Genomicandtranscriptomiclandscapesofacutepromyelocy3cleukemia

KankanWang

StateKeyLaboratoryofMedicalGenomics

Rui-JinHospitalShanghaiJiaotongUniversitySchoolofMedicine

UnderstandingthepathogenesisandtreatmentofAPLusinghigh-throughputtechnologies

CH3

RNA polymerase

ChIP-seq RNA-seq

PML/RARαAPLEffectsofATRAandATO(co-regulators)

? ?

Hallmarksofacutepromyelocy3cleukemia

HowdoesPML/RARαdrivethedevelopmentofAPL?

•  PML/RARα retains the DNA binding domain of wild-type RARα.

•  PML/RARα acts as a strong transcriptional repressor.

•  Myeloid commitment is hardly affected in various RAR-deficient mice.

Genome-widePML/RARαbindingsites

ChIP-chip/ChIP-seq

Wang et al. Cancer Cell 2010 Martens et al. Cancer Cell 2010

Genome-widebindingpicturesofPML/RARα

ATRA ATRA

!  PU.1-mediated transactivation was restored!  Genes repressed by PML/RARα were reactivated

!  Increased in H3K9K14 acetylation!  Induced transcriptional changes correlate

with H3K9K14 acetylation

PU.1andRAREhalfsitesarethemostsignificantmo3fsinthePML/RARαChIPregions

Wang et al. Cancer Cell 2010

Wang et al. Cancer Cell 2010

RecruitmentofPML/RARαtochromaEnpre-boundbyPU.1

PML/RARαpredominantlyrepressesPU.1-regulatedgenes

RepressionofPU.1-dependenttransacEvaEonthroughbothPU.1andRAREhalfsites

PML/RARαtendstocolocalizewithRXRpreferen3allyonpromoterregions

Martens et al. Cancer Cell 2010

ATRAtreatmentincreasesthelevelofH3K9K14acbutnotH3K9me3andH3K27me3

Martens et al. Cancer Cell 2010

PML/RARα

PML/RARα

HCK PMSB8

Tissue factor

PU.1

PU.1

PMSB9

PMSB10 PML/RARα PU.1

PU.1 PU.1

POL II

Detailed analysis of key targets

Genome-wide PML/RARα binding

PML/RARα

CDKN2D

Indirect binding Binding to multiple PU.1 Coordinated regulation Binding to ER8

Hemorrhage

Yan JS, et al, PNAS. 2010

Antigen presentation

Yang XW, et al, Oncogene. 2014

Differentiation & Cell cycle

Wang YW, et al, Cell Death Dis. 2014

Tumor suppression

Zou DD, et al, Leukemia Res. 2010

Regulatoryfeaturesandfunc3onsofPML/RARαkeytargetsinAPL

ER8

PML/RARαtransac3vatesthe3ssuefactorpromoterthroughanindirectinterac3on

Yan et al. PNAS 2010

PML/RARαexertedbothrepressiveandac3va3ngfunc3ons

Unpublished data

Func3onallyimportanceofbothPML/RARα-repressedand-ac3vatedtargets

Unpublished data

RepressionwasassociatedwithRARα,whereasac3va3onwasindependentofRARα

Unpublished data

Dis3nctATRAeffectsonPML/RARα-ac3vatedand-repressedgenes

Unpublished data

PML/RARαpreferen3allyexistedwithinsuper-enhancerregions

Unpublished data

Transcriptome analysis of the mechanism of ATRA action

•  RNA-seq/microarray

•  Ribo-minusRNA-seq

RA 0.1 µM

ATO 0.5 µM

12 h 48 h

12 h 48 h

12 h 48 h

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

ATO 0.5 µM

RA 0.1 µM

RA 0.1 µM

ATO 0.5 µM

12 h 48 h

12 h 48 h

12 h 48 h

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

2

1

0

-1

-2

ATO 0.5 µM

RA 0.1 µM

ATRAregulatesgeneexpressionatthetranscriptomiclevel,whereasATOfunc3onsattheproteomiclevel

