Special Emphasis Pathway in Cancer BiologySpecial Emphasis Pathway in Cancer Biology

Paroxysmal Nocturnal Hemoglobinuria Paroxysmal Nocturnal Hemoglobinuria (PNH)(PNH)

Monica Bessler, MD, PhDWashington University School of Medicine St. LouisDivision of Hematology

Email mbessler@im.wustl.eduWeb: http://bmf.im.wustl.edu

Bone Marrow Failure

Normal bone marrow Bone marrow failure

Bondurant et al.Wintrobe 1993

Hematopoiesis Occurs in the Bone Marrow

Acquired Inherited

Secondary bone marrow failure FanconiÕs anemia Radiation Dyskeratosis congenita (DC) Drug and chemicals (e.g. Benzene) Shwachman Diamond Syndrome Idiosyncratic reactions (e.g. Chloramphenicol) Diamond Blackfan Anemia (DBA) Virues (e.g. EBV, Hepatitis, CMV) Amegakaryocytic thrombocytopenia Immune diseases (e.g. Graft-versus-host disease) Severe congenital neutropenia Pregnancy Cartilage-Hair Hypoplasia Reticular Dysgenesis Idiopathic bone marrow failure Thrombocytopenia with absent radii Paroxysmal Nocturnal Hemoglobinuria (PNH) Pearson syndrome Non hematologic syndromes (e.g. DownÕs syndrome) Familial aplastic anemia

Causes of Bone Marrow Failure

Published by AAAS

C. Mayr et al., Science 315, 1576 -1579 (2007)

Fig. 1. Chromosomal translocations involving HMGA2, and the influence of let-7 on protein expression

F9

NIH 3T324hrs 48 hrs

oncogene

Tumor supressor

Let 7 tumor spressor

Published by AAAS

C. Mayr et al., Science 315, 1576 -1579 (2007)

Fig. 2. Luciferase reporter assays showing the influence of miRNA-target pairing

miRNA to let-7a

miRNA to mlet-7a

F9

NIH 3T3

HeLa

Published by AAAS

C. Mayr et al., Science 315, 1576 -1579 (2007)

Fig. 3. Soft-agar assay for anchorage-independent growth

NIH 3T3

Fig. S1

Paroxysmal Nocturnal Hemoglobinuria

I. Hemoglobinuria

II.Bone Marrow Failure

III.Thrombosis

Clinical Manifestations of PNH

Age Distribution of Patients with PNHAge Distribution of Patients with PNH

J Nishimura et. al. Medicine 2004

Tiananmen SquareTiananmen Square

Institute of Hematology & Blood Diseases HospitalChinese Academy of Medical ScienceTianjinChina

Peking Union Medical CollegeBeijingChina

Median Survival of Patients Diagnosed with PNH is 10 -15 Years

Thromboembolism in PNH

GPI-linked Proteins on Human Blood Cells

MonocytesB CellsT CellsNK CellsGranulocytesRed CellsPlateletsCD55CD58 1

CD59CD109(Gova/b-Ag)PrPcGP500

CD55 CD58 1

CD59 CD14 CD16 CD24(NAB1-Ag)CD48 CD66bCD66c CD87CD109 CD157LAP NB1 PrPc ADP-RTp50-80 GPI-80

CD55CD58 1

CD59CD14{CD16 2}CD48CDw52CD87CD109CD157Group-8PrPcGPI-80

CD55CD58 1

CD59CD24 CD48CDw52 {CD73}{CDw108}PrPc

CD55CD58 1

CD59{CD16 2} CD48CDw52{CD73}CD87{CD90}CDw108{CD109}PrPcADP-RT

CD55CD58 1

CD59CD16 2

CD48CDw52PrPc

HematopoieticStem Cell

CD59 CD109CD90

CD55, CD59(Cromer Ag)CD58, PrPc, AChE (Cartwrigt-Ag), CDw108(John-Milton-Hagen Ag)Dombroch residueHolley Gregory AG

Increased Sensitivity of Red Cells to Activated Complement

PNH Control

S HS S HSPNH patient

HamTest: 1938

Deficiency of GPI-Linked Proteins Deficiency of GPI-Linked Proteins on PNH-Granulocyteson PNH-Granulocytes

Diagnosis of PNH by Flow Cytometry

Patient

Control

Sideward scatter

CD59

Red Cells PMN Monocytes Lymph.

