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Children’s Cancer and Blood Foundation LaboratoriesDivision of Hematology-OncologyDepartment of PediatricsWeill Medical College of Cornell UniversityNew York
New approaches to modulate abnormal erythropoiesis and improve the
transfusion regimen in ß-thalassemia and sickle cell disease
Erythroid Marrow Expansion-Splenomegaly
IneffectiveErythropoiesis
Anemia
Iron Overload
ß-Thalassemia
Mutations in the beta-globin gene
Alpha/Heme Aggregates
Thrombosis
Adult Hemoglobin
Heme
Heme
Consequences of Reduced ß-Globin Chain Production
Normal vs. stress erythropoiesis
Normal
Proliferation
Differentiation
Lenox et al, Blood 2005.Menon et al. JCI 2006.Marinkovic et al. JCI 2007.Harandi et al. JCI 2010.Ulyanova et al. Blood 2011.
Stress erythropoies
is
Proliferation
Differentiation
Proliferation
Differentiation
Stress vs. ineffective erythropoiesis
Normal
Stress erythropoies
is
Ineffectiveerythropoies
is
:Chronic Stress Erythropoiesis
Ineffective erythropoiesis
Cell death
Anemia
Chronic stress erythropoiesis:
Increased cell proliferationReduced cell differentiation
pJak2 pJak2
pStat5
Epo
Jak2 Jak2
EpoR
Stat5
Erythropoiesis: a brief summary
Cell Replication
Protection from apoptosis
Progenitor erythroid cells
Normal Erythropoiesis
Red cell
Cooley’s Anemia
Apoptosis
Jak2: a gene that controls red cell production
: pJak2
Libani I. & al, Blood 2008
Red cell
Cooley’s Anemia
Potential effect of Jak2 inhibitors on ineffective erythropoiesis
Jak2 Inhibitor
Ineffective Erythropoiesis : pJak2
Libani I. & al, Blood 2008
Hypothesis
● Can we modulate Jak2 activity to prevent or revert splenomegaly and EMH?
● Can we use Jak2 inhibitors to ameliorate the transfusion regimen?
● Can we use Jak2 inhibitors for other transfusion dependent-related disorders, such as sickle cell anemia?
Day0
pJak2 inhibitor/ TG101209
Experimentaldesign
Analysis
Day 11
Administration of a pJak2 inhibitor on mice affected bythalassemia intermedia
TG101209 (TG: 100 or 150 mg/kg/day)or Placebo for10 days
Luca Melchiorri, Carla Casu& Pedro Ramos
Reduction of splenomegaly and amelioration ofextramedullaryhematopoiesis without major impact
on anemia in animals affected by ß-thalassemiaintermedia
Placebo(7) Tg(100)(5) Tg(150)(6)0
5
10
15
20
25
Hemoglobin g/dL
RBC(x10e6ul)
Retic(x10e5 ul)
*
*
***
*
Luca Melchiorri, Carla Casu& Pedro Ramos
Placebo
TG 100
TG 150
Placebo (7) Tg (100)(5) Tg (150)(6)0
20
40
60
80
100
120
*****
Spleen size (%placebo ctl)
WT
Administration of a pJak2 inhibitor to mice affected by ß-thalassemiaintermedia and major in presence of blood
transfusion
Administration of a pJak2 inhibitor in mice affected byß-Thalassemia
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Analysis
Day
Hb
Blood Transfusi
on
Luca Melchiori, CralaCasu& Pedro Ramos
TG101209 (TG: 150 mg/kg/day) or Placebofor10 days
Placebo+Txf(5) Tg (100)+Txf(4) Tg (150)+Txf(5)0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00 Hemoglobin
RBC (#x10e6/uL)
**
Placebo+Txf(5) Tg (100)+Txf(4) Tg (150)+Txf(5)0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00b-thal RBCs (#x10e6/ul) WT RBCs (#x10e6/ul)
*
Hematological parameters in mice affected by ß-thalassemia intermedia after transfusion and TG101209
treatment
Carla Casu& Pedro Ramos
Placebo+Txf(5) Tg (100)+Txf(4) Tg (150)+Txf(5)0
10
20
30
40
50
60
70
80
90
100
Sple
en w
eig
ht
(% o
f non-
Txf
Ctr
) ** **
CTRL no-TXF
WT
Luca Melchiorri, Carla Casu& Pedro Ramos
Jak2 inhibitor increases efficacy of transfusion in transfusion dependent mice affected by ß-
thalassemia major
Non-Txf Placebo + Txf Tg (150) + Txf0
2
4
6
8
10
12
14 Hemoglobin RBC (#x10e6/ul)
**** **
***
th3/th3
Placebo + TX
Jak2 inhib./TG101209 + TX
Non-Txf Placebo + Txf Tg (150) + Txf0
20
40
60
80
100
120
Sple
en w
eig
ht
(% o
f non-
Txf
th3/t
h3)
***
***
**
WT
Could we use a similar approach to ameliorate the transfusion regimen in sickle cell
anemia?
