Transcript PNH - Siteman Cancer Center

Special Emphasis Pathway in Cancer Biology
Paroxysmal Nocturnal Hemoglobinuria
(PNH)
Monica Bessler, MD, PhD
Washington University School of Medicine St. Louis
Division of Hematology
Email [email protected]
Web: http://bmf.im.wustl.edu
Bone Marrow Failure
Normal bone marrow
Bone marrow failure
Hematopoiesis Occurs in the Bone Marrow
Bondurant et al.
Wintrobe 1993
Causes of Bone Marrow Failure
Acquired
Inherited
Secondary bone marrow failure
Radiation
Drug and chemicals (e.g. Benzene)
Idiosyncratic reactions (e.g. Chloramphenicol)
Virues (e.g. EBV, Hepatitis, CMV)
Immune diseases (e.g. Graft-versus-host disease)
Pregnancy
FanconiÕ
s anemia
Dyskeratosis congenita (DC)
Shwachman Diamond Syndrome
Diamond Blackfan Anemia (DBA)
Am egakaryocytic thrombocytopenia
Severe congenital neutropenia
Cartilage-Hair Hypoplasia
Reticular Dysgenesis
Thrombocytopenia with absent radii
Pearson syndrome
Non hematologic syndromes
(e.g. DownÕ
s syndrome)
Familial aplastic anemia
Idiopathic bone marrow failure
Paroxysmal Nocturnal Hemoglobinuria (PNH)
Fig. 1. Chromosomal translocations involving HMGA2, and the influence of let-7 on protein expression
Let 7 tumor spressor
oncogene
Tumor supressor
F9
NIH 3T3
24hrs
48 hrs
C. Mayr et al., Science 315, 1576 -1579 (2007)
Published by AAAS
Fig. 2. Luciferase reporter assays showing the influence of miRNA-target pairing
F9
miRNA to let-7a
NIH 3T3
HeLa
miRNA to mlet-7a
C. Mayr et al., Science 315, 1576 -1579 (2007)
Published by AAAS
Fig. 3. Soft-agar assay for anchorage-independent growth
NIH 3T3
C. Mayr et al., Science 315, 1576 -1579 (2007)
Published by AAAS
Fig. S1
Paroxysmal Nocturnal Hemoglobinuria
Clinical Manifestations of PNH
II.
I.
Hemoglobinuria Bone Marrow Failure
III.
Thrombosis
Age Distribution of Patients with PNH
J Nishimura et. al. Medicine 2004
Tiananmen Square
Institute of Hematology & Blood
Diseases Hospital
Chinese Academy of Medical
Science
Tianjin
China
Peking Union Medical College
Beijing
China
Median Survival of Patients Diagnosed with PNH is
10 -15 Years
Thromboembolism in PNH
GPI-linked Proteins on Human Blood Cells
Hematopoietic
Stem Cell
CD59
CD90
CD109
Platelets Red Cells Granulocytes Monocytes B Cells T Cells NK Cells
CD55
CD58 1
CD59
CD109
(Gova/bAg)
PrPc
GP500
CD55, CD59 CD55 CD58 1
(Cromer Ag) CD59 CD14
CD58, PrPc, CD16 CD24
(NAB1-Ag)
AChE
CD48 CD66b
(Cartwrigt
CD66c CD87
-Ag),
CD109 CD157
CDw108
NB1
(John-Milton LAP
-Hagen Ag) PrPc ADPDombroch RT
p50-80 GPI-80
residue
Holley Gregory AG
CD55
CD58 1
CD59
CD14
{CD16 2}
CD48
CDw52
CD87
CD109
CD157
Group-8
PrPc
GPI-80
CD55
CD58 1
CD59
CD24
CD48
CDw52
{CD73}
{CDw108}
PrPc
CD55
CD58 1
CD59
{CD16 2}
CD48
CDw52
{CD73}
CD87
{CD90}
CDw108
{CD109}
PrPc
ADP-RT
CD55
CD58 1
CD59
CD16 2
CD48
CDw52
PrPc
Increased Sensitivity of Red Cells to
Activated Complement
PNH
Control
HamTest: 1938
S HS S HS
PNH patient
Deficiency of GPI-Linked Proteins
on PNH-Granulocytes
Diagnosis of PNH by Flow
Cytometry
PMN
Sideward scatter
Control
Patient
Red Cells
CD59
Monocytes
Lymph.
