Thalassemia: New Insights that Lead to Novel
Therapies in 2016or:What does thalassemia have in common with hematological
malignancies?
?מה משותף בין תלסמיה וממאירויות המטולוגיות
דבורה רונד' פרופ
New Insights into Cell Biology of
Thalassemia
What is the process of ineffective
erythropoiesis in thalassemia?
Can it be improved?
Inhibition of JAK2?
Macrophage directed therapy?
Heat shock proteins?
AND MORE!! (activin ligand traps)
Ineffective Erythropoiesis
•Erythroid hyperplasia
•Left shift: No ortho-
chromic normoblasts
Four Key Steps of Pathological
Erythropoiesis in Thalassemia (Animal and Human)
• Expansion of erythroid precursors (primarily basophilic and polychromatophilic
normoblasts)
• Accelerated differentiation to polychromatophilic stage
(not well understood)
• Arrest of maturation and proliferation at
the polychromatophilic stage
• Apoptosis at polychromatophilic stage
Four Key Steps of Dyserythropoiesis
in Thalassemia (Animal and Human)
• Expansion of erythroid precursors (primarily
basophilic and polychromatophilic
normoblasts)
• Accelerated differentiation to polychromatophilic stage
(not well understood)
• Arrest of maturation and proliferation at
the polychromatophilic stage
• Apoptosis at polychromatophilic stage
Erythroid Expansion
• Rivella: “In thal, RBC are like leukemic blasts”:
proliferate, fail to differentiate, invade other organs
and inhibit their function
• In thalassemia, hypoxia due to anemia
Epo and persistent phosphorylation of JAK-2
• Phosphorylation of JAK2 (among other things)
ID1 transcription factor (ID1=inhibitor of
differentiation 1): inhibits differentiation, promotes
cell cycling and cell migration
Possible Therapy: JAK-2 inhibitor?
In theory: could
improve thal int
if reduces
splenomegaly
and hepcidin
suppression Phase II trials
of JAK2
inhibition
are underway
JAK-2 Inhibition in Thalassemia?
• Libani et al, 2008: Murine thalassemia
model, JAK-2 inhibitors reduce spleen
size, but do not increase Hb
• In theory, if titrate the dose properly in thal
intermedia, could reduce spleen size,
reduce RBC sequestration and increase
RBC lifespan
What Else do Thalassemia and
P.Vera have in common?
• Besides erythroid expansion and
splenomegaly and potential use of JAK2
inhibitors?
What Else do Thalassemia and P.Vera have
in common?
Macrophages!
Bone Marrow Microenvironment:
Essential in Hematopoiesis
• Erythroid island: central macrophage
which interacts with surrounding
erythroblasts
Bone Marrow Macrophages provide iron,
regulatory signals and phagocytize nucleii
of maturing RBC: “Nurse cells”
Extruded RBC nucleus
“Nurse Cells” Exert Negative Effect on
Hematopoiesis in Thalassemia
(and in P. Vera!)
• This was discovered by eliminating
macrophages by injection of liposomal
clodronate (Bonefos)
• A single injection of this drug
significantly decreases BM and splenic
macrophages
• Did this in thalassemia mouse model
Liposomal Clodronate Eliminates
Macrophages, Improves Thalassemic Mice
• 20-40 hrs after Clodronate injection, Hb increased and spleen decreased as much as 32%, persisted for 2 months of chronic Rx
• Increased numbers of differentiated RBC and reduction of number of cycling RBC in spleen
• Increased hepcidin production and decrease serum iron
• Not due to lack of iron (iron loaded mice: same effect)
• Conclusion: Macrophages impair erythroid development in beta thalassemia
Liposomal Clodronate also Improves
P. Vera Mice
• The same treatment given to mice with
a murine model of P. Vera: lower Hb
and smaller spleens!!
• Also tried on normal human erythroid
primary cell cultures
Clodronate Effect on Normal Primary
Human Erythroblast Cultures
• Macrophages promoted erythroid proliferation and survival, while reducingerythroblast differentiation
• Need cell-cell contact with macrophages
• Macrophages supported pathologic erythropoiesis in BOTH thalassemia and P. Vera!
• No therapeutic drug yet, but…..a “druggable” target!
Ramos and Rivella Nat Med, April 2013
Four Key Steps of Dyserythropoiesis
in Thalassemia (Animal and Human)
• Expansion of erythroid precursors (primarily
basophilic and polychromatophilic
normoblasts) (Possibly due to increased Epo
and increased phosphorylation of JAK2?)
• Accelerated differentiation to polychromatophilic stage
(not well understood)
• Arrest of maturation and proliferation at
the polychromatophilic stage
• Apoptosis at polychromatophilic stage
Normal erythropoiesis:
•GATA-1 is master erythroid transcription factor
•Stage of hemoglobin production: late stages
Thalassemic erythropoiesis:
•Block at polychromatophilic stage,
diminished further maturation
Accelerated Apoptosis in Thalassemia
Erythroid Precursors
• 1993: Yuan and Schreier demonstrated apoptoticDNA “ladder” in erythroid cells of thalassemia pts compared to thal trait and normals
• 2000: Mathias et al reported ineffective erythropoiesis of thalassemia erythroid cells is due to apoptosis at polychromatophilic stage
• 2001: Paradoxically, Zermati et al: Caspase 3 activation (activates apoptosis) is required for normal terminal erythroid differentiation
Apoptosis is Essential for Terminal
Erythroid Differentiation
• Zermati 2001: Normal erythroid differentiation
requires transient activation of caspase 3; if
add caspase inhibitor: arrest of maturation
and apoptosis at polychromatophilic stage
• Polychromatophilic stage is when intense
globin synthesis takes place
Normal hemoglobin production:
equal amounts - and -chains
THALASSEMIA: CHAIN IMBALANCE
Thalassemia RBC and Normal RBC
Inside-out
RBC ghosts:
Aggregates of
excess unpaired
globin chains
Shinar et al,
Blood, 1987
Effect of Excess Alpha Chains: not just on Membrane
Normal erythropoiesis:
•GATA-1 is master erythroid transcription factor
•Epo dependent stage (till basophilic normoblasts)
•Stage of hemoglobin production: late stages
GATA-1 Transcription Factor:Essential for Normal Erythroid Development
• GATA-1 is cleaved by transiently active
caspase 3 during late erythroid
development
• In normal erythroid precursors, GATA-1 is
protected by a chaperon protein HSP70,
which translocates to the nucleus and
protects GATA-1 from caspase-mediated
proteolysis at the wrong time.