Placebo Tg (100)0
2
4
6
8
10
12Hemoglobin RBC (#x10e6/uL)
**
Placebo Tg (100)0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0 Wt cells (#x10e6/ul) Sickle Cells (#x10e6/ul)
*
Combination of TG101209 with transfusion reduces the number of sickle RBC cells in
SickleCell mice
Pedro Ramos & Carla Casu
0
20
40
60
80
100
120
spleen weight (% Placebo ctrl)
*
Placebo +TX Tg (100) +TX
WT
Transfusion aloneDonor RBCs
Transfusion alone
Jak2 Inhibitors: Potential Applications
ß-Thalassemia Intermedia
Sickle Cell Anemia
ß-Thalassemia Major
Erythroid progenitors
Jak2 inhib. + Transfusion
Jak2 inhib. + Transfusion
Circulating thalassemic
RBCs
Acknowledgments
Department of Pediatrics - WCMC, New York, USA
Sara GardenghiPedro RamosM.Franca MarongiuLuca MelchioriElla GuyLori BystromLaura BredaCarla CasuNiva RaoTom RenaudRobert W. Grady Patricia J. Giardina
The Edith Wolfson Medical Center, Holon,IsraelEliezer A. Rachmilewitz
Chaim Sheba Medical Center,Tel-Aviv University, IsraelNinette AmariglioGideon Rechavi
David Geffen School of Medicine,UCLA, USAElizabeta Nemeth
Duke University School of Medicine, Durham, USANancy C Andrews
Johns Hopkins University, Baltimore, USACindy N Roy
New York Blood Center, USAYelena GinzburgXiu Li AnNarla Mohandas
SponsorsNIH-The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)Cooley’s Anemia Foundation (CAF)Roche Foundation for Anemia Research (RoFar)
● Jak2 plays a major role in controlling IE, splenomegaly and EMH in ß-thalassemia.
● Using Jak2 inhibitors might be possible to prevent or revert splenomegaly and EMH.
● Use of Jak2 inhibitors might ameliorate the transfusion regimen in conditions associated with profound ineffective erythropoiesis (i.e. ß-thalassemia major).
● Use of a tailored dose of Jak2 inhibitors might also be beneficial in transfusionalsickle cell anemia...to be continued
Conclusions
Ineffective erythropoiesis:excessive cell proliferation coupled with
hemichrome formation
Might Jak2 inhibitors be even more effective in combination with therapies that modulate iron
metabolism?
Ineffective erythropoiesis: New therapies?
Hepcidin, the iron hormone regulator, acts on Ferroportin, the iron exporter
Hamp1 is expressed at relatively low levels in iron
overloaded th3/+ mice
0
1.0
2.0
+/+ 2M 5M 12M
Liv
er
Ham
p1 m
RN
A
level
th3/+
1.5
0.5
P <0.001
0
400
800
1200
1600
Org
an
tota
l ir
on
con
ten
t (u
g)
Liver
Spleen
+/+
th3/+2M
+/+
th3/+5M
+/+
th3/+
12M
P < 0.001
P < 0.05
P < 0.05
P < 0.01
Iron overload increases with time in th3/+ mice
Iron overload and anemia worsen over time in thalassemic mice
Gardenghi et al. Blood 2007, 109: 5027-35.
Anemia worsens with time in th3/+ mice
Hem
og
lob
in
(g/d
L)
15.0
13.0
11.0
9.0
7.0
Months2.0 12.0
+/+
th3/+
Sara Gardenghi
th3/+
Tg-Hamp1/th3
th3/+ control
TgHamp/th3
Increased Hepcidin expression leads to extended RBCs lifespan and improved
morphology
Decreased erythroid iron intake and reduced hemichrome formationsLess apoptosis, extended RBCs lifespan and improved morphologyAmelioration of erythropoiesis and splenomegaly
Sara Gardenghi& Pedro Ramos
Florid erythropoiesis & apoptosisSplenomegalyIron overload
Hemoglobin Levels in Post Splenectomized and Untransfused
ß-Thalassemia Intermedia Patients
Time (years post splenectomy)
Hem
og
lob
in (
g/d
l)
0
2
4
6
8
10
12
1 62 43 50
N = 11
Splenectomy and Anemia in ß-Thalassemic Patients
Sergios Zacharoulis and Patricia J. Giardina ASH, 2004
Normal Patient
Ki67
Ki67+
Glycophorin-A
& spectrin
Increased erythroid cell proliferation in human ß-thalassemic erythroid cells
Amy Chadburn, YiFang-Liu and Patricia J. Giardina