AATF Genome : Importance In Oncogenesis

AATF Genome: Importance In Oncogenesis

Dr. Deepak Kaul Ph.D(AIIMS); F.R.C. Path (London); FNA; FWAASProfessor & HeadDepartment of Experimental Medicine & Biotechnology,Postgraduate Institute of Medical Education & Research,Chandigarh-160012 (India)

E-Mail : dkaul235@gmail.com

Cell

Nucleus

Hyperosmotic

Hypoxia

“ER” stress

Glucose Deprivation

DNA damage

CELLULAR STRESS

Nucleolus

AATFSensor

Cell Cycle

DNA Repair

Apoptosis

Autophagy

Stre

ss

Cytoplas

m

Nucleolus Discovered by German Physiologists : Rudolph Wagner & Gabriel Valentin

Exons 2-11

Exon-1

Exon-12

Intron

miR -2909

AATF GenomeDr .Deepak Kaul

(Discovered 2009)

5’

3’

ImmunityCancer

MetabolismPhysiological processes

(NCBI Accession No.: GSE54949)

Functional Genomics

Par-4; Dlk / Zip; NRAGE

AATF protein

HDAC1 AATFSP1SP1

AATF

G1 S G2 M

RbE2F

AATF

P21

AATF

P53

p65

KLF4

Apoptosis

AATF Interactome & Cell Decision

AATF Protein : Nature

DNA Methylation addiction

Molecular Sweet Tooth

Cell: Oncogenic Transformation

CELL : DNA Methylation Addiction

Unbridled Growth & Immortality

Role of AATF in the methylation dependent down-regulation of Tumor- suppressor genes ?

Trimeric AATF (70 kDa)

Monomeric AATF(23 kDa)

10060453020

128

Mol wt (kDa)

SDS-PAGE0

10

20

30

40

50

60

70

80EB3

MOLT4

JURKAT

HELa-229

IMR-32

Rel

ativ

e A

ATF

Exp

ress

ion

(Tra

nscr

iptio

nal L

evel

)

Nature of AATF Gene in Cancer Cells

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

EXON 2 EXON 4-12EXON 3EXON 1

A T G A C C T T G G G A G C A

A T G A C C T - G G G A G C A

A T G A C C T - G G G A G C A

A T G A C C T - G G G A G C A

A T G A C C T - G G G A G C A

A T G A C C T - G G G A G C A

744 BpNCBI

T-ALL (JURKAT)

T-ALL (MOLT-4)

B-Cell Lymphoma (EB3)

Neuroblastoma (IMR-32)

Cervical cancer (HELA 229)

MAGPQPLALQLEQLLNPRPSEADPEADPEEATAARVIDRFDEGEDGEGDFLVVGSIRKLASASLLDTDKRYCGKTTSRKAWNEDHWEQTLPGSSDEEISDEEGSGDEDSEGLGLEEYDEDDLGAAEEQECGDHRESKKSRSHSAKTPGFSVQSISDFEKFTKGMDDLGSSEEEEDEESGMEEGDDAEDSQGESEEDRAGDRNSEDDGVVMTFSSVKVSEEVEKGRAVKNQIALWDQLLEGRIKLQKALLTTNQLPQPDVFPLFKDKGGPEFSSALKNSHKALKALLRSLVGLQEELLFQYPDTRYLVDGTKPNAGSEEISSEDDELVEEKKQQRRRVPAKRKLEMEDYPSFMAKRFADFTVYRNRTLQKWHDKTKLASGKLGKGFGAFERSILTQIDHILMDKERLLRRTQTKRSVYRVLGKPEPAAQPVPESLPGEPEILPQAPANAHLKDLDEEIFDDDDFYHQLLRELIERKTSSLDPNDQVAMGRQWLAIQKLRSKIHKKVDRKASKGRKLRFHVLSKLLSFMAPIDHTTMNDDARTELYRSLFGQLHPPDEGHGD

MAGPQPLALQLEQLLNPRPSEADPEADPEEATAARVIDRFDEGEDGEGDFLVVGSIRKLASASLLDTDKRYCGKTTSRKAWNEDHWEQTLPGSSDEEISDEEGSGDEDSEGLGLEEYDEDDLGAAEEQECGDHRESKKSRSHSAKTPGFSVQSISDFEKFTKGMDDLGAVRRRKTKRVAWKKGMTRKTPKARVRKTGLEIETVRMMVW- -stop- -

Common mutant

sequence in the above

mentioned cell lines

759 Bp

Wt

Mt

560 Amino acids

212 Amino acids

AATF gene

AATF Gene - Micro-dissection

AATF

PRO

TEIN

SE

QU

ENCE

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Elution

IB: P

ar4

IB:S

p1

IB:D

NMT3

B

Mutant AATF

DNMT3B

Sp1

Par4

Mol wt.

