Ruolo del microRNA-223 nella mielopoiesi normale e leucemica Clara Nervi Simposio SIES 41° Congresso Nazionale della Società Italiana di Ematologia Bologna 16 ottobre 2007 FONDAZIONE PARCO BIOMEDICO SAN RAFFAELE
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Ruolo del microRNA-223nella mielopoiesi normale e leucemica
Clara Nervi
Simposio SIES
41° CongressoNazionale della Società Italiana di Ematologia
Bologna 16 ottobre 2007
FONDAZIONE
PARCO BIOMEDICO
SAN RAFFAELE
PluripotentStem cells
MultipotentProgenitor
CommittedPrecursors
Maturecells
LT-HSC
Lymphoid
Myeloid
B cell
T cell
Erythrocytes
Platelets
Monocyte
GranulocyteRARa
GATA-1GATA-2 MEP
GMP
Lineages-specific transcription factors play key roles in hematopoiesis and lineage differentiation
AML-1Scl/Tal-1
LM02, Mll, TelHoxB4
PU-1
C/E
BPa
C/EBPa
FOG-1/GATA-1
IkarosPU.1
ST-HSC MPP
Epigenetic modifications of DNA (methylation) and chromatin (acetylation and methylation of histones) affect the transcription of genes and regulate proper cell function, proliferation/differentiation and tissuespecificity
5’ 3’
CpG
Inactive chromatin state
MeK9H3
MeK4H3
HP1 HP1 HP1 HP1
HMT
HDAC
MBPDNMTs
Transcriptional SilencingX
TF
From: Zardo et al, Cell Res 2005
3’
HATTF
AcK9H3
MeK4H3 TF
Active chromatin state
Transcriptional
activation
5’
CpG
microRNAs (21-28 nt):
¬3000 microRNA genes identified in C.elegans,Drosophila, mouse and humans (>500) Act as negative regulators of the expression of genesinvolved in:
developmentdifferentiationproliferationapoptosisstress response
Role in oncogenesis
Nucleus
Cytosol
miRNA gene
Pri-miRNA
microRNAs production and function
Dro
sha
DGCR8
Pre-miRNA
Incorporation intothe RISC Complex
ss-miRNA
TRBPDicer
AGO
miRNAduplex
targetmRNA
perfect pairing
mRNAcleavage
3’
Translational repression
Incomplete pairing
3’
The expression of miRNAs is tissue-specific and highly regulated according to the
cell’s developmental lineage and stage
CD34+: stem/progenitor cells: 4% of BM cells
CD34-: in the BM 60-75% are myeloid cells at different stages of neutrophylic differentation
PB mononucleated cells: 50-70% are mature granulocytes
It is miR-223 expression associated with human granulocytopoiesis?
miR-142-5
CD
34+
CD
34-
PB
MN
CB
cel
lsT
cel
ls
miR-223
miR-181a
U6
BMTissue expression ofmiRNAs cloned from mousebone marrow (Chen at alScience 2004)in human hematopoietic cells
Acute Promyelocytic Leukemia
+ RA
Outcome results in APL prior and after all-trans-retinoic acid (ATRA)
Years
1 2 3 4 5 60
miR-223
miR-142-5
miR-181a
U6
0 24 72RA 0 4 24 48 72
APL-1 APL-2
miR-223 is up-regulated by RA in primaryAPL blasts undergoing granulocytic differentiation
in vitro and in vivo
A mini-circuitry comprising miR-223, CCAAT-boxbinding transcription factors NFI-A and C/EBPa
regulates granulocytopoiesis
+RA
Granulocytic differentiation
miR-223 gene
miR-223
NFI-A mRNA
C/EBPa
3’UTRAAAAA 3’
5’
NFI-A
AAAAA 3’
miR-223 gene
C/EBPa
NFI-A miR-223
NFI-A mRNA3’UTR5’
Fazi F. et al. Cell 123:819, 2005
0
5
10
15
20
25
% o
f p
osi
tiv
e ce
lls
vector
Lenti-223
CD11b+ CD14+
0
0,5
1
1,5
2
2,5
vector
Lenti-223
G-CSFr GM-CSFr
mR
NA
s
Lenti-223Vector
Vec
tor
Len
ti-22
3miR-223
U6
U
Stable ectopic expression of miR-223 increases thegranulocytic differentiation of APL blasts
Lenti-223
5’LTR GFP 3’LTR
pre-miR-223 TU1PU1
PPGK
5’LTR GFP 3’LTR
pre-miR-223 TU1PU1
PPGK
1. NFI-A and miR-223 has novel regulators of granulocyticdifferentiation
2. Key role for the transcriptional regulation of miR-223expression in human myelopoiesis.
3. A titrated miR-223 production reprograms myeloiddifferentiation in the APL NB4 cell line
Conclusions (1)
Role for a de-regulated miR-223 transcription andexpression in myeloid leukemias?
