El nicho medular y la inflamacion en las NMP

Lorena Arranz, PhD

Common features of stem cell niches

Adapted from Lane, Williams & Watt,

Nat Biotechnol 2014

The stem cell niche regulates the function, fate and numbers of the stem cell

/ Cytokines

Cytokine/

The haematopoietic stem cell niche in the bone marrow

Adapted from Crane, Jeffery & Morrison, 2017

Nes-GFPdim

Nes-GFPbright

Cytokines/

Growth factors

Cell-to-cell contacts

Soluble Factors

CXCL12 – Retention

SCF – Quiescence

Others:

TGFβ

CXCL4

G-CSF

GM-CSF

IL-1β

IL-8

IL-6

Adipocyte

Neutrophil

Treg

Model of sympathetic regulation of HSC

Adapted from Mendez-Ferrer et al, Nature 2008

Egress

Quiescence

Survival

Thiele et al, 2006 Thiele et al, 2006

Myeloproliferative

neoplasms

originate in a

lesion on a HSC

… and result in

fibrosis

/osteosclerosis

of the BM

Bone marrow Schwann cell

NA SNS

CXCL12 Nestin+ MSC

3-AR

mutant HSC

Study of HSC niche alterations in myeloproliferative disease

Does the mutant HSC alter the niche?

How do these alterations change the mutant HSC behavior?

Myeloproliferative neoplasms (MPN)

Levine et al, 2007

Tsiftsoglou et al, 2008

JAK2 inhibitors do not eradicate MPN

The 2016 WHO classification for myeloid neoplasms and

acute leukaemia

MPN

CML, BCR-ABL1+

CNL

PV

PMF

ET

CEL NOS

MPN, unclassifiable

Mastocytosis

Myeloid/lymphoid neoplasms with eosinophilia and

rearrangement of PDGFRA, PDGFRB, or FGFR, or

with PCM1-JAK2

MDS/MPN

CMML

aCML, BCR-ABL1-

JMML

MDS/MPN with ring sideroblasts and thrombocytosis

(MDS/MPN-RS-T)

MDS/MPN, unclassifiable

MDS

Myeloid neoplasms with germ line predisposition

AML and related neoplasms

Mutation JAK2-V617F+ PV (95%)

PMF (50%)

ET (50%)

Kralovics et al, 2005

Jones et al, 2005

Chronic inflammation in MPN

PV

ET MF Chronic

inflammation

Chronic inflammation results from leucocyte, megakaryocyte,

and platelet activation, and proinflammatory cytokines

Premature atherosclerosis

Second cancer

Atherosclerosis

Second cancer

MDS/AML

Cytokine

storm TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

Adapted from Hasselbalch & Bjørn, Mediators Inflamm 2015

JAK2-V617F+

Hallmarks of myeloproliferative neoplasms (Polycythemia vera)

Spleen

Control

JAK2-V617F

Control

JAK2-V617F

LT-HSC ST-HSC MPP 0

2

4

6

8

10

*

*

*

Bone marrow

JAK2-V617F

JAK2-V617F

BM

HS

C (

x10

5)

Peripheral blood

5 10 0

0.5

1.0

1.5

2.0

2.5

**

t (wk)

***

5 10 t (wk)

0

5

10

15

20

***

5 10 t (wk)

0

5

10

15

20

25

Control JAK2-V617F

Neu

trop

hils

pe

r

ml of

blo

od

(x10

6)

Haem

oglo

bin

per

dl of blo

od

(g)

Ery

thro

cyte

s p

er

ml of

blo

od

(x10

9)

Mx-Cre JAK2-V617F

pIpC induction (i.p.)

In vivo transplantation

JAK2-V617F BM WT recipient

Mx-Cre JAK2-V617F donor

JAK2-V617F HSC

pIpC induction (i.p.)

Mx-Cre JAK2-V617F mouse model resembles human PV

Decreased number of nestin+ cells in the BM of MPN patients

Control human MPN

Decreased number of nestin+ cells in the BM of MPN patients and mice

Murine skull bone marrow

200µm Control JAK2-V617F

Nes-GFP

JAK2-

V617F

0

2

4

6

8

10

**

mR

NA

(ra

tio)

C MPN

Nestin (mouse)

0

5

10

15

20

* C

NESTIN (human)

0

500

1000

1500

2000

2500

**

BM Nes-GFP+ cells

JAK2-

V617F

C 0

100

200

300

400

500

BM mesensphere-forming cells BM CFU-F

** 0

1000

2000

3000

4000

5000

*

JAK2-

V617F

C JAK2-

V617F

C

Mx-cre;JAK2-V617F Nes-gfp

Mx-cre;JAK2-V617F;Nes-gfp

Tiedt et al, 2008

Kubovcakova et al, 2013

pIpC induction (i.p.)

