I have the following financial relationships: Contracted ...€¦ · Malcovati et al, Best Pract Res Cl Ha 2013, Papaemmanuil et al, NEJM 2011, Malcovati. et al, Blood 2015, Conte

DISCLOSURE

▪ I have the following financial relationships:

Contracted Research: Celgene

Ineffective Erythropoiesis in MDS

Eva Hellström Lindberg

Karolinska Institutet, Karolinska University Hospital

Stockholm, Sweden

MDS Foundation Symposium ASH 2019

CME question

• Which subtypes of MDS frequently show hyperplastic erythropoiesis?

• MDS with fibrosis

• 5q- syndrome

• MDS with ring sideroblasts

• CMML

Learning objectives

• The impact of anemia

• Erythropoiesis

• Mechanisms of anemia in MDS

• Modeling erythropoiesis in SF3B1 mutated MDS with

ring sideroblasts

• Targeting ineffective erythropoiesis

Symptomatic anemia

and chronic transfusion

need

• 309 consecutive MDS and

MDS-MPN patients

• 11,350 red cell and 1956

platelet units over 777 person-

years of follow-up,

• Overall transfusion intensity of

14.6 and 2.5 units/person-year,

respectively

Rydén, et al. Leukemia. 2019 Feb;33(2):522-527

Lower-risk MDS – RBC transfusions over time

Rydén, et al. Leukemia. 2019 Feb;33(2):522-527

EU MDS Registry data

Stauder, et al, Leukemia (2018), Garelius, et al, JIM (2117)

ESA treatment before TD

prolongs time to first transfusion

QoL significantly impaired in

anemic patients

Erythropoiesis

HSC

MEP

BFU-E

CFU-E

PRO-EB BASO-EB POLY-EB ORTHO-EB

RETIC RBC

CD34

CD117

CD45

CD71

CD36

CD105

CD235a and hemoglobin

SCF, IL-3, IL-6

EPO

IRONGATA2

GATA1

Receptors

Growth

factors

Transcription

Factors

Bone marrow microenvironment

Adler et al. J. Nat Rev Endocrinol 2014 National Cancer Institute, at the National Institutes of Health

Erythroblastic islands

Chasis and Mohandas Blood 2008

Patterns of ineffective hematopoiesis in MDS

Hyperplastic erythropoiesis

Hypoplastic erythropoiesis

Low BM blasts High BM blasts

Hyperplastic bone marrow

Hypoplastic bone marrow

Mutations

Cytogenetic

aberrations

Ineffective

hematopoisis

Anemia and aberrant erythropoiesis

the most common feature in MDS

▪ Underrepresented erythropoiesis (few erythroblasts)

→Hypoplastic MDS, low general cellularity,± immune-mediated cytopenia

▪ As examplified by many germline conditions; DBA, GATA2 mut, etc

▪ But also in immune-mediated aplastic anemia

→Advanced MDS, increased blast percentage (MDS EB-2)

→Mixed MDS-MPN, often maximal cellularity

→MDS och MDS-MPD with fibrosis

→5q- syndrome, in particular in later stages

▪ Hyperplastic ineffective erythropoiesis

→Normo / hyperplastic bone marrow with erythroid dysplasia / apoptosis

→MDS-RS

→Some forms of MDS-SLD and MLD, and MDS EB-1

→Myelodysplastic syndrome with erythroid predominance

Low-risk MDS with isolated del(5q)


Normo / hypoplastic erythropoiesis




Mutations

Cytogenetic

aberration

del(5q)

The 5q- syndrome

• 3-4% of all MDS, female preponderance

• Hypolobated megakaryocytes, thrombocytosis

• Isolated deletion of 5q

• involving 5q32-33

• ~ 1.5Mb, 44 gene

• Including RPS14, CSNK1A1, and miR-145

• Del(5q) alone causes macrocytic anemia

• No progression to higher-risk MDS / AML unless

clonal evolution (mainly TP53, RUNX1, additional

cytogenetic aberrations)

Van den Berghe, 1974, Schanz et al, JCO 2011, Ebert et al. 2008 Nature, Schneider,

et al, Cancer Cell 2014, Barlow et al, Nature Medicine 2010, Jädersten JCO 2011

5q-syndrome: cellular and molecular characteristics

RPS14

Nilsson et al, Blood, 2000, Ebert et al. 2008 Nature, List et al, NEJM 2005 & 2006, Schneider, et al, Cancer Cell 2014, Barlow et

al, Nature Medicine 2010, Tehranchi et al NEJM 2010, Scharenberg BrH 2018, Ribezzo, et al, Leukemia 2019

Stem cell disorder

Haploinsufficiency of RPS14

mediates erythroid apoptosis

through p53 activation

Haploinsufficiency of CSNK1A1

explains clonal dominance

Combined haploinsufficiciency of

RPS14, CSNK1A1, and miR-145

recapitulate the clinical phenotype

Lenalidomide (LEN) induce CR

through inhibiting the 5q clone

However, del(5q) HSC, megakaryo-

cytes, and TP53 mut cells persist

during LEN-induced CR

MDS with ring sideroblasts (MDS-RS)


+RS

Hypoplastic erythropoiesis




SF3B1

Mutation

Cytogenetic

aberrations

Björkman S, Blood 1956

HSC

MEP

BFU-E

CFU-E

PRO-EB BASO-EB POLY-EB ORTHO-EB

MDS-RS

▪ Hyperplastic erythropoiesis

▪ Ring sideroblasts (RS) in the bone marrow

▪ Severe anemia, transfusion dependency and low risk of

transformation to AML

▪ SF3B1 mutations in >80% of patients

Malcovati et al, Best Pract Res Cl Ha 2013, Papaemmanuil et al, NEJM 2011, Malcovati. et al, Blood 2015, Conte et al, Br J Haematol 2015

NBMRARSRA

Mitochondrial dysfunction in MDS erythroblasts

Tehranchi et al., Blood, 2003, 2005, CCR 2005

NBM RARS + G-CSF 4hRARS• Marked cytochrome c release

• Caspase-9 and 3 activation

• Erythroblasts express G-CCF R

• G-CSF inhibits cyt c release

Early Erythroblasts in RARS express an

aberrant form of mitochondrial ferritin (MtF)

0

20

30

40

50

10

0 4 7 11 14Day

MtF+Cells(median)

NBM

RA

RARS

Cazzola etal,Blood 2003,Tehranchi etal,Blood 2005

Spliceasome mutations in MDS

Papaemmanuil, NEJM, 29 Sept 2011

Yoshida, Nature, 2011

Clinical significance of SF3B1

mutations in myelodysplastic

syndromes and

myelodysplastic/myeloproliferative

neoplasms

Malcovati L*, Papaemmanuil E*

BLOOD, on-line, 2011

Papaemmanuil, NEJM, 2011

EPO+G-CSF act through unspecific but highly

effective inhibition of erythroid apoptosis

- does not inhibit iron accumulation

- Increases release of aberrant RBC

0

20

30

40

50

10

0 4 7 11 14Day

MtF+Cells(median)