101.487.552.7

47.827.818.8Cell Extract

Anti-AATFIP

IB

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Exploration of Mutant AATF Interactome-I

IP:DNMT3B

IB: AATFIP:PAR4

IP:SP1

IP:DNMT3B

IP:SP1IP:PAR4

IB: AATF

45 kDa (Mt:dimeric form)

Cell Extract

Anti-SP1

IPAnti-PAR4

Anti-DNMT3B

Elution

IB

23 kDa (Mt:monomeric form)

Mutant AATF

DNMT3B

Sp1

Par4

Monomeric Mt AATF Interactome

Dimeric Mt AATF Interactome

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Exploration of Mutant AATF Interactome-II

IB: SP1 78kDa

IB: DNMT3B 98 kDa

IP:DNMT3B

IP:AATF

IP:PAR4

IP:PAR4

IP:AATF

IP:SP1

IB:PAR4IP:AATF

IP:SP1IP:DNMT3B

38 kDa

Cell Extract

Anti-SP1

IP

Anti-PAR4

Anti-DNMT3B

Elution

IB

Anti-AATF

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Exploration of Mutant AATF Interactome-III

Se-ries1

0

5

10

15

20

25

B-ALL

HeLa 2

29

Rel

ativ

e E

xpre

ssio

n of

DN

MT3

B (T

rans

latio

nal L

evel

)

IMR 32

T-A

LL

DNMT3B (98 kDa)

PBMCs

**

**

**

**

Before pull down with

DNMT3B antibody

After pull down with

DNMT3B antibody

Trimeric form

Dimeric formMonomeric

form

52.787.5

47.827.8

18.8

101.4

Mol wt(kDa)

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

DNMT3B Expression and its interaction with Truncated-AATF protein

Series10

20

40

60

p21

p53

Cyclin D

C-myc

β-actin

Control siRNA-DNMT3B

Rel

ativ

e Tr

ansl

atio

nal

expr

essi

on

Contro

lsiR

NA-DNMT3

B

Series10

20

40

60

80

100

120

Tran

slat

iona

l exp

ress

ion

of

DN

MT3

B (A

.U.)

DNMT3B

β actin

Control siRNA-DNMT3B

Contro

lsiR

NA-DNMT3

B

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

DNMT3B Knockout Cells : Gene Regulation

Selection

& Analysis

AATF (Mt) Cloning

AA

PCR Product

BGH pA

f1 ori

SV40 oriEM

-7SV40 pA

pUC ori

Pbla

P EF-1α

T7 A

sp71

8 I

Kpn

IB

amH

ISp

e I

Bst

X I T

T

P P

TOPOTOPO

Eco

RVB

stX

IN

ot I

Xba

I

V5 epitope 6XHis Pm

e I

5’ UTR Coding Region 3’UTR

PCR Amplification

Ligation

Transformation

Plasmid Isolation

into Competent E. coli Cells

PCR Product Purification

Poly A Tailing

AATF (Mt)

pEF6/V5-His-TOPO

Series10

20

40

60

80

p53

Par-4

C-myc

Bcl-2

β-actin

Control Transfected AATF (Mt)

Rela

tive

Tran

slatio

nal e

xpre

ssio

n

Contro

lTr

ansfe

cted

AATF

(Mt)

Series10

20

40

60

80

100

120

Tran

slatio

nal e

xpre

ssio

n of

AAT

F (A

.U.)

Mt AATF (23kDa)

β actin

Control

Contro

l

Transfected AATF (Mt)

Tran

sfecte

d

AATF

(Mt)

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Mutant AATF : Functional Genomics

PROMOTER-1000 0

SP1

DNMT3B

Par-4Mt

AATF

-75 9.94

p21 Gene

POSITION SCORE

SP1

DNMT3B

Par-4Mt

AATF

PROMOTER-1000 0

p53 Gene

POSITION SCORE -602

12.13 -605

10.7

-61010.2

CpG Islands

SP1 site

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Methyl Groups

1006045

30

20

12

Mol wt (kDa)

LC3I (18 kDa)LC3II (16 kDa)

Contro

l

Trans

fected

AATF (Mt)

8

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Mutant AATF & Cell Decision

S. Sharma, D. Kaul et. al. Cell Biol. Int. (2014):1-8

Promoter

p53

Bcl2

Beclin-1 Autophagy

p21;p53

p*AATF(M t)