Transcriptome Proteome

Zheng et al. PNAS 2005

Molecularnetworksunderlyingthecombina3onofATRAandATOinNB4cells

CD

44

SE

LL

CD

11b

CD

18

ICA

M1

Surf

ace

Mole

cule

s

Restoration of NB

Plasma Membrane

Cytoplasm

IFNAR2

JAK1 TYK2

ISGF3GSTAT1 STAT2

IFNGR

JAK1 JAK2

STAT1IRF7ISGF3

STAT1

IFN PathwayG-CSF-R Receptor

GRB2

SOS2

Ras

Raf

MAPK6

Receptor

TRAF2Daxx

ASK1

MKK3

Rac

P38

MKK4/7

JNK2

PTPN9 MEK1

MAPK-JNK-p38 PathwayG-CSF-R

SHP2

TFs/CoFs

MEKK1

Protein Synthesis PML-RARα Degradation Cell Cycle

Nucleus

TRADDFADD

CASP10 CASP8

CASP3CASP7

cytcAPAF1

CASP9

BCL2

Mitochondria Apoptosis

Apoptosis

IRF1

CASP1

TRAIL-R

FADD

PDCD6IPBCL2A1

TNF-R

STK24

BAK1

M

CDK4CCND2

CDK2CCNE1

CDK2CCNA2

CDK7CCNH

CDK1CCNA2

CDK1CCNB1

CDKN2DCDKN1A

CDC25B

CHEK1

PCNA

GADD45B

CDC25A

G1 G2S

CDKN1B

Stress Cytoskeleton

AHSA1

HSPCA

DNAJA1

HSPA8

DNAJB1

ReceptorG proteinAC

ATP cAMP

PKA

cAMP-PKA Pathway

AKAP13

AKAP1

PDE4DIP

AKAP9

ReceptorG proteinPLCB

PIP2

IP3

DG2

ITPR

RYR3Ca2+

CALM

Ca2+

PKC

Calcium Pathway

PKA

ANK3

ERNUCB2

AKAP9

S100A8

S100A9PLSCR1

CGREF1

SP100

SP110 SP140

PSME2 PMLothers

PML-RARαP/CAFNCOA3

TIF

CBPRARA Pathway

EP300

ID1

BHLHB2ID2 CEBPZ

CEBPBCEBPGCEBPD

CEBPE HHEXTFs/CoFs

40S

AUG

Proteins

EIF4A1

EEF1B2

EEF1A1EEF1D

EEF1G

60S

EIF4B

EIF4G1

80S PML-RARα

SUMO1UBE2L6UBE1L UBE4B

UBE3C

PSMB9

PSMB8

PSMB10

PSMC3

PSMD13

PSMC2

SumoylatedPML-RARα

SENP5

MAP2

MYH3MYOM1

FBN2

UbiquitiatedPML-RARα

Degradation of PML-RARα

proteasome

Cell Cycle ArrestIFI16b

GAS7ARHI

PEG10

CCNG2

PIBF1

SE

LP

LG

CD

44

SE

LL

CD

11b

CD

18

ICA

M1

Surf

ace

Mole

cule

s

Restoration of NB

Plasma Membrane

Cytoplasm

IFNAR2

JAK1 TYK2

ISGF3GSTAT1 STAT2

IFNGR

JAK1 JAK2

STAT1IRF7ISGF3

STAT1

IFN PathwayG-CSF-R Receptor

GRB2

SOS2

Ras

Raf

MAPK6

Receptor

TRAF2Daxx

ASK1

MKK3

Rac

P38

MKK4/7

JNK2

PTPN9 MEK1

MAPK-JNK-p38 PathwayG-CSF-R

SHP2

TFs/CoFs

MEKK1

Protein Synthesis PML-RARα Degradation Cell Cycle

Nucleus

TRADDFADD

CASP10 CASP8

CASP3CASP7

cytcAPAF1

CASP9

BCL2

Mitochondria Apoptosis

Apoptosis

IRF1

CASP1

TRAIL-R

FADD

PDCD6IPBCL2A1

TNF-R

STK24

BAK1