NBM

RA

RARS

Cazzola etal,Blood 2003,Tehranchi etal,Blood 2005,Ljung,Haematologica 2004

RANormal

RARS RARS-GF

Size

Chromicity

Erythrocytes

Spliceasome mutations in MDS

Papaemmanuil, NEJM, 29 Sept 2011

Yoshida, Nature, 2011

Clinical significance of SF3B1

mutations in myelodysplastic

syndromes and

myelodysplastic/myeloproliferative

neoplasms

Malcovati L*, Papaemmanuil E*

BLOOD, on-line, 2011

Papaemmanuil, NEJM, 2011

The consequences of SF3B1 mutations affects

erythropoiesis during terminal maturation

Papaemmanuil NEJM 2011, Conte, Br J Haematol, 2015,

ABCB7

The iron transporter ABCB7 in RARS

medates erythroid failure

ABCB7 mutated in XLSA-T, these patients have RS in BM

Nikpour M, Br J Haematol. 2010, Nikpour Leukemia 2013

0

50

100

150

200

250

300

BFU‐E CFU‐GM total

rela

%v

e n

um

be

r o

f C

FU

s

control

mock

ABCB7

0

40

80

120

160

200

240

280

Control +LV PLKO.1 +LV shR-E1

shRNA in normal BM induces a RARS

phenotype Overexpression in RARS BM restores

a normal phenotype phenotype

Altered exon usage and nonsense-mediated decay

CFU data

Overall Survival

HR .35, P=.007

HR .32, P=.005 HR .26, P=.045

RA

RS

/RC

MD

-RS

All W

HO

cate

go

ries

Malcovati, Blood, 2015

SF3B1 mutations associated with lower-risk

MDS with ring sideroblasts

Experimental models of MDS-RS

▪ SF3B1 mutation conditional knock-in

mice do not support RS formation

- Murine orthologues of genes associated

with RS in humans not mis-spliced

- Poor conservation of splice sites

between the species

▪ CD34+ suspension cultures do not mimic the

mature MDS-RS phenotype

- Proliferation of erythroblasts

- Aberrant mitochondrial ferritin accumulation

- Limited production of mature red blood cells

or RS

25Mupo et al, Leukemia 2017, Obeng et al, Cancer Cell 2016, Tehranchi et al, Blood 2003 and 2005, Nikpour et al, Leukemia 2013, Claessens et al, Blood 2002

SF3B1K700E

X

CD34+

X

What is the cell of origin in MDS-RS?

Long Term Co Cultures

CFU

In Vitro

12

Functional assays

LTC-CFC

In Vivo

8 24

Engraftment and RS formation

NSG

Targeted sequencing

Mortera Blanco, Blood, 2017

Recurrent SF3B1 mutations are a part of the lymphoid lineage


Only RARS HSC give rise to LTC-IC and engraft in NSG mice

L ym p h o id M ye lo id L ym p h o id M ye lo id L ym p h o id M ye lo id L ym p h o id M ye lo id

0 .0 1

0 .1

1

1 0

1 0 0

% o

f H

SC

ce

lls

en

gra

fte

d i

n N

SG

mic

e

P a tie n t 4

P a tie n t 5

P a tie n t 1 1

P a tie n t 1 3

H S C C M P G M P M E P

Patient n.% of RS of total

cells (NSG mice)

4 1.5

5 19.1

11 4.3

13 3.2

M D S -R S N B M M D S -R S N B M M D S -R S N B M

0

1 0 0

2 0 0

3 0 0

4 0 0

Nu

mb

er o

f c

olo

nie

s p

er 4

00

ce

lls

E ry th ro id

M y e lo id

G M P M E PC M P

**

**

Nu

mb

er o

f c

olo

nie

s p

er 7

50

ce

lls

pla

ted

H S C C M P G M P M E P H S C C M P G M P M E P

0

2 0 0

4 0 0

6 0 0

8 0 0

M D S -R S N B M

00 0 0 0 0


LTC-IC

Modelling of terminal erythropoiesis in vitro

Mortera-Blanco et al. Biomaterials 2010 and 2011, Elvarsdottir, Leukemia 2019

• Polyurethan foam, pore size of human

bone marrow

• Coated with collagen type I

• Cut into scaffold cubes that could be

aspirated and fixated

Mortera-Blanco et al. Biomaterials 2011

3D culture facilitates expansion of CD34+ cells with self-renewal capacity

6 december 2019Namn Efternamn 30

Elvarsdottir, Leukemia 2019

31

CD34+ 3D facilitates erythropoiesis more effectively than MNC cultures

NBM

MDS-RS


Erythroblastic islands detected after 4 weeks of MNC and CD34+ 3D cultures

32

NBM

3D MNC 3D CD34+2D MNC

MDS-RS

3D MNC 3D CD34+2D MNC


6 december 2019Namn Efternamn 33

Erythroblastic island inside fixed scaffold

Nucleus (Draq5)

Macrophage/monocyte (CD68)

Erythrocyte (CD235a)

C

3D MNC

2D MNC

3D CD34+

A B

0 2 3 40

20

40

60

Week

VA

F %

0 2 3 40

20

40

60

Week

%R

S

0 2 3 40

20

40

60

Week

VA

F %

0 2 3 40

20

40

60

Week

%R

S

0 2 3 40

20

40

60

Week

VA

F %

0 2 3 40

20

40

60

Week

%R

S

MDS 1

MDS 2

MDS 3

MDS 4

MDS 5

MDS 6

Maintenance of SF3B1 VAF and RS formation

Scaffold cultures now used to study targeted drugs


New treatment options for MDSTGF-β superfamily ligand traps

35

Extracellular

domain of ActRIIB

modified to reduce

activin binding

Fc domain of the

human IgG1

antibody

Luspatercept ▪ Increased hemoglobin levels and RBC

counts in

- Post menopausal women

- MDS murine model

▪ Phase 2/3 studies in MDS patients

- Significantlt increased hemoglobin and

reduced transfusion burden

- Greater response rate in MDS-RS patients

Suragani et al, Nat Med 2014, Platzbecker et al, Lancet Oncol 2017, Fenaux & Platzbecker NEJM, accepted