Dimeric form

Par4

DNMT3B

Monomeric

form

SP1

AATF

AATF

Immortality

DNA Methylation addiction

Molecular Sweet Tooth

Cell: Oncogenic Transformation

CELL : Molecular Sweet Tooth

Exosome Secretion Apoptotic Mimicry

Immuno-privilegeAerobic - Glycolysis

Lactate

Glucose

pyruvateO2

CO2

Lactate95%5%

Protein

RNAmiRNA

PSPS

AATF Genome

GUUAGGGCCAACAUCUCUUGG

miR-2909 precursor

miR-2909Discovered

by Prof. D. Kaul (2009)

Exon 1 Exon 2 to 11 Exon 12

(NCBI Accession No.: GSE54949)

ImmunityCancerMetabolismPhysiological processes

Blood Cells Mol Dis. 2015;55:89-93 Blood Cells Mol Dis. 2015;54:342-7 Mol Immunol. 2015;64:210-7 Cell Biol Int. 2015;39:326-33 Mol Cancer. 2014;13:175 Mol Cell Biochem 2014;392:49-63 Gene 2014;536:326-331 Gene 2013;522:60-64 Cell Biochem Funct 2012;30:500-504

Kaul,D.et.al.

Series10

2.5

5

Rel

ativ

e E

xpre

ssio

n of

miR

-290

9

(

Cha

nge

in Δ

Ct va

lue)

HEK 293PBMCs

HeLaPC-3

Se-ries1

0

2.5

5

HEK 293PC-3

PBMCsHeLa

miR

-290

9 E

xpre

ssio

n

in

Exo

som

es

Series10

2.5

5

miR

-290

9 E

ctop

ic -

Exp

ress

ion

Control v

ector

transfe

cted ce

lls

miR-2909 vecto

r

transfe

cted ce

lls

PBMCs

HeLa

Protein

RNAmiRNA

PSPS

Kaul et al. Mol Cell. Biochem. 2014; 392:49-63

UCP2

CCL5 UCP2 IL170

0.5

1 Control AATF Transfected

IL17

CCL5

HistonePro

tein

Exp

ress

ion

**

**

**

Control AATF Transfected0

2.5

5

Rel

ativ

e E

xpre

ssio

n of

m

iR-2

909

**

0

2

4

Tran

slat

iona

l E

xpre

ssio

n of

AAT

F

**

AATF

Histone

CCL5 (9 kDa)

Control miR-2909 Transfected

0

0.2

0.4

Tran

slat

iona

l E

xpre

ssio

n of

CC

L5

Histone

**

Control AATF Transfected0

4

8

Tran

scrip

tiona

l E

xpre

ssio

n of

p53

**

Kaul et al. Blood Cells Mol Dis. 2015;55:89-93

UCP2

Promoter

-62 -48 -36

KLF4 Binding Site

0

3

6

UC

P2

Exp

ress

ion

(Tra

nscr

iptio

nal L

evel

) ** **

UCP2 Histone

IL17

IL17 UCP20

3

6

ControlmiR-2909miR-2909 + antagomiR-2909

Prot

ein

Expr

essi

on

** **

** **

Position

Kaul et al. Blood Cells Mol Dis. 2015;55:89-93

miR-2909

KLF4CYLD UCP2

Aerobic - Glycolysis

Inclusion Criteria Exclusion Criteria

Control : Without hematological malignancy - 25

Test: With hematological malignancy B-ALL – 18 T-ALL – 7

Age: Less than 14 years Immunophenotyping:

B- Cell lineage Markers T-Cell lineage MarkersCD19, CD22, CD79a CD7, CD2, CD3, CD5CD10, CD20, CD24

Adults: Patients more than 14 years of age

MixedLineage

Subject Selection

(N=50)

Paediatric Acute Lymphoblastic Leukemia

B-Contro

l

B-ALL

T-Contro

l

T-ALL

0

2

4

6

8

Rel

ativ

e m

iR-2

909

expr

essi

on( f

old

chan

ge)

**

**

*

Se-ries1

-10

-8

-6

-4

-2

0

2

4

Rel

ativ

e K

LF4

mR

NA

exp

ress

ion

(f

old

chan

ge)

**

B-C

ontr

ol

B-A

LL

T-C

ontr

ol

T-A

LL

β-actin β-actin

KLF4

KLF4

T-Con-trol

T-ALL0

200

400

600

800

KLF

4 P

rote

in e

xpre

ssio

n(%

of c

ontro

l) *

B-Con-trol

B-ALL0

20

40

60

80

100

120K

LF4

Pro

tein

exp

ress

ion

(% o

f con

trol)

*

51KDa 56 KDa

Activation Domain Repressor Domain DNA binding Domain3 Zinc fingers

N 3’UTR5’UTR C

T-ALL : KLF-4 Genome

D. Malik, D. Kaul et al. Molecular Cancer 2014, 13:175

D. Malik, D. Kaul et al. Molecular Cancer 2014, 13:175

Cell Proliferation

MYC CCND1SP1

AATF

Apoptosis

AATF SP1

KLF4

miR-2909

KLF4(M)

BCL3

CCND1

p 2

1

Cell Proliferation

MYC

3’ UTR

p 21T-AL

LB-

ALL

CYLDCa2+

channel signaling

Survival & Apoptosis

Antiviral response

Inflammation

T-cell developme

nt & Activation

Spermato-genesis

Rela

tive

expr

essio

n of

CYL

D

(Cha

nge

in Δ

C t val

ue)

T-Control T-ALL Patient0

2

4

**

Arora et al. Blood Cells Mol Dis. 2015, 54:132-8

Promoter

-124

CYLDSP1

T-Control T-ALL0

50

100

150

200

SP1

prot

ein

expr

essio

n

(% o

f co

ntro

l)

**

T-Control T-ALL0

100

200

300

400

500

600

CYLD

pro

mot

er a

ctivi

ty

β-G

al A

ctivi

ty(n

mol

es o

f ONP

G h

ydro

lyzed

(nm

ol/m

g pr

otei

n)

110kDa

35kDa

T-Control

T-ALL

Arora et al. Blood Cells Mol Dis. 2015, 54:132-8

T-ALL_3_Exon_7

T-Control_3_Exon_7

CAP_Gly DUBCAP_Gly CAP_Gly

127 203 232 303 472 540 956

hCYLDDIIPALSESVTQERRPPKLAFMSR GVGDKGSSSHNKPKATGSTSDPGNRNRSELFYTLNG

MALT1 Cleavage

CAP_Gly CAP_Gly

127 203 232 318

109 KDa

35 KDaDegraded CYLD

Wild Type CYLD

P

?

Promoter

-82

MALT1SP1

Control KLF-4 Transfected0

20

40

60

80

100

120

MAL

T1 p

rom

oter

act

ivity

β-G

al A

ctivi

ty (n

mol

es o

f ONP

G h

ydro

lyzed

(nm

ol/m

g pr

otei

n)

**

Kaul et al. Mol Immuno. 2015, 64(1):210-7

T-Control T-ALL0

2.5

5

MAL

T1 e

xpre

ssio

n

(Tra

nsla

tiona

l Lev

el)

**

β actin

MALT1

Arora et al. Blood Cells Mol Dis. 2015, 54:132-8

Gain of functio

n

Loss of functio

n

MA

LT1

Exp

ress

ion

of

(Tra

nsla

tiona

l Le

vel)

0

1.5

3**

**

MALT1

β-actin

Arora et al. Blood Cells Mol Dis. 2015, 54:132-8

110 KDa

70 KDa

35 KDa

CYLD Protein Expression

ControlMALT1 Transfected

MALT1 + siRNA MALT1

110 KDa

Contro

l

MALT1 T

ransfe

cted

MALT1 +

siMALT

10

1.5

3

miR

2909

Exp

ress

ion

(Tra

nscr

iptio

nal l

evel

) ** **

Series10

10

20

30 ** **

Control miR-2909 Transfected

miR-2909 + antagomiR-2909

Transfected

Kaul et al. Mol Immuno. 2015, 64(1):210-7

MA

LT1

Exp

ress

ion

(Tra

nscr

iptio

nal l

evel

)

Kaul D et al. Mol. Immunol .2015, 64(1):210-17

Selection

& Analysis

CYLD (Mt; 35KDa) Cloning

AA PCR Product

BGH pA

f1 oriSV40 ori

EM-7

SV40 pA

pUC ori

Pbla

P EF-1α

T7

Asp

718

IK

pn I

Bam

H I

Spe

IB

stX

I TT

P P

TOPOTOPO

Eco

RV

Bst

X I

Not

IXb

a I V5

epitope 6XHis Pme

I

PCR Amplification

Ligation

Transformation

Plasmid Isolation

into Competent E. coli Cells

PCR Product Purification

Poly A Tailing

T-ALL: CYLD (Mt)

pEF6/V5-His-TOPO

Control Transfected0

5

10

15

Control

CYLD (N terminal)