PNH red cells are of clonal origin

(Oni et al. 1970)

G6PD A

G6PD B

Female PNH Total RBCs

PNH RBCsMale normal RBCs

Male normal RBCs

GPI-Anchor

I

O OC=O

PEthN

P EthN

C-C-C

Man

GlcN

Man

P

Membrane

C=O

NH2

PEthN

Man

Protein

Phosphoethanolamine

P EthN

Man

GlcN

PI

Mannose

Glucosamine

Inositolphosphate

Pathway of GPI-anchor Biosynthesis

COOH COOH

NH 2

NH2

PIGSPIGTPIGUPIGKGAA1

DG

Ac

PIGL

DPM1DPM2DPM3MPDU1

GDP

GDP

Dol-P

Dol-P

PIGN

GlcNAcMan

PEth

PI

Ac

PIGAPIGCPIGHGPI1GPIPDMP2

?AcUDP UDP acetate

acyl-CoA

Dol-PDol-P

PIGB

PIGFPIGO DG

Endoplasmic reticulum

Dol-PDol-P

Dol-PDGPIGMPIGX

PIGW

PIGVGPI7

GPI-biosynthesis in PNH cell lines

[3H] mannose label

Block in the Pathway of GPI-anchor Biosynthesis

COOH COOH

NH 2

NH2

PIGSPIGTPIGUPIGKGAA1

DG

Ac

PIGL

DPM1DPM2DPM3MPDU1

GDP

GDP

Dol-P

Dol-P

PIGN

GlcNAcMan

PEth

PI

Ac

PIGAPIGCPIGHGPI1GPIPDMP2

?AcUDP UDP acetate

acyl-CoA

Dol-PDol-P

PIGB

PIGFPIGO DG

Endoplasmic reticulum

Dol-PDol-P

Dol-PDGPIGMPIGX

PIGW

PIGVGPI7

GPI-N-acetylglucosaminyltransferase

ER

P

I

P

I

Glc -NAc

NAc

PIGH

hGPI1Glc -NAc

NAc

UDP

PIGA

PIGC

PIGP

DPM2

PIGAPIGCPIGH

PIGPhGPI1

DPM2GPIQ?

Correction of the PNH Phenotype in LCL after PIGA

cDNA Tranfection

Anti-CD59-FITC

The PIGA Gene and the Mutations found in PNH

Luzzatto, 2000

PIGA maps to

the X-Chromosome

Major areas of investigationMajor areas of investigation

• Why does a PNH hematopoietic stem cell missing so many different proteins take over normal hematopoiesis ?

•What is the reason for clotting ?

• Can we design a more targeted treatment for patients with PNH ?

Erythroid Differentiation of PIGA- Erythroid Differentiation of PIGA- ES CellsES Cells

21 34 5 6

Cre

lacZ

loxP loxP

lacZ

loxPiga

wt Piga

Piga

Mouse Model for PNH

Blood Cells Deficient in Blood Cells Deficient in GPI-Linked ProteinsGPI-Linked Proteins

GPI-linked proteins

Granulocytes

Red cells∆ PIGA Control PNH Control

Mouse Human∆ PIGA

GPI-linked proteins

PNH Control∆ PIGA Control

S HS S HS(Heterologous SerumHS, Heat inactivated)

Acidified Serum Lysis Ham-Dacie - Test

Tremml et. al. 1999S HS S HS

PNH Blood Cells in Mouse and Man

GPI-linked marker

Lineagespecificmarker

Granulo-cytes

Red blood cells

MalePNH

FemalePNH

wtcontrol

Patient with PNH

Healthyindividual

Mouse Human

Red Blood Cells

0

20

40

60

80

100

1 2 4 8 12

Age (months)

% G

PI-

Ce

lls

Granulocytes

Age (months)

0

20

40

60

80

100

1 2 4 8 12

% G

PI-

Ce

lls

T Cells

0

20

40

60

80

100

1 2 4 8 12Age (months)

% G

PI-

Ce

lls

B Cells

0

20

40

60

80

100

1 2 4 8 12

Age (months)

% G

PI-

Ce

llsBlood Cells Deficient inBlood Cells Deficient in

GPI-Linked Proteins in Mice GPI-Linked Proteins in Mice

03/07

500

1000

2000

3000

8/93 1/99 5/00 9/01 2/03 6/04 11/05

AA PNH

Lac

tate

deh

ydro

gen

ase

(LD

H)

Normal range

ATG+Cy x3

Un

its

RB

C

12

84

Red cell transfusions

EculizumabEculizumabC5 blockadeC5 blockade

Case report: Case report: 31 year old female PNH patient # 9931 year old female PNH patient # 99