Table 1: Morphological and genetic features of primary leukemia samples
Patient N° Morphology by FAB Karyoty pe
1
AML/M0
46,XY,der(12)t(12;?)(q23;?)/46,XY
2 AML/M0 Complex aberrations*
3 MDS-AML/M1 46,XY,del(7)(q31)
4 AML/M1 47,XX,+11
5 MDS-AML/M1 not available
6 MDS-AML/M1 46,XY,t(3;7;10)(q27;p?;p?),del(7)q31
7 AML/M2 not available
8 AML/M2 47,XX,add(12)(q23),+mar
9 AML/M2 46,XX
10 AML/M2 not available
11 AML/M2 46,XX,t(8;21)(q22;q22)/47, idem, +15
12 AML/M2 46,XY,t(8;21)(q22;q22)
13 AML/M2 46,XX,t(8;21)(q22;q22)
14 AML/M2 46,XY,t(8;21)(q22;q22)
15 AML/M3 46,XY,t(15;17)(q22;q21)
16 AML/M3 46,XY,t(15;17)(q22;q21)
17 AML/M3 46,XY,t(11;17)(q23;q21)
18 AML/M3 46,XY,t(15:17)(q22;q21)
19 AML/M4eo 46,XY
20 AML/M4 46,XY,t(4;16)(q25;q22)
21 AML/M4 not available
22 AML/M4 not available
23 AML/M4 -M5 not available
24 AML/M5 not available
25 AML/M5a 45,XY,del(7), -16
26 AML/M6 46,XY
27 AML/M6 not available
28 CML Blast crisis 45,XX,-7,t(9;22)(q34;q11)
29 Leukemic Mantle Cell Lymphoma 46,XY
30 ALL 46,XY,del(12)(p13;pter)/46,XY
31 ALL 46,XX
miR-223 expression levels in human primaryhematopoietic cells and in acute myeloid leukemias
(AML)
t(8;21)
M0 M1 M2 M3 M4 M5
L-M
CL
CM
L-B
C
132 3 4 5 7 8 9
10
11
12
14
15
16
17
19
20
21
22
24
25
26 28
291 6
AML
27
M6
AL
L
18
23
30
31
t(8;21)
M0 M1 M2 M3 M4 M5
L-M
CL
L-M
CL
CM
L-B
C
132 3 4 5 7 8 9
10
11
12
14
15
16
17
19
20
21
22
24
25
26 28
291 6
AML
27
M6M6
AL
LA
LL
18
23
30
31
0.0
0.5
1.0
1.5
PB
PB
CB
CD34+MNC
BM
BM
0.0
0.5
1.0
1.5
PB
PB
CB
CD34+MNC
BM
BM
miR-223 expression is down-regulated inAML1/ETO-positive cell lines
AML1
miR
-22
3A/E
U937
_-tubulin
A/E
-HA
Mo
ck
HL
60
NB
4K
56
2
Mo
ck
siA
/E
WT
SKNO-1 SKNO-1
AM
L1
t(8;21)
AML1/ETO+
-426 gttgtggtta -415AML-1
AML1 binding site is present on the pre-miR-223 promoter
+1 +203-1500
C/EBP-730 gctaaatcaattgccaattag -709
C/EBP
NFI-A
pre-miR-223
+137
TGTGGTAML1 target genes
HDAC/DNMTs
corepressor
complex
XAML1 ETO
HDAC
Gelmetti V. et al. MCB 1998Fazi F. et al Blood 2007
-426 +1
AML -1
-203 +203-1500 -426 +1
AML -1
-203 +203-1500pre-miR-223
oligo1
+137
miR-223 is a direct transcriptional target of AML1/ETO oligo12
-2000
Inpu
t
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
Inpu
t
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
No-A
b
-HA
-AM
L1
Input
-ET
O
Mock
A/E
-HA
SK
NO
-1
U937
No-A
b
-HA
-AM
L1
Input
-ET
O
Mock
A/E
-HA
SK
NO
-1
U937
Mock
A/E
HA
SK
NO
1
U937
Mock
A/E
HA
SK
NO
1
U937
AML1/ETO recruits chromatin remodeling enzymes at this AML1-site on pre-miR-223 promoter
Inpu
t
No-A
b
-HD
AC
1
-MeC
P2
-DM
T3b
Inpu
t
-DM
T3a
-DM
T1
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
Inpu
t
No-A
b
-HD
AC
1
-MeC
P2
-DM
T3b
Inpu
t
-DM
T3a
-DM
T1
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2
GAPDH
oligo1
oligo2Moc
kA
/EH
AS
KN
O1
U93
7
Moc
kA
/EH
AS
KN
O1
U93
7
Moc
A/E
HA
SK
NO
1
U937
Moc
A/E
HA
SK
NO
1
U937
+1 +203-1500pre-miR-223AML-1 X
AML1 ETO
HDAC
TGGTGT +137
Heterochromatic silencing of pre-miR223 promoter by AML1/ETO
Con
trol
5-a
zacy
tidin