**

35kDaTransfected

Dip G1: 56.1 %Dip G2: 0.00 %Dip S: 43.9 %

Dip G1: 100 %Dip G2: 0 % Dip S: 0 %

Control Transfected

GO/G1 S G2/M0

20

40

60

80

100

120ControlTransfected

% o

f tot

al c

ell n

umbe

r**

Rel A/p65

Histone H3

pIΚBα

β-actin

Nuclear Extract

Cytoplasmic Extract

Cont

rol

Tran

sfec

ted

64 kDa

18 kDa

36 kDa

42 kDa

Arora et al. Blood Cells Mol Dis. 2015, 54(1):132-8

CYLD

exp

ress

ion

(T

rans

latio

nal L

evel

)

IL-6 c-Myc Bcl-3 TNF-α Par-4 AATF Cyclin-E Cyclin-D Notch10

4

8

12

Control

Transfected

Expr

essio

n of

var

ious

gen

es(T

rans

crip

tiona

l Lev

el)

******

****

**

**

**

**

Arora,M & Kaul,D. et al. Blood Cells Mol Dis. 2015, 54:132-8

Series1

-3

-1.5

0

1.5

3

**

Rela

tive

expr

essio

n of

miR

-290

9

(C

hang

e in

ΔC t v

alue

)

Contro

l

35kD

a CYLD

Trans

fected

CD1a CD2 CD4 CD5 CD7 CD80

20

40

60

80

100ControlTransfected

Perc

enta

ge o

f cel

ls

CD13 CD10 CD19 CD20 CD3 HLADR0

20

40

60ControlTransfected

Perc

enta

ge o

f cel

ls

CD33 CD34 CD38 CD79a CD117 CD3 TdT0

20

40

60ControlTransfected

Perc

enta

ge o

f cel

ls

**

**

****

**

**

**

**

**

****

Arora et al. Blood Cells Mol Dis. 2015, 54:132-8

B-Cell Lineage ALL

CD10; CD19; CD20; CD22; CD79a; TdT; HLA-DR; CD34

T-Cell Lineage ALL

CD2; CD3; CD5; CD7 TdT; HLA-DR; CD34

35kDa CYLD

MALT1

SP1

miR-2909

NF-kB

Cellular Proliferation

Impaired Apoptosis

Unregulated Immaturity cellular

markers

3’UTR

Cellular Proliferation

Increased ApoptosisKLF4 (M)

T-ALL

Arora,M. & Kaul,D. et al. Blood. Cells. Mol. Dis. 2015, 54(1):132-138

TCAATP

Pyruvate

Glycolysis

Lactate

p53

NO

iNOS

miR-2909

KLF4

UCP2

AATF

CYLD

Bmi-1

x

xx

Kaul,D. et. al. Blood Cells. Mol. Diseases 55 (2015) 89-93Kaul,D. et. al. Mol. Immunology 64 (2015) 210-217Arora,M. & Kaul,D. et al. Blood. Cells. Mol. Dis. 2015, 54(1):132-138Malik,D & Kaul,D. et al. Mol. Cancer 13 ( 2014) 175-191

AATF

Paediatric Acute Lymphoblastic leukemia

DNMT3B Inhibitors

AntagomiR-2909

FROM OUR STUDY….. Therapeutic Lessons

+

Effective Cancer Therapy

can be achieved by

Early Apoptosis

15.1% 9.5% 8.2% 10.4%

Late Apoptosis

30% 48.3% 54.3% 63.3%

HEK293 cells : Apoptosis Assay at the end of 48 hrs. Incubation period .

Cells +DNMT3B-siRNA +AntagomiR-2909

Cells +DNMT3B-siRNA

Cells +AntagomiR-2909

Control Cells

Cell Fate

Research Group

Ph.D Scholars

• Shaveta Sharma

• Mansi Arora

• Sugandha Sharma

• Anuradha Garg

• Nalini Chauhan

• Sameena Bani

• Hitaishi Kaushik

Technical / Bioinformatics Support Staff

• Dr. Ashvinder Raina

• Sangeeta Supehia

• Ajit Singh

• Ritu Soni

• S.P. Bedi

Post Doctoral Fellows • Dr. Gazalla Khan

• Dr. Maya Ramdas

• Dr. Mansi Arora

• Dr. Deepti Malik

Research Collaborators

Dr. Neelam Verma Professor & HeadDeptt. of HaematologyPGIMER, Chandigarh

Late Prof. Rakesh MarwahaEx. Chief Clinical Oncology Advance Paediatric CentrePGIMER, Chandigarh

The City Beautiful: Chandigarh