0

20

40

60

Apoptotic Preapoptotic Live

BM Nes-GFP+ cells

Control JAK2-V617F

*

BM nestin+ cell reduction in

MPN occurs by early apoptosis

(%)

BM nestin+ cells do not contribute

to fibrosis/ osteosclerosis in MPN

Early apoptosis of BM nestin+ cells contributes to MPN pathogenesis

Haem

ato

cri

t (%

) 0

2

4

6

**

Neutr

oph

ils p

er

ml of

blo

od

(x10

6)

0

5

10

15

20H

aem

oglo

bin

per

dl of blo

od

(g) *

0

20

40

60 *

iDTA Nes-creERT2;iDTA

In vivo depletion of BM nestin+ cells accelerates MPN

Control iDTA

Nes-creERT2;iDTA

Early apoptosis of BM nestin+ cells contributes to MPN pathogenesis

0.0

0.5

1.0

1.5

LS

K (

% B

MN

C) *

Cxcl12fl/fl

Nes-creERT2;

Cxcl12fl/fl

In vivo deletion of

Cxcl12 in nestin+ cells

accelerates MPN

Control human MPN

Neuropathy of the bone marrow HSC niche in human MPN

TH DAPI

Sympathetic neuropathy of the bone marrow HSC niche in MPN

Gfap

Control

JAK2-V617F

Nes-GFP

Control

JAK2-V617F

Th Merge

BM sympathetic neuropathy occurs before nestin+ cell apoptosis

2 4 8

0

50

100

150

200

2 4

0

100

200

300

*

Gfap

Are

a B

M (

% c

ontr

ol)

p=0.06

time (weeks)

*

Th

Are

a B

M (

% c

ontr

ol)

time (weeks)

Gfap

Control (4 weeks) JAK2-V617F (4 weeks)

Th

2 4 80

10

20

30

40

*

time (weeks)

Nes-GFP+ cells

Apop

totic (

%)

Nes-GFP

Contribution of HSC-derived IL-1 to MPN pathogenesis

0

50

100

150

200 **

IL-1

(p

g

per

mg p

rote

in)

0

10

20

30

40

50

TN

F-

(p

g

per

mg p

rote

in)

C JAK2-

V617F

C JAK2-

V617F

BMEF

0

2

4

6

8

0.00

0.05

0.10

0.15

0.20

0.25

IL-1

mR

NA

(ra

tio)

Casp1

mR

NA

(ra

tio)

*

0.0

0.1

0.2

0.3

0.4

(%)

*

BM LSK cells

C JAK2-

V617F

C JAK2-

V617F

C JAK2-

V617F

0

100

200

300

400

0

10

20

30

Nestin-GFP+

IL1

ra m

RN

A (

ratio)

IL1

r m

RN

A (

ratio)

C JAK2-

V617F

C JAK2-

V617F

* *

Early stimulation of IL-1β

pathway in MPN

Contribution of HSC-derived IL-1 to MPN pathogenesis

BM CD90+ cells

0

1000

2000

3000

4000

5000per

ml (x

10

6)

0.0

0.5

1.0

1.5

2.0

2.5

(%)

0.0

0.5

1.0

1.5

2.0BM CD105+ cells

0

2

4

6

8

10(%

)

BM LSK cells PB plateles

Ca

sp1

mR

NA

(ra

tio)

JAK2V617F

IL1ra +

- + - -

+ - +

JAK2V617F

IL1ra +

- + - -

+ - +

*** *

*

**

* p=0.08 * *

IL-1β plays a pathogenic

role in MPN, and its

targeting may have

clinical implications

Contribution of HSC-derived IL-1 to MPN pathogenesis

HSC-derived IL-1 contributes

to neuroglial damage in MPN

Could the mutant HSC directly cause BM Schwann cell

death in MPN?

JAK2V617F LSK JAK2V617F LSK

IL1ra JAK2V617F LSK Control LSK JAK2V617F LSK

IL1ra

DAPI

TUNEL

0

1

2

3

TU

NE

L+ c

ells

(fo

ld)

** *

JAK2V617F

IL1ra - + + - + - - - - + MSC Schwann

Control Schwann cells

Control Schwann

Control MSC

Control LSK

JAK2V617F LSK +

Treatment with a 3-adrenergic agonist or a neuroprotective drug blocks MPN progression

GFAP control

vehicle

4-methylcatechol

MxCre;JAK2-V617F, 4-methylcatechol

MxCre;JAK2-V617F, vehicle

MxCre;JAK2-V617F, BRL37344

Control

0.0

0.5

1.0

1.5

2.0

Neutr

oph

ils p

er

ml of

blo

od

(x10

6)

* * **

Neuroprotection/

neurocompensation

prevent early neutrophilia

Neuroglial damage as therapeutic target

4-methylcatechol – Rescue, protection

BRL37344 – Compensation

Treatment with a 3-adrenergic agonist drug blocks MPN progression

BR

L3

7344

Trichromic stainingReticulin fibres

ve

hic

le

JAK2-V617F , vehicle JAK2-V617F, BRL37344

4 8 12 160

20

40

60

80

Neutr

oph

ils p

er

ml of

blo

od

(x10

6)

** ***

time (weeks) 4 8 12 16

0

2000

4000

6000

Pla

tele

ts p

er

ml of

blo

od

(x10

6)

