Children’s Cancer and Blood Foundation Laboratories Division of Hematology-Oncology

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.   Heme    Heme   ß-Thalassemia Adult Hemoglobin Alpha/Heme Aggregates Mutations in the beta-globin gene Consequences of Reduced ß-Globin Chain Production Anemia Ineffective Erythropoiesis Iron Overload Erythroid Marrow Expansion-Splenomegaly Thrombosis

3. Normal vs. stress erythropoiesis Proliferation Proliferation Proliferation Differentiation Differentiation Differentiation Stress erythropoiesis Normal 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.

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

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

6. Epo EpoR Jak2 Jak2 Stat5 pJak2 pJak2 Protection from apoptosis Cell Replication pStat5 Erythropoiesis: a brief summary

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

8. Cooley’s Anemia Ineffective Erythropoiesis Jak2 Inhibitor Red cell Potential effect of Jak2 inhibitors on ineffective erythropoiesis : pJak2 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. Administration of a pJak2 inhibitor on mice affected bythalassemia intermedia Experimentaldesign TG101209 (TG: 100 or 150 mg/kg/day)or Placebo for10 days pJak2 inhibitor/ TG101209 Day 11 Day0 Analysis Luca Melchiorri, Carla Casu& Pedro Ramos Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days Tg101209(Tg) or Placebo Daily oral gavage (100 or 150 mg/kg/day) for 10 days th3/+ th3/+ th3/+ th3/th3 th3/th3 th3/th3 Day -7 Day 0 Day -7 Day 0 Day -7 Day 0 +/- Transfusion (TX) +/- Transfusion (TX) +/- Transfusion (TX)

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

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 TG101209 (TG: 150 mg/kg/day) or Placebofor10 days Hb Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Blood Transfusion Analysis Luca Melchiori, CralaCasu& Pedro Ramos

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

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

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 WT * Placebo +TX Tg (100) +TX * ** Pedro Ramos & Carla Casu

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

19. Acknowledgments 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 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 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. Conclusions • 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

22. Ineffective erythropoiesis: New therapies? Ineffective erythropoiesis: excessive cell proliferation coupled with hemichrome formation Might Jak2 inhibitors be even more effective in combination with therapies that modulate iron metabolism?

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

24. Iron overload and anemia worsen over time in thalassemic mice Iron overload increases with time in th3/+ mice Hamp1 is expressed at relatively low levels in iron overloaded th3/+ mice P < 0.05 2.0 Organ total iron content (ug) Spleen 1.5 P < 0.01 P < 0.001 P < 0.05 Liver Liver Hamp1 mRNA level 1.0 P <0.001 0.5 Anemia worsens with time in th3/+ mice +/+ 15.0 0 13.0 +/+ 2M 5M 12M +/+ +/+ +/+ th3/+ th3/+ th3/+ Hemoglobin (g/dL) 11.0 th3/+ 9.0 th3/+ 12M 5M 2M 7.0 Gardenghi et al. Blood 2007, 109: 5027-35. 2.0 Months 12.0 Sara Gardenghi

25. Increased Hepcidin expression leads to extended RBCs lifespan and improved morphology th3/+ th3/+ control Florid erythropoiesis & apoptosis Splenomegaly Iron overload Tg-Hamp1/th3 Decreased erythroid iron intake and reduced hemichrome formations Less apoptosis, extended RBCs lifespan and improved morphology Amelioration of erythropoiesis and splenomegaly TgHamp/th3 Sara Gardenghi& Pedro Ramos

26. Hemoglobin Levels in Post Splenectomized and Untransfused ß-Thalassemia Intermedia Patients 12 N = 11 10 8 Hemoglobin (g/dl) 6 4 2 0 0 1 2 4 6 3 5 Time (years post splenectomy) Splenectomy and Anemia in ß-Thalassemic Patients Sergios Zacharoulis and Patricia J. Giardina ASH, 2004

27. 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