aatf genome : importance in oncogenesis dr. deepak kaul ph.d(aiims); f.r.c. path (london); fna;...
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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
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