Enhancing progenitor cell self-renewal: a new approach to stimulating red cell production

1. Enhancing progenitor cell self-renewal: a new approach to stimulating red cell production Development of novel therapies for Diamond-Blackfan anemia and other erythropoietin- resistant anemias Professor Harvey Lodish Whitehead Institute for Biomedical Research Departments of Biology and Biological Engineering, MIT

2. Although I have helped start several successful biotechnology companies, at heart I am a cell and developmental biologist focused on understanding basic life processes • 1979 Damon Biotech • 1979 BioInformation Associates • 1981 Genzyme Sold to Sanofi for $20.2 billion • 1989 Arris (now Axys) Pharmaceuticals • 1993 Millennium Pharmaceuticals Sold to Takeda for $9 billion • 2005 Allozyne • 2013 Rubius Since 2006 I have been the Founding Chair of the Scientific Advisory Board of the Massachusetts Life Sciences Center, the group charged with oversight of the state’s 10- year $1 billion investment in life sciences.

3. Gaucher Disease – Symptoms • Gaucher is a progressive, debilitating and sometimes life-threatening disease. • Symptoms can include:easy bleeding and bruising, fatigue, anemia, weak bones, bone and joint pain, and enlargement of the spleen or liver. • Symptoms can appear at any age.

4. Cerezyme: novel technologies 1980- 1985 • A personalized medicine for a rare disease: replacing the missing enzyme in Gaucher Disease • A recombinant protein • A protein targeted to a specific type of cell • Based on glycoengineering

8. Red Blood Cells (Erythrocytes): • The most common type of blood cell - ~45 – 50% of blood volume; one quarter of all human cells • Lack DNA, a nucleus, and other internal structures characteristic of normal human cells • Transports oxygen from the lungs to body tissues and waste carbon dioxide back to the lungs • Cytoplasm is filled with the red protein hemoglobin that binds oxygen • Adult humans produce ~ 2.4 million red cells per second • Red cells circulate for 100 - 120 days before being degraded; each circulation takes about 20 seconds

9. Blood cell formation - from a stem cell to multiple types of blood cells

10. Multiple hormones regulate red cell formation (erythropoiesis)

11. Erythropoietin (Epo) regulates red cell production.Epo synthesis is induced in the kidney in response to hypoxia (low oxygen in the blood).

12. Epo, synthesized in the kidney in response to low oxygen in the blood, is the singular hormone that stimulates formation of red blood cells from CFU-E progenitors.Epo is widely used to treat anemia caused by cancers and kidney failure

13. Epo is also a drug of abuse

14. Many anemias do not respond to Epo treatment because the number of CFU-E progenitors is low • Genetic bone marrow failure diseases such as Diamond Blackfan anemia • Severe trauma, sepsis • Severe anemia of malaria • ~18% of kidney dialysis patients

15. Diamond–Blackfan anemia (DBA) is a congenital bone marrow failure disorder due to death of erythroid progenitors • DBA patients have decreased numbers of erythroid progenitors in the bone marrow. • Mutation in any of any of several genes for ribosomal proteins can cause DBA. Nathan et al., The Journal of Clinical Investigation, 1978

16. Epo (very high in DBA) SCF DBA patient In Diamond Blackfan Anemia and other bone marrow failure disorders, proliferation of CFU-Es is defective and many die even in the presence of high Epo levels early BFU-E late BFU-E HSC GEMM CFU-E Erythroblasts Erythrocytes Epo (low during steady state) SCF Normal  

17. Prednisone (a corticosteroid) treatment for Diamond Blackfan Anemia • Up to 80% respond initially • Severe side effects • Patients that require too high doses can not be maintained on prednisone • Need for deeper understanding for what is important for erythroid response to glucocorticoids in DBA • Knowledge may lead to novel therapies not only for DBA but for the many other Epo- resistant anemias • Corticosteroids stimulate normal and DBA red cell production equally well. But in 2009 we did not know how corticosteroids stimulate red cell production