DVT

Hemolytic / Classical PNH AA/ PNH

PNHPNHPNH

Per

iphe

ral b

lood

ce

ll co

unt

Normal Normal Normal

Cytopenia

Relationship of PNH with Aplastic Anemia (AA)

Adapted from Rotoli & Luzzatto 1989

Trisomy 8 in MDS/PNH(Longo et. al. 1994)

Chromosome 8

Cytogenetic Abnormalities in PNH (Araten 2001)

U.P.N. Age % PNH Karyotypic abnormality

MSK 18 31M 7 47, XY, +6 [6/20] MSK 30 20M 5 47,X,+5,del(5)(q11.2)(6/15) MSK 8 57M 100 47,XY,+X[20/20]

MSK 20 16M 98 46,XY,t(17;19)(q11;q13)[6/20] MSK 17 42M 59 45,XY,-7[14/25] MSK 49 32M 36 47, XY+6[12/20] MSK 23 27M 70 46,XY,del(8p)[2/30] MSK 21 26M 82 47,XY,+8[2/20] MSK 19 63M 80 46,XY,del(5)(q15q31)[2/17] HH 56 19F 60 46,XX,del(13)(q12q14)[5/10]

MSK 16 45M 100 47,XY,+8[6/30]

Cytogenetic Abnormalities in PNHCytogenetic Abnormalities in PNH

PNH clone

Trisomy 8% B

lood

Cel

ls

Trisomy 8

PNH clone%B

lood

Cel

ls

%B

lood

Cel

ls

Trisomy 8

MDS within PNH PNH and MDS PNH within MDS

PNH clone

Leukemogenesis in PNH Mice After ENULeukemogenesis in PNH Mice After ENU

Leukemia in PNH mice

Tumor PNH WT Ovary (Adenoma) 3 1 Lung (Adenoma) 1 3 Adenocarcinoma 2 0

6/15 4/15

Leukemia

Myeloid hyperplasia in the bone marrow 2 2

Myeloid hyperplasia of the spleen 2 1

Liver infiltration 2 2 Parasternal infiltration 2 1 2/15 2/15

The Rate of Transformation is SimilarThe Rate of Transformation is Similarin AA and PNHin AA and PNH

MDS / AML in Patients with Aplastic Anemia (AA) Study Tichelli

(1988) DePlanqu e

(1989) Socie (1993)

Paquet te (1995)

Doney (1997)

Ohara (1997)

137 468 860 155 227 119 No of patients

% MDS / AML (Incidence) 7 1 (15-25) 4 2 (13) 10 (11) 10

MDS / AML in Patients with PNH

Study Hillmen (1995)

Socie (1995)

Spaet-Schwalbe (1995)

Moyo (2004)

Nishimura (2004) Japan Duke

Ware (1991)

Eibrink (2005)

80 220 40 49 209 176 49 11 No of patients

% MDS / AML (Incidence) 0 1 (5) 0 6 8 16 2 36

Possible Interrelationship Between PNH, Possible Interrelationship Between PNH, Aplastic Anemia, Myelodysplastic Syndrome,Aplastic Anemia, Myelodysplastic Syndrome,

and Acute Myeloid Leukemiaand Acute Myeloid Leukemia

PIGA(-) CellsNormal Cells

Normal MarrowNormal Marrow

Model for the Development of PNH Model for the Development of PNH

MutationPIGA(-) Cells + Second Hit MutationBM Injury

Adaptive MutationsAdaptive Mutations

Survival advantage hypothesisSurvival advantage hypothesis

PN

HP

NH

PN

HP

NH

Clinical OutcomeClinical Outcome

2 hit hypothesis2 hit hypothesis

BM InjuryBM Injury

Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

Inoue, N. et al. Blood 2006;108:4232-4236

Figure 3. Effects of the chromosome 12 abnormalities in 2 patients with PNH

Regulation of Ink4a/Arf during Aging

Tzatsos &Bardeesy Cell Stem Cell 2008

HMGA2 (a second factor for expansion of a PNH clone?)

• Expression of high mobility group AT-hook 2 (HMGA2), which is regulated by let-7 miRNAs (Mayr C et al, Science, 2007), contributes to proliferation of cells.

• Chromosomal abnormalities involving in HMGA2, which remove let-7-complementary sites in 3’-untranslated region (UTR), have been reported in patients with PNH (Inoue N et al., Blood, 2006) and related disorders including myeloproliferative disorders (Guglielmelli P et al., Stem Cells, 2007) and AML with myelodysplasia (Odero MD et al., Leukemia, 2005).

Mouse HