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

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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

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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 states 10- year 1 billion
  investment in life sciences.

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Gaucher Disease Symptoms

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

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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

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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

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Blood cell formation - from a stem cell to
multiple types of blood cells
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Multiple hormones regulate red cell formation
(erythropoiesis)
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Erythropoietin (Epo) regulates red cell
production. Epo synthesis is induced in the
kidney in response to hypoxia (low oxygen in the
blood).
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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
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Epo is also a drug of abuse
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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
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DiamondBlackfan 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
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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
Erythrocytes
Erythroblasts
CFU-E
Epo (low during steady state)
SCF
Normal
?
?
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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

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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
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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)
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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
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Cell divisions
BFU-E CFU-E Erythroblast
Glucocorticoids
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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, 474477 (2015).
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Chemical Screening

• Workflow

Isolate BFU-Es from mouse fetal livers
Treat BFU-Es with compounds
Count cells every other day until day12
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PPARa agonist GW7647

• Originally developed by GlaxoSmithKline for
  dyslipidemia
• Potent and highly selective PPARa agonist (EC50
  values are 6, 1100 and 6200 nM for human PPARa,
  PPAR? and PPARd 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

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PPARa (Peroxisome Proliferation Activated
Receptor a) 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
Peroxisome Proliferator Hormone Response Element
(PPRE)
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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
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PPAR? agonist GW7647 synergizes with low
concentrations of dexamethasone (Dex) to promote
red cell formation from BFU-E cells
P lt 0.05 P lt 0.01 Plt 0.001
PPARa agonist
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PPAR? agonist synergizes with Dex to increase
human red cell production
4-fold
Erythroid cells formed from each CD34 cell
days
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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
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GW7647 reverses the anemia of Nan mice
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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
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Russell Elmes
Xiaofei Gao Sherry Lee
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September 13, 2014 Whitehead Scientific Retreat
Waterville Valley NH
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A proud 31 year tradition continues July 19,
2014