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?