Chaperones Help New Proteins Fold
Properly and Fix Misfolded Proteins
Chaperones for Globin Chains:
Alpha Hemoglobin Stabilizing ProteinDiscovered in 2007 (Weiss), never really proven to be
involved in the phenotype of beta thal
Thalassemia: Nuclear GATA-1 Transcription
Factor not Protected by HSP70 Chaperon
• In thalassemia, HSP70 remains in the cytoplasm
bound to FREE ALPHA GLOBIN CHAINS! And
cannot translocate to the nucleus
• Therefore GATA-1 is exposed to premature
caspase cleavage APOPTOSIS of
erythroblasts
• Experimental use of uncleavable GATA-1 or
HSP 70 targeted to the nucleus, restored normal
differentiation.
J-B Arlet et al. Nature (2014)
J-B Arlet et al. Nature , 1-5 (2014) doi:10.1038/nature13614
HSP70 and GATA-1 localization in fresh bone
marrow from β-TM patients
Compare control to -thal patient: HSP70 is in cytoplasm,
not in nucleus and same for GATA-1
Experiments with normal peripheral
blood erythroid precursors
• SAME RESULTS!
Conclusions:
• Chaperon HSP70 required in the nucleus
to protect GATA-1 at a critical stage
• In thalassemia, excess alpha globin chains
associate with HSP70, trapping it in
cytoplasm
• Result is apoptosis at polychromatophilic
stage when excess alpha chains are made
HSP70: Therapeutic Target?
• At the present time there is no therapy
directed at HSP70 but this is a potential
target
• Must use caution since HSP70 is
important for many cellular functions
Activin Ligand Trap Therapy
• Will soon be available on experimental
basis in Israel!
• What is it and how does it work??
Activin, a Member of TGF Superfamily,
has Type I and II receptors
Sotatercept: Developed to Treat
Osteoporosis
• Developed to Treat Osteoporosis by Targeting Activin IIA Signalling
• Sotatercept binds activin IIA and thereby prevents its prosurvival effects on osteoclasts.
Another analogue Luspatercept, targets Activin IIB. (This is the drug we will have soon)
• Unexpectedly, patients with osteoporosis who were given Sotatercept had a 12% rise in Hb! And the same happened in normal controls.
In an animal model, RAP-011 (murine analogue of
Sotatercept) had a beneficial effect in thalassemia
Mechanism? Blocks GDF11!
• The unexpected finding of rise in Hb from Sotatercept led to an extensive investigation of the mechanism and GDF11 was found to be responsible pathway (it was found to be the only member of TGF-beta superfamily that was upregulated at the appropriate time point in mice)
• GDF11 was not known to be involved in erythropoiesis
• It was involved in developing nervous system, pancreas, intestine, kidneys.
Activin, a Member of TGF Superfamily,
has Type I and II receptors
GDF11 is an activin receptor IIA ligand
Block GDF11:
Positive Effect on Erythropoiesis
• Dussiot 2014: Nature Medicine
(experiments were quite complex but elegant)
• Increased terminal erythroid differentiation
and correction of maturation arrest by
inducing apoptosis of immature
erythroblasts via Fas-Fas pathway
Block GDF11: Effect on Erythropoiesis
• Improved RBC morphology, fewer alpha
globin precipitates on RBC membranes
since it caused decrease in alpha globin
transcription and increase in the mouse
equivalent of gamma globin
• The beneficial effect was organ dependent,
that is, affected spleen more than bone
marrow cells
• Decreased splenomegaly
HPLC: application to anemia
diagnosis
• New method of quantifying levels of
minor hemoglobins and identifying other
hemoglobin forms
• Can diagnose “peaks” corresponding to
different types of hemoglobin: helps in
identification
Block GDF11: Effect on Erythropoiesis
• GDF-11 was overexpressed in
thalassemia patient serum and in splenic
erythroblasts from thalassemic mice
• Also the pathological expression of GDF-
11 was dependent on oxidative stress
• Blocking GDF-11 increased hepcidin
production (independent of GDF15)
Reducing Ineffective Erythropoiesis also
Increases Hepcidin Production, Reduces Iron
Overload
Potential for Improving Thalassemia?
• Remember that these experiments were
done in mice in model of thalassemia
intermedia
• In the meantime, the drug is being tested
in beta thalassemia patients with
transfusion requirement but not too high
transfusion need. Seems logical to
assume the pts need some residual beta
globin function to benefit.
Conclusion with a Question:
Q: Will we be able to turn HUMAN thalassemic RBC
into normal ones?
A: Probably.
Using JAK2 inhibitors, macrophage depletion, HSP70
manipulation, activin ligand traps, and other drugs that
are under development.
The question is only: WHEN?