e23%48%46%
% of global methylation
4% 8% 60% 40%66%63%
Methylated CpG
Unmethylated CpG
- GAPDH -
- oligo1 -
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
No
-Ab
-H3A
c
-H4A
c
Inp
ut
WT siA/EMock
SKNO-1
HL60 Mock A/E-HA
U937 SKNO-1 (AML1/ETO+)
+1
-448 +137-203
AML1
TGTGGT+1
-448 +137-203
AML1
TGTGGT
transcription
Chromatin status on pre-miR-223 gene promoter
Myeloid Progenitor
MeCP2DNMTs X
HDAC
+1
-448
ETO
AML1TGTGGT
+137
-203
Silen
cingMeCP2DNMTs X
HDAC
+1
-448
ETO
AML1TGTGGT
+137
-203
Silen
cingAML1/ETO+
Myeloid Progenitors
Fazi F. et al Cancer Cell in press
5-azacytidine and siRNA-A/E relieves miR-223 silencing and restores myeloid differentiation of
SKNO-1 AML1/ETO+ cells m
iR-223
-NFI-A
-tubulin
WT Mock siA/E
AZA +-+- +-
SKNO-1
miR
-223
-NFI-A
-tubulin
WT Mock siA/E
AZA +-+- +-
SKNO-1
% p
osi
tive c
ell
s
CD11b CD14
WT siA/EMock WT siA/EsiA/EMock
AZA +-+- +-
SKNO-1
+-+- +-%
posi
tive c
ell
s
CD11b CD14
WT siA/EMock WT siA/EsiA/EMock
AZA +-+- +-
SKNO-1
+-+- +-
Ectopic expression of miR-223 reprograms myeloiddifferentiation in AML cell lines carrying or not the AML1/ETO
Lenti-223
5’LTR GFP 3’LTR
pre-miR-223 TU1PU1
PPGK
5’LTR GFP 3’LTR
pre-miR-223 TU1PU1
PPGK
Len
ti-2
23
Vecto
r
SKNO-1HL60
Len
ti-2
23
Vecto
r
SKNO-1HL60HL60
0
1
miR
-223
Vector
Lenti-223
SKNO-1
NFI-A--tubulin-
HL60
0
1
miR
-223
Vector
Lenti-223
Vector
Lenti-223
SKNO-1
NFI-A--tubulin-
HL60
4
6
8
10
% o
f p
osi
tiv
e c
ell
s SKNO-1
CD11b CD14 CD11b CD14
HL60
4
6
8
10
% o
f p
osi
tiv
e c
ell
s SKNO-1
CD11b CD14 CD11b CD14
HL60
1
2
M3
M4-M
5
miR-223
fold
inductio
n
1
2
CD11b
Vector
Lenti-223
Vector
Lenti-223
M3
M4-M
5
1
2
M3
M4-M
5
M3
M4-M
5
miR-223miR-223
fold
inductio
n
1
2
CD11bCD11b
Vector
Lenti-223
Vector
Lenti-223
M3
M4-M
5
M3
M4-M
5
Ectopic expression of miR-223 reprograms myeloid differentiation in primary blasts from AML patients
AML/M3 AML/M4-M5
Lenti
-223
Vecto
r
1. microRNA expression can be de-regulated epigenetically
2. a novel oncogenic action for a leukemia fusion protein: theepigenetic silencing of a tissue and developmental stagespecific microRNA
3. de-regulated microRNA production can be linked todifferentiation block underlying myeloid leukemiapathogenesis, independently from the presence of aspecific genetic lesion.
4. miRNAs as additional epigenetic molecular targets fortherapeutic intervention in leukemias.
Conclusions (2)
Collaborators
Francesco FaziGiuseppe ZardoLinda StarnesLorena TravagliniVanessa Gelmetti
Dept of Histology and Medical EmbryologyUniversity of Rome “La Sapienza”&San Raffaele Biomedical Park Foundation
Irene BozzoniAlessandro FaticaAlessandro Rosa
Dept of Genetics and Molecular BiologyUniversity of Rome “La Sapienza”
Department of Biopathology, University of Rome “Tor Vergata”
Giuseppe CiminoMarco ManciniDaniela Diverio
Franco GrignaniSerena Racanicchi
University of Perugia
Department of Cellular Biotechnologies and Hematology, University “La Sapienza”
Francesco Lo CocoEmanuele Ammatuna
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