PNH red cells are of clonal origin
(Oni et al. 1970)
G6PD A
G6PD B
Male normal Male normal Female PNH
RBCs
RBCs
Total RBCs
PNH RBCs
GPI-Anchor
NH2
P EthN Phosphoethanolamine
Protein
Man
Mannose
GlcN
Glucosamine
EthN
C=O
P
Man
Man
P EthN
Man
EthN
GlcN
P
I
Membrane
P
C-C-C
OO
C=O
I
P Inositolphosphate
Pathway of GPI-anchor Biosynthesis
GDP
Dol-P DPM1
DPM2
DPM3
MPDU1
GDP
PIGA
PIGC
PIGH
GPI1
Dol-P
GPIP
Dol-P
DMP2
Dol-P
acylDol-P
?
DG PIGV
UDP Ac UDP acetate CoA PIGM
PIGN
PIGX
PIGL
PIGW
Dol-P
Dol-P
PIGB
PIGF
DG
PIGO
GPI7
Ac
Endoplasmic reticulum
DG
PIGS
PIGT
PIGU
PIGK
GAA1
Ac
NH2
GlcNAc
Man
PEth
PI
NH 2
COOH
COOH
GPI-biosynthesis in
PNH cell lines
[3H] mannose label
Block in the Pathway of GPI-anchor
Biosynthesis
GDP
Dol-P DPM1
DPM2
DPM3
MPDU1
GDP
PIGA
PIGC
PIGH
GPI1
Dol-P
GPIP
Dol-P
DMP2
Dol-P
acylDol-P
?
DG PIGV
UDP Ac UDP acetate CoA PIGM
PIGN
PIGX
PIGL
PIGW
Dol-P
Dol-P
PIGB
PIGF
DG
PIGO
GPI7
Ac
Endoplasmic reticulum
DG
PIGS
PIGT
PIGU
PIGK
GAA1
Ac
NH2
GlcNAc
Man
PEth
PI
NH 2
COOH
COOH
GPI-N-acetylglucosaminyltransferase
Correction of the PNH Phenotype in
LCL after PIGA cDNA Tranfection
Anti-CD59-FITC
The PIGA Gene and the Mutations found in PNH
Luzzatto, 2000
PIGA maps to
the X-Chromosome
Major areas of investigation
• Why does a PNH hematopoietic stem cell missing so
many different proteins take over normal hematopoiesis ?
•What is the reason for clotting ?
• Can we design a more targeted treatment for patients
with PNH ?
Erythroid Differentiation of PIGA- ES Cells
Mouse Model for PNH
1
2
34 5
wt Piga
loxP loxP
loxPiga
lacZ
Cre
D Piga
lacZ
6
Blood Cells Deficient in
GPI-Linked Proteins
Mouse
Human
∆ PIGA ∆ PIGA Control
PNH
Control
Red cells
Granulocytes
GPI-linked proteins
∆ PIGA Control
GPI-linked proteins
Acidified Serum Lysis
Ham-Dacie - Test
PNH
Control
S HS S HS
(Heterologous Serum
HS, Heat inactivated)
Tremml et. al. 1999
S HS S HS
PNH Blood Cells in Mouse and Man
Mouse
Male
PNH
Female
PNH
Human
wt
control
Patient with
PNH
Healthy
individual
Red
blood
cells
Lineage
specific
marker
Granulocytes
GPI-linked marker
Blood Cells Deficient in
GPI-Linked Proteins in Mice
100
% GPI- Cells
80
60
40
20
0
80
60
40
20
0
1
2
4
8
12
1
Age (months)
80
80
% GPI- Cells
100
100
60
40
20
0
1
2
4
Age (months)
2
4
8
12
Age (months)
B Cells
% GPI-Cells
% GPI- Cells
Granulocytes
Red Blood Cells
100
8
12
T Cells
60
40
20
0
1
2
4
Age (months)
8
12
Lactate dehydrogenase
(LDH)
Units RBC
Case report:
31 year old female PNH patient # 99
Red cell transfusions
12
8
4
DVT
3000
2000
1000
Eculizumab
C5 blockade
ATG+Cy
x3
500
0
8/93
Normal range
1/99
AA
5/00
9/01
2/03
6/04
PNH
11/05
3/07
Peripheral blood
cell count
Relationship of PNH with Aplastic Anemia (AA)
Normal
PNH Normal PNH
Normal
PNH
Cytopenia
Hemolytic / Classical PNH
AA/ PNH
Adapted from Rotoli & Luzzatto 1989
Cytogenetic Abnormalities in PNH
Cytoge ne ticAbnorm alitie sin PNH
(Araten 2001)
Trisomy 8 in MDS/PNH
U.P.N.
