1. Children’s Cancer and Blood Foundation Laboratories Division of Hematology-Oncology Department of Pediatrics Weill Medical College of Cornell University New York New approaches to modulate abnormal erythropoiesis and improve the transfusion regimen in ß-thalassemia and sickle cell disease

2. 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

3. Normal vs. stress erythropoiesis NormalProliferationDifferentiation Lenox et al, Blood 2005. Lenox et al, Blood 2005. Menon et al. JCI 2006. Menon et al. JCI 2006. Marinkovic et al. JCI 2007. Marinkovic et al. JCI 2007. Harandi et al. JCI 2010. Harandi et al. JCI 2010. Ulyanova et al. Blood 2011. Ulyanova et al. Blood 2011. Stress erythropoiesis ProliferationDifferentiationProliferationDifferentiation

4. Stress vs. ineffective erythropoiesis Normal Stress erythropoiesis Ineffective erythropoiesis : Chronic Stress Erythropoiesis :

5. Ineffective erythropoiesis Cell death Anemia Ineffective erythropoiesis Cell death Anemia  Chronic stress erythropoiesis: Increased cell proliferation Reduced cell differentiation  Chronic stress erythropoiesis: Increased cell proliferation Reduced cell differentiation

6. pJak2pJak2 pStat5 EpoJak2 Jak2 EpoR Stat5 Erythropoiesis: a brief summary Cell Replication Protection from apoptosis

7. Progenitor erythroid cells Progenitor erythroid cells Normal Erythropoiesis Normal Erythropoiesis Red cell Red cell Cooley’s Anemia Cooley’s Anemia Apoptosis Jak2: a gene that controls red cell production : pJak2 Libani I. & al, Blood 2008 Libani I. & al, Blood 2008

8. Red cell Red cell Cooley’s Anemia Cooley’s Anemia Potential effect of Jak2 inhibitors on ineffective erythropoiesis Jak2 Inhibitor Jak2 Inhibitor Ineffective Erythropoiesis Ineffective Erythropoiesis : pJak2 Libani I. & al, Blood 2008 Libani I. & al, Blood 2008

9. 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?

10. Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days th3/th3 +/- Transfusion (TX) Day 0 Day -7 th3/+ Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days th3/th3 +/- Transfusion (TX) Day 0 Day -7 th3/+ Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days th3/th3 +/- Transfusion (TX) Day 0 Day -7 th3/+ Day0 pJak2 inhibitor/ TG101209 Experimentaldesign Analysis Day 11 Administration of a pJak2 inhibitor on mice affected bythalassemia intermedia 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 Luca Melchiorri, Carla Casu& Pedro Ramos

11. wt *** Placebo Tg (100) Tg (150) wt *** Placebo Tg (100) Tg (150) wt *** Placebo Tg (100) Tg (150) wt Placebo Tg (100) Tg (150) Erythroid apoptosis Reduction of splenomegaly and amelioration ofextramedullaryhematopoiesis without major impact on anemia in animals affected by ß-thalassemiaintermedia * * ** * * Luca Melchiorri, Carla Casu& Pedro Ramos Luca Melchiorri, Carla Casu& Pedro RamosPlacebo TG 100 TG 150 ** *** Spleen size (%placebo ctl) WT

12. Administration of a pJak2 inhibitor to mice affected by ß- thalassemiaintermedia and major in presence of blood transfusion

13. Administration of a pJak2 inhibitor in mice affected byß-Thalassemia 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 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Analysis Day Hb Blood Transfusion Blood Transfusion Luca Melchiori, CralaCasu& Pedro Ramos Luca Melchiori, CralaCasu& Pedro Ramos TG101209 (TG: 150 mg/kg/day) or Placebofor10 days

14. * * * Hematological parameters in mice affected by ß-thalassemia intermedia after transfusion and TG101209 treatment Carla Casu& Pedro Ramos Carla Casu& Pedro Ramos ** ** CTRL no-TXF WT

15. Luca Melchiorri, Carla Casu& Pedro Ramos Luca Melchiorri, Carla Casu& Pedro Ramos Jak2 inhibitor increases efficacy of transfusion in transfusion dependent mice affected by ß-thalassemia major ** ** ** ** * th3/th3 Placebo + TX Jak2 inhib./TG101209 + TX *** *** ** WT