*** ***

time (weeks) 0

1

2

3

mR

NA

(ratio)

JAK2V617F

CD45

AR - - - - + + + + - - - - + + + +

3 2

CD45+ cells do no

express 3-AR

0

250

500

750

1000

1250

pe

r m

l of b

loo

d (

x10

6)

*

Control

3-AR KO

Platelets

Protective role of

3-AR in MPN

0.0

0.5

1.0

1.5

BMEF Cxcl12

(ng

per

2 f

em

ora

) *

0

500

1000

1500

2000

2500

*

BM Nes-GFP+

cells

Compensation of BM neural damage prevents MSC loss and early inflammation

Control, BRL37344 JAK2-V617F, vehicle JAK2-V617F, BRL37344 JAK2-V617F, 4-methylcatechol Control, vehicle

0

100

200

300

400

0

20

40

60

80

pg

per

mg o

f pro

tein

BMEF IL-1

pg

per

mg o

f pro

tein

BMEF IL-1

** * *

*

*** * *

Compensation of BM neural damage prevents mutant HSC expansion

Control, BRL37344 JAK2-V617F, vehicle JAK2-V617F, BRL37344 JAK2-V617F, 4-methylcatechol Control, vehicle

(%)

0.0

0.1

0.2

0.3

BM CFU-C

** *

0.0

0.5

1.0

1.5

2.0

2.5

BM LSK

(%)

*** ** ***

0 10 20 30 401

10

100

1 in 192,641

1 in 42,167

p0.05

Nega

tive

mic

e (

%)

Bone marrow nucleated cells (x104)

BRL37344

Vehicle

MPN-initiating cells

MP

N

Glial and neural

damage

CXCL12

IL-1

mutant HSC

Nestin+ MSC

apoptosis

CXCL12

Cytokine storm Fibrosis &

osteosclerosis

SNS rescue/protection

4-methylcatechol

3-AR

H

EA

LT

H

BONE MARROW Schwann cell

normal HSC

NA SNS

CXCL12 Nestin+

MSC

Rescue of sympathetic neuropathy of BM HSC niche blocks MPN

Arranz et al, Nature 2014

SNS compensation

3-adrenergic agonists

No fibrosis or

osteosclerosis

CXCL12

3-AR

3-AR

agonists

Chronic inflammation in MPN

PV

ET MF Chronic

inflammation

Chronic inflammation initiates already in the HSC compartment and

through IL-1β release.

IL-1β-derived neural damage is required for further progression of MPN.

Premature atherosclerosis

Second cancer

Atherosclerosis

Second cancer

MDS/AML

Cytokine

storm TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

TNF-α, IL-1β,

IL-6, IL-8,

IL-11, HGF

Adapted from Hasselbalch & Bjørn, Mediators Inflamm 2015

JAK2-V617F+

IL-1β

Injury/ Infection

IL-1

NF-κB

PU.1

normal HSC

Monocyte

Endothelial cell

Myeloid-

primed HSC

Expansion

Myeloid

output

Inflammation

Reviewed in Arranz*, Arriero & Villatoro,

Blood Rev 2017

Adapted from Pietras et al,

Nat Cell Biol 2016

JMML (Ptpn11-E76K)

mutant MSPCs/

osteoprogenitors

normal HSC

CCL3

Monocyte

HSC

expansion

IL-1

Monocyte

recruitment

Adapted from Dong et al,

Nature 2016

Pathological mechanisms of IL-1β on HSC function

AML (MLL-AF9)

normal HSC mutant HSC

IL-1

IRAK4

UBE2O

MLL

MLL Fusion

Gene

expression

MLL

degradation

Reviewed in Arranz*, Arriero & Villatoro,

Blood Rev 2017

Adapted from Liang et al, Cell 2017

Pathological mechanisms of IL-1β on HSC function

Hematological

malignancy

Preclinical

model

Drug Mechanism Reference

MPN JAK2-V617F IL-1RA IL-1RA is a competitive

inhibitor of IL-1R1 that

binds to IL-1 impeding its

interaction with IL-1R1

Nature 2014

CML BCR-ABL IL-1RA and

Imatinib Blood 2016

JMML Ptpn11-E76K

in MSPCs

CCR1A or

CCR5A

Antagonists that block

CCL3 binding to CCR1 or

CCR5, respectively

Nature 2016

AML MLL-AF9

IRAK1/4 or

IRAK4

inhibitors

Inhibition of IL-1R-

associated kinases that

impedes signaling

downstream IL-1R1

Cell 2017

Reviewed in Arranz*, Arriero & Villatoro,

Blood Rev 2017

Treatments targeting IL-1 pathway efficient in preclinical models

Basel University Hospital (Switzerland)

Jürg Schwaller

Radek Skoda

CNIC Technical Units

CNIC Comparative Medicine

CNIC DDRC Department

Stem Cell Niche Pathophysiology Lab: Abel Sánchez-Aguilera

Daniel Martín-Pérez

Joan Isern

Javier Langa

Simón Méndez-Ferrer

Acknowledgements

Funding sources

Thank you