Age
% PNH
(Longo et. al. 1994)
MSK 18
MSK 30
MSK 8
MSK 20
MSK 17
MSK 49
MSK 23
MSK 21
MSK 19
HH 56
MSK 16
31M
20M
57M
16M
42M
32M
27M
26M
63M
19F
45M
7
5
100
98
59
36
70
82
80
60
100
Chromosome 8
Trisomy 8
Trisomy 8
PNH clone
PNH within MDS
%Blood Cells
PNH clone
47, XY, +6 [6/20]
47,X,+5,del(5)(q11.2)(6/15)
47,XY ,+X[20/20]
46,XY ,t(17;19)(q11;q13)[6/20]
45,XY,-7[14/25]
47, XY +6[12/20]
46,XY ,del(8p)[2/30]
47,XY ,+8[2/20]
46,XY ,del(5)(q15q31)[2/17]
46,XX,del(13)(q12q14)[5/10]
47,XY ,+8[6/30]
PNH and MDS
%Blood Cells
% Blood Cells
MDS within PNH
Karyoty pic abnormality
Trisomy 8
PNH clone
Leukemogenesis in PNH Mice After ENU
Tumor
Ovary (Adenoma)
Lung ( Adenoma)
Adenocarcinoma
Leukemia
Myeloid hyperplasia in
the bone marrow
Myeloid hyperplasia of
the spleen
Liver infiltration
Parasternal infiltration
Leukemia in PNH mice
PNH
3
1
2
6/15
WT
1
3
0
4/15
2
2
2
2
2
2/15
1
2
1
2/15
The Rate of Transformation is Similar
in AA and PNH
MDS / AM L in Patients with Aplastic Anemia (AA)
Study
Tichelli
DePlanqu e
Socie
Paquette
Doney
Ohara
(1988)
(1989)
(1993)
(1995)
(1997)
(1997)
137
468
860
155
227
119
7
1 (15-25)
4
2 (13)
10 (11)
10
No of patients
% MDS / AML
(Incidence)
MDS / AML in Patients with PNH
Hillmen
Socie
Spaet-Schwal be
Moyo
(1995)
(1995)
(1995)
(2004)
No of patients
80
220
40
49
209
% MDS / AML
(Incidence)
0
1 (5)
0
6
8
Study
Nishimura (2004)
Japan
W are
Eibrink
(1991)
(2005)
176
49
11
16
2
36
Duke
Possible Interrelationship Between PNH,
Aplastic Anemia, Myelodysplastic Syndrome,
and Acute Myeloid Leukemia
Model for the Development of PNH
PNH
Survival advantage hypothesis
Normal Marrow
BM Injury
PNH
2 hit hypothesis
Adaptive Mutations
Normal Cells
BM Injury
Mutation
Clinical Outcome
PIGA(-) Cells
PIGA(-) Cells + Second Hit Mutation
Figure 3. Effects of the chromosome 12 abnormalities in 2 patients with PNH
Inoue, N. et al. Blood 2006;108:4232-4236
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.
Regulation of Ink4a/Arf during Aging
Tzatsos &Bardeesy Cell Stem Cell 2008
HMGA2 (a second factor for expansion of a PNH clone?)
