ChIP-on-chip

Gene

Spot name

P value

Binding ratio

RefSeq no.

PRCC

PRCC

4.76E-09

4.148045

NM_005973

BUB3

BUB3

3.12E-08

3.44207

NM_004725

FAM96A

FLJ22875

6.57E-08

3.331196

NM_032231

PL6

PL6

7.37E-08

3.33461

NM_007024

PAFAH2

PAFAH2

9.78E-08

3.393245

NM_000437

RANGAP1

RANGAP1

2.23E-07

3.227676

NM_002883

TARBP2

TARBP2

3.01E-07

3.093956

NM_004178

ZNF133

ZNF133_0,0

3.22E-07

3.041622

 

ZNF10

ZNF10_0,0

3.34E-07

3.153393

 

HPS6

FLJ22501

3.5E-07

2.930667

NM_024747

TMEM48

FLJ10407

5.76E-07

2.944545

NM_018087

ING3

ING3

6.53E-07

2.8508

NM_019071

MDH1

MDH1

9.83E-07

2.839357

NM_005917

ZNF361

ZNF361

1.17E-06

2.861604

NM_018555

RC74

FLJ10871

2.41E-06

2.710062

NM_018250

C6orf166

FLJ10342

2.45E-06

2.726724

NM_018064

CFL1

CFL1

2.51E-06

2.596959

NM_005507

LTA4H

LTA4H

2.6E-06

2.778543

NM_000895

AAMP

AAMP

2.96E-06

2.645006

NM_001087

CPE

CPE

3.37E-06

2.645677

NM_001873

ZNF331

BC009433_0,0

3.64E-06

2.663466

 

ACTR1A

ACTR1A

3.65E-06

2.625543

NM_005736

Cep 290

FLJ13615

3.87E-06

2.657315

NM_025114

SNX1

SNX1

4.14E-06

2.608905

NM_003099

MDS025

MDS025

4.39E-06

2.798992

NM_021825

TARBP2

TARBP2_0,1

4.69E-06

2.52562

 

PPP1R12B

PPP1R12B

5.26E-06

2.550444

NM_032105

DDX23

U5-100K

6.8E-06

2.552142

NM_004818

ZNF337

ZNF337_0,0

8.26E-06

2.436899

?

NPR2L

NPR2L

8.31E-06

2.564293

NM_006545

SKD3

SKD3

1.23E-05

2.447516

NM_030813

TRAP150

TRAP150

1.25E-05

2.529101

NM_005119

IFRD1

IFRD1

1.39E-05

2.431396

NM_001550

ZNF547

NM_173631_0,0

1.47E-05

2.341599

 

SCNM1

MGC3180

1.77E-05

2.52025

NM_024041

HSPC138

HSPC138

1.8E-05

2.431735

NM_016401

MRPL24

MRPL24

1.88E-05

2.527329

NM_024540

USP5

USP5

1.92E-05

2.402084

NM_003481

DNAJC17

FLJ10634

1.95E-05

2.437994

NM_018163

ACAD8

ACAD8

2.12E-05

2.339718

NM_014384

PVRL2

PVRL2

2.12E-05

2.571582

NM_002856

UBE2V1

UBE2V1

2.28E-05

2.902147

NM_022442

ZNF225

ZNF225

2.29E-05

2.513603

NM_013362

SNAPC3

SNAPC3_0,0

2.3E-05

2.341246

 

INVS

INVS

2.31E-05

2.461536

NM_014425

PDE6D

PDE6D

2.43E-05

2.432897

NM_002601

GTF2H4

GTF2H4_0,0

2.59E-05

2.345813

 

TSNAXIP1

LOC55815

2.86E-05

2.373108

NM_018430

COX7C

COX7C

3.26E-05

2.254306

NM_001867

C14orf133

FLJ12707

3.54E-05

2.309728

NM_022067

FTL

FTL

3.66E-05

2.306027

NM_000146

MGC4161

MGC4161

4.03E-05

2.279359

NM_024303

PEMT

PEMT

4.42E-05

2.300557

NM_007169

H1RNA

H1RNA_X16612_TATA345_chr14_0,0

4.87E-05

2.22743

 

AHSA1

C14orf3

5.03E-05

2.224114

NM_012111

CD3D

CD3D

5.28E-05

2.314604

NM_000732

SPK

SPK

5.29E-05

2.196515

NM_004819

PRPF31

PRPF31

5.71E-05

2.36207

NM_015629

ZNF133

ZNF133

5.72E-05

2.182406

NM_003434

STCH

STCH

5.84E-05

2.2189

NM_006948

CYB561D2

101F6

6.14E-05

2.307171

NM_007022

FLJ21742

FLJ21742

7.27E-05

2.307891

NM_032207

ING3

ING3_0,0

7.5E-05

2.152426

 

CNOT4

CNOT4

9.65E-05

2.118939

NM_013316

PET112L

PET112L

9.98E-05

2.284023

NM_004564

ZNF436

BC056400_0,0

0.000105

2.123893

 

AUTL1

AUTL1

0.00011

2.121149

NM_032852

FYCO1

FYCO1

0.000116

2.634725

NM_024513

COPS7B

COPS7B

0.000123

2.160383

NM_022730

SEDLP

SEDLP

0.000131

2.117285

NM_015890

HSPC144

HSPC144

0.000132

2.227479

NM_014174

HSP105B

HSP105B

0.000136

2.172044

NM_006644

ZFYVE19

FLJ14840

0.000137

2.157679

NM_032850

RBL1

RBL1_0,0

0.000147

2.0961

 

ARL6

DKFZP434L1123

0.000152

2.197188

NM_032146

TNFAIP1

TNFAIP1

0.000156

2.467579

NM_021137

RPL18A

RPL18A

0.000157

2.100565

NM_000980

C11orf10

C11orf10

0.00016

2.083368

NM_014206

BRD8

BC008076_0,0

0.000182

2.347046

 

VRK3

LOC51231

0.000184

2.118904

NM_016440

FNTB

FNTB

0.000186

2.238582

NM_002028

BTRC

BTRC

0.000194

2.06488

NM_033637

PHB

PHB

0.000196

2.141411

NM_002634

GSPT1

GSPT1

0.000197

2.099706

NM_002094

KBTBD7

DKFZP434E2318

0.000208

2.070793

NM_032138

RBM6

RBM6

0.000243

2.01901

NM_005777

ZCCHC8

DKFZp434E2220

0.000248

2.135749

NM_017612

PHF6

MGC14797

0.000249

1.984857

NM_032335

SEC11L3

LOC90701

0.000252

2.091293

NM_033280

CD3EAP

ASE-1

0.000255

2.174561

NM_012099

IFRD2

IFRD2

0.000255

2.047137

NM_006764

QTRTD1

FLJ12960

0.000269

2.208297

NM_024638

ZNF317

BC009367_0,0

0.000272

2.002955

 

ZNF155

ZNF155_0,0

0.000273

2.254842

 

RNU42B

RNU42B_NR_000013_chr17_0,0

0.000276

2.013662

 

SMAP

IMAGE145052

0.000282

2.027713

NM_014267

HKE2

HKE2

0.000293

1.981017

NM_014260

STARD10

SDCCAG28

0.000302

1.961713

NM_006645

CLDND1

C3orf4

0.000309

1.975918

NM_019895

RPLP0L

RPLP0L

0.000311

1.982819

NM_016183

SPHK2

SPHK2

0.000318

1.948548

NM_020126

ACAD8

ACAD8_0,0

0.00033

2.04361

 

OSCAR

OSCAR

0.000351

2.27924

NM_130771