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
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.
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.
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
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
Blood cell formation - from a stem
cell to multiple types of blood cells
Multiple hormones regulate red cell formation
(erythropoiesis)
Erythropoietin (Epo) regulates red cell
production.
Epo synthesis is induced in the kidney in response to
hypoxia (low oxygen in the blood).
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
Epo is also a
drug of abuse
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
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
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
HSC
GEMM
early
BFU-E
late
BFU-E
CFU-E
Erythroblasts
Erythrocytes
Epo (low during steady state)
SCF
Normal

Epo (very high in DBA)
SCF
DBA patient
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
Corticosteroids activate the glucocorticoid receptor (GR),
which binds to DNA and can either turn off or turn on
certain genes
GC
ON
OFF
GR
GC
GC
GC
Gene Repression
GR
GR
Activator complex
GR
GR
GR
GR
Dimerization
OFF
ON
Gene Activation (direct)
Cytoplasm
Nucleus
A corticosteroid (Dex) increases the number of erythroblasts
formed from each BFU-E ~ 40-fold, but does not affect
erythroblast formation from CFU-E progenitors
Dex 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
25,000
100
10,000
+ Dex
1,000
590
Negative
control
590
100
10
1
0
2
4
6
8
Days in culture
10
12
Erythroid cells formed from
cell
one CFU-E
CFU-E cell
expansion
Erythroid cells formed from
formed from 1
BFU-E cells
cell
oneErythroid
BFU-E
100,000
+ Dex
Negative
control
10
1
0
2
4
6
Days in culture
Serum-free medium containing Stem Cell Factor (SCF), IGF-1, and Epo Blood, 117: 3435 - 3444 (2011)
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
Glucocorticoids
CFU-E
Erythro
blast
Strategy for testing compounds for their ability to stimulate BFU-E selfrenewal and production of increased numbers of red blood cells:
Developing potential therapies for Diamond Blackfan Anemia and other
Epo- resistant anemias
Mouse BFUEs from E14.5
fetal livers
Human
CD34+
erythroid
differentiation
system
Test mice in
vivo
Lee et. al., Nature 522, 474–477 (2015).
Chemical Screening
• Workflow:
Isolate BFU-Es
from mouse fetal
livers
Treat BFU-Es with
compounds
Count cells every
other day until
day12
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
PPARα (Peroxisome Proliferation Activated Receptor α) also
binds to DNA and can either turn off or turn on certain genes
Co-Repressor
PPARa
RXR
Co-Activator
PPARa
RXR
AGGTCA-N-AGGTCA
TCCAGT-N-TCCAGT
Peroxisome Proliferator
Hormone Response Element
(PPRE)
Adipogenesis
Lipid metabolism
Inflammation
Total cell numbers from each BFUE cell
PPAR agonist GW7647 synergizes with dexamethasone
(DEX) to significantly increase erythroid expansion of mouse
BFU-E cells
days
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
PPAR agonist synergizes with Dex to increase human red cell
Erythroid cells formed from each
CD34+ cell
production
4-fold
days
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
GW7647 reverses the anemia of Nan mice
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
• PPAR agonists: Fenofibrate, GW7647
Russell Elmes
Xiaofei Gao Sherry Lee
September 13, 2014 – Whitehead Scientific Retreat
Waterville Valley NH
33
A proud 31 year tradition continues: July 19, 2014