Cytokine measurements in extracted medium of scaffolds

MNCs NBM CD34+ NBM MNCs MDS-RS CD34+ MDS-RS

1 2 4

0

2000

4000

6000

8000

10000

Week

pg/m

L

MCP-1**

MNCs CD34+

0

20

40

60

80

pg/m

L

GDF11

****

MNCs CD34+

0

50

100

150

200pg/m

L

VEGF

***

37

Which cells mediate the response to ESAs and other therapeutic compounds

in SF3B1 MDS with ring sideroblasts?

Research question

Isabel Hofman poster # 2993, Sunday

38

Experimental workflow

ddPCR for SF3B1mt VAF

At diagnosis During response

+EPO

Group 2

Untreated

(Stable anemia

without treatment)

n = 7

Group 4

Non-responders

to EPO

n = 5

Group 3

Responders

to EPO

n = 5

Group 1

Normal donors

n = 5MDS-RS

patients

CD38+CD34+

At relapse

FACS sorting of HSPCs

Isabel Hofman poster # 2993, Sunday

39

MDS-RS with stable anemia have lower VAF% in CD34+ BM cells

VAF% in

total BM

VAF% in

CD34+ BM cells

Non-responder

Untreated

Responder

U n tre a te d T re a te d

0

1 0

2 0

3 0

4 0

5 0

M e d ia n V A F % in to ta l b o n e m a rro w

ns

U n tre a te d T re a te d

0

1 0

2 0

3 0

4 0

5 0

V A F % C D 3 4 +

U n tre a ted

T re a ted

***

VA

F o

f S

F3

B1

%

Unpublished data Isabel Hofman poster # 2993, Sunday

Concluding remarks

• Severe anemia has an immense impact on the quality of life of elderly MDS patients

• Lower-risk MDS patients are high-consumers of packed RBC

• The mechanism of ineffective erythropoiesis differs largely between MDS subtypes and is

related to response to various treatments

• SF3B1 mutations arise at the primitive lymphomyeloid stem cell stage and lead to terminal

erythroid apoptosis

• New in vitro and in vivo models of erythropoiesis in MDS can help to dissect cellular and

molecular disease mechanisms as well as the effects of pro-erythroid therapies

• A key research question is whether pro-erythroid treatment mainly affects mutated or wild-

type bone marrow cells

Mei et al, Leukemia 2018, Giudice et al, Cytokine 2019, Song et al, Nat. Comm. 2019, Hsu et al ,Blood 2019 40

Acknowledgements

MSKCCElli Papaemmanuil & team

University of PaviaMario Cazzola

Luca Malcovati

King´s, London Ghulam Mufti

HannoverBrigitte Schlegelberger

Gudrun Göring

Karolinska InstitutetMarios Dimitriou

Teresa Mortera Blanco

Magnus Tobiasson

Martin Jädersten

Isabel Hofman

Edda Elvarsdottir

Maria Creignou

Gabriele Todisco

Monika Jansson

Gunilla Walldin

Lotte Björklund

Indira Barbosa

David Chang

Sten-Eirik Jacobsen

Petter Woll

Seishi Ogawa. Kyoto-KI

OxfordJackie Boultwood

Andrea Pellagatti

EU MDSTheo de Witte

Alex Smith, et al

CME question

• Which subtypes of MDS frequently show hyperplastic erythropoiesis?

• MDS with fibrosis

• 5q- syndrome

• MDS with ring sideroblasts

• CMML

Documents

I have the following financial relationships: Contracted ...€¦ · Malcovati et al, Best Pract Res Cl Ha 2013, Papaemmanuil et al, NEJM 2011, Malcovati. et al, Blood 2015, Conte