16. Could we use a similar approach to ameliorate the transfusion regimen in sickle cell anemia?

17. ** * Combination of TG101209 with transfusion reduces the number of sickle RBC cells in SickleCell mice Pedro Ramos & Carla Casu Pedro Ramos & Carla Casu * Placebo +TX Tg (100) +TX WT

18. Transfusion alone Donor RBCs Transfusion alone Jak2 Inhibitors: Potential Applications ß-Thalassemia Intermedia Sickle Cell Anemia ß-Thalassemia Major Erythroid progenitors Jak2 inhib. + Transfusion Circulating thalassemic RBCs

19. AcknowledgmentsAcknowledgments Department of Pediatrics - WCMC, New York, USA Sara Gardenghi Pedro Ramos M.Franca Marongiu Luca Melchiori Ella Guy Lori Bystrom Laura Breda Carla Casu Niva Rao Tom Renaud Robert W. Grady Patricia J. Giardina Department of Pediatrics - WCMC, New York, USA Sara Gardenghi Pedro Ramos M.Franca Marongiu Luca Melchiori Ella Guy Lori Bystrom Laura Breda Carla Casu Niva Rao Tom Renaud Robert W. Grady Patricia J. Giardina The Edith Wolfson Medical Center, Holon, Israel Eliezer A. Rachmilewitz Chaim Sheba Medical Center,Tel-Aviv University, Israel Ninette Amariglio Gideon Rechavi David Geffen School of Medicine,UCLA, USA Elizabeta Nemeth Duke University School of Medicine, Durham, USA Nancy C Andrews Johns Hopkins University, Baltimore, USA Cindy N Roy New York Blood Center, USA Yelena Ginzburg Xiu Li An Narla Mohandas The Edith Wolfson Medical Center, Holon, Israel Eliezer A. Rachmilewitz Chaim Sheba Medical Center,Tel-Aviv University, Israel Ninette Amariglio Gideon Rechavi David Geffen School of Medicine,UCLA, USA Elizabeta Nemeth Duke University School of Medicine, Durham, USA Nancy C Andrews Johns Hopkins University, Baltimore, USA Cindy N Roy New York Blood Center, USA Yelena Ginzburg Xiu Li An Narla Mohandas Sponsors NIH-The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Cooley’s Anemia Foundation (CAF) Roche Foundation for Anemia Research (RoFar) Sponsors NIH-The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Cooley’s Anemia Foundation (CAF) Roche Foundation for Anemia Research (RoFar)

21. ● 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

22. 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?

23. Hepcidin, the iron hormone regulator, acts on Ferroportin, the iron exporter

24. Hamp1 is expressed at relatively low levels in iron overloaded th3/+ mice 0 1.0 2.0 +/+2M5M12M Liver Hamp1 mRNA level th3/+ 1.5 0.5 P < 0.001 Organ total iron content (ug) Liver Spleen +/+ th3/+ 2M +/+ 5M +/+ 12M P < 0.001 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 Hemoglobin (g/dL) 15.0 13.0 11.0 9.0 7.0 Months2.012.0 +/+ th3/+ Sara Gardenghi Sara Gardenghi

25. 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 formations Less apoptosis, extended RBCs lifespan and improved morphology Amelioration of erythropoiesis and splenomegaly Sara Gardenghi& Pedro Ramos Florid erythropoiesis & apoptosis Splenomegaly Iron overload

26. Hemoglobin Levels in Post Splenectomized and Untransfused Hemoglobin Levels in Post Splenectomized and Untransfused ß-Thalassemia Intermedia Patients Time (years post splenectomy) Time (years post splenectomy) Hemoglobin (g/dl) 0 2 4 6 8 10 12 1624 35 0 N = 11 N = 11 Splenectomy and Anemia in ß-Thalassemic Patients Sergios Zacharoulis and Patricia J. Giardina ASH, 2004

27. NormalPatientKi67 Ki67+Glycophorin-A & spectrin Increased erythroid cell proliferation in human ß-thalassemic erythroid cells Amy Chadburn, YiFang-Liu and Patricia J. Giardina Amy Chadburn, YiFang-Liu and Patricia J. Giardina