Mouse HMGA2
•
•
Expression of high mobility group AT-hook 2 (HMGA2), which is regulated by let-7 miRNAs
(Mayr C et al, Science, 2007), contributes to proliferation of cells.
Chromosomal abnormalities involving in HMGA2, which remove let-7-complementary sites
in 3’-untranslated region (UTR), have been reported in patients with PNH (Inoue N et al.,
Blood, 2006) and related disorders including myeloproliferative disorders (Guglielmelli P et
al., Stem Cells, 2007) and AML with myelodysplasia (Odero MD et al., Leukemia, 2005).
Introduction of truncated cDNA clone of Hmga2 into pPGKPuro
Complementary
sites of let-7a
1
(
2
3
45
67
)
cDNA
BC052158 5’-UTR coding
XhoI
3’-UTR
ClaI
SalI
XhoI
Replace puro with the truncated cDNA of Hmga2
ClaI
SalI
Cleaved recombinant with SalI for injection
SalI
SalI
PGK-PROM
5’-UTR
Coding
Specific primer pair
PGK-PA
Homologous recombination for
conditional truncation of Hmga2 3’-UTR
Exon 1
wt Hmga2
IRES/NEOlox-Hmga2
2
3
4
5
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IRES/NEO
loxP
loxP
Expression of Hmga2 in Truncated-Hmga2+ TG mouse
1.4
qRT-PCR
Relative expression
TG (n=3 or 4)
1.2
WT (n=3)
P<0.01
1
0.8
0.6
P<0.05
P=0.0509
0.4
P<0.02
0.2
0
BM
Western
Blotting
Spleen
Thymus
Liver
Actin
42 kDa
HMGA2
17 kDa
TG WT
BM
TG WT
Spleen
TG WT
Thymus
TG WT
Liver
Peripheral blood count
•
Age- and sex-matched 19 truncated Hmga2+ TG and wild-type BL6 mice
Competitive repopulation assay
vs
Donors
Truncated-Hmga2+
(CD45.2)
Prepare BM cells
1.
2.
3.
50%
10%
100%
PEP
(CD45.1)
vs
vs
vs
50%
90%
0%
5 x 106 cells
Total 1000 rad
Injection
(BM transplantation; BMT)
Recipients
PEP
Observe chimerisms of PB cells 6 weeks and 10 to 12 weeks after BMT
Proportions of donor-derived cells after competitive repopulation assay
60
6w (n=2)
50
12w (n=3)
40
10% Hmga2
90% Recipient
30
20
10
50% Hmga2
50% Recipient
Granulocytes
Monocytes
T cells
B cells
%CD45.2+ cells
0
Granulocytes
Monocytes
T cells
B cells
100% Hmga2
Granulocytes
Monocytes
T cells
B cells
Breed truncated Hmga2+ mice with Piga- mice to
see effect of Hmga2 on growth advantage of PNH
hematopoietic cells
X
Pigawith
1% GPI60% GPI- etc.
Piga- (without truncated Hmga2)
Truncated-Hmga2+/-
Truncated-Hmga2+/-Piga-
2 mice
1 mouse
(need more breeding)
•
•
•
•
Time courses of changes in proportion of GPI- cells
Blood cell count
Competitive repopulation assay
Spontaneous or ENU-induced leukemogenesis
Acknowledgements
Washington University St. Louis:
Kazu Ikeda
Jeffrey Hicken
Marek Jasinsky
Peter Keller
Bing Han Ike Pantazopoulos
Shashikant Kulkarni
Philip J. Mason
Division of Hematology
Morey Blinder
Joshua Fields
Bone Marrow Transplant Team
John DiPersio
Peter Westervelt
Hereditary Cancer Core
Chissie Kamp
Jennifer Ivanovich
Division of Rheumatology
Celia Fang
John Atkinson
Dept. Pathology
Richard Burack
Susan Treese
Center for Clinical Studies
Participating Medical Centers
For
TRIUMPH
SEPHERED
EMBRACE
All patients with PNH for participating