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