M

CDK4CCND2

CDK2CCNE1

CDK2CCNA2

CDK7CCNH

CDK1CCNA2

CDK1CCNB1

CDKN2DCDKN1A

CDC25B

CHEK1

PCNA

GADD45B

CDC25A

G1 G2S

CDKN1B

Stress Cytoskeleton

AHSA1

HSPCA

DNAJA1

HSPA8

DNAJB1

AHSA1

HSPCA

DNAJA1

HSPA8

DNAJB1

ReceptorG proteinAC

ATP cAMP

PKA

cAMP-PKA Pathway

AKAP13

AKAP1

PDE4DIP

AKAP9

ReceptorG proteinPLCB

PIP2

IP3

DG2

ITPR

RYR3Ca2+

CALM

Ca2+

PKC

Calcium Pathway

PKA

ANK3

ERNUCB2

AKAP9

S100A8

S100A9PLSCR1

CGREF1S100A8

S100A9PLSCR1

CGREF1

SP100

SP110 SP140

PSME2 PMLothers

SP100

SP110 SP140

PSME2 PMLothers

PML-RARαP/CAFNCOA3

TIF

CBPRARA Pathway

EP300

ID1

BHLHB2ID2 CEBPZ

CEBPBCEBPGCEBPD

CEBPE HHEXTFs/CoFs

40S

AUGAUG

Proteins

EIF4A1

EEF1B2

EEF1A1EEF1D

EEF1G

60S60S

EIF4B

EIF4G1

80S PML-RARα

SUMO1UBE2L6UBE1L UBE4B

UBE3CUBE2L6UBE1L UBE4B

UBE3C

PSMB9

PSMB8

PSMB10

PSMC3

PSMD13

PSMC2 PSMB9

PSMB8

PSMB10

PSMC3

PSMD13

PSMC2

SumoylatedPML-RARα

SENP5

MAP2

MYH3MYOM1

FBN2

MAP2

MYH3MYOM1

FBN2

UbiquitiatedPML-RARα

Degradation of PML-RARα

proteasome

Cell Cycle ArrestIFI16b

GAS7ARHI

PEG10

CCNG2

PIBF1

SE

LP

LG

IRF1

Zheng et al. PNAS 2005

CircularRNAgenera3ngfromthefusionsiteofPML/RARαisoncogenicandcontributestocellulartransforma3on

Zepp et al. Cell 2017

DynamicregulatedcicrRNAprofilinguponATRAtreatmentofAPLcells

Unpublished data

cicrRNAregula3onindependentoftheirhostlinearmRNA

Unpublished data

Unpublished data

Requirementofcirc-RELL1inATRA-induceddifferen3a3onofAPLcells

Summary "  Genome-wide binding pictures of PML/RARα

-  PML/RARα selectively targets PU.1-regulated genes.

-  PML/RARα is colocalized with RXR on chromatin.

-  PML/RARα exerts both activation and repression roles of in driving APL.

"  Regulatory features and functions of key targets of PML/RARα.

"  Transcriptomic view of the mechanism of ATRA action

-  ATRA induces transcriptional remodeling and a series of signaling pathways in APL cells.

-  ATRA dynamically regulates a number of cicrRNAs upon ATRA treatment.

Acknowledgements Omics platforms Yun Tan Ping Wang Yizhen Li Xuehua Zhu Functional studies Wen Jin Xianwen Yang Xuefei Ma Yewei Wang Bioinformatics analysis Hai Fang Ming Zhao Jiantao Shi Huanwei Wang