18. GR GC Activator complex ON OFF GC GR GR GR GR GR ON GR Corticosteroids activate the glucocorticoid receptor (GR), which binds to DNA and can either turn off or turn on certain genes Gene Repression GC GC Dimerization OFF Gene Activation (direct) Cytoplasm Nucleus

19. A corticosteroid (Dex) increases the number of erythroblasts formed from each BFU-E ~ 40-fold, but does not affect erythroblast formation from CFU-E progenitorsDex stimulates the likelihood of BFU-E self renewal during each cell division, allowing over time more CFU-E progenitors to be formed and thus more erythrocytes BFU-E CFU-E 25,000 + Dex + Dex Negative control Erythroid cells formed from one CFU-E cell Erythroid cells formed from one BFU-E cell Negative control 590 Serum-free medium containing Stem Cell Factor (SCF), IGF-1, and Epo Blood, 117: 3435 - 3444 (2011)

20. Glucocorticoids stimulate BFU-E self-renewal, leading over time to increased formation of CFU-E progenitors, and then to increased erythroblast production 0 1 2 3 4 5 6 7 Cell divisions BFU-E CFU-E Erythroblast Glucocorticoids

21. Strategy for testing compounds for their ability to stimulate BFU-E self-renewal and production of increased numbers of red blood cells: Developing potential therapies for Diamond Blackfan Anemia and other Epo- resistant anemias Mouse BFU-Es from E14.5 fetal livers Human CD34+ erythroid differentiation system Test mice in vivo Lee et. al., Nature 522, 474–477 (2015).

22. Chemical Screening • Workflow: Isolate BFU-Es from mouse fetal livers Treat BFU-Es with compounds Count cells every other day until day12

23. PPARα agonist GW7647 • Originally developed by GlaxoSmithKline for dyslipidemia • Potent and highly selective PPARα agonist (EC50 values are 6, 1100 and 6200 nM for human PPARα, PPARγ and PPARδ receptors respectively) • More potent and specific than fenofibrate • Exerts cardioprotective effects in a mouse model of acute ischemia/reperfusion myocardial injury • Has lipid-lowering effects following oral administration in vivo • Exhibits anti-inflammatory properties

24. PPARα (Peroxisome Proliferation Activated Receptor α) also binds to DNA and can either turn off or turn on certain genes Co-Repressor PPARa RXR Adipogenesis Lipid metabolism Inflammation Co-Activator PPARa RXR AGGTCA-N-AGGTCA TCCAGT-N-TCCAGT PeroxisomeProliferator Hormone Response Element (PPRE)

25. PPAR agonist GW7647 synergizes with dexamethasone (DEX) to significantly increase erythroid expansion of mouse BFU-E cells Total cell numbers from each BFU-E cell days

26. PPAR agonist GW7647 synergizes with low concentrations of dexamethasone (Dex) to promote red cell formation from BFU-E cells P < 0.05 * P < 0.01 ** P< 0.001 *** PPARa agonist

27. PPARagonist synergizes with Dex to increase human red cell production 4-fold Erythroid cells formed from each CD34+ cell days

28. PPARa agonist GW7647 synergizes with Dex to increase red cell production in cultures following knockdown of ribosomal protein s19 (rps19).Mutation in one gene for rps19 is a frequent cause of DBA

29. GW7647 reverses the anemia of Nan mice

31. Activators of specific nuclear receptors enhance BFU-E self renewal and production of red blood cells. These have immediate potential for treatment of erythropoietin-resistant anemias including Diamond Blackfan Anemia • Low concentrations of corticosteroid agonists: Prednisone, • Inhibitors of Prolyl hydroxylase 2: Amgen and Fibrogen drugs • PPARa agonists: Fenofibrate, GW7647

32. Russell Elmes Xiaofei Gao Sherry Lee

33. September 13, 2014 – Whitehead Scientific Retreat Waterville Valley NH

34. A proud 31 year tradition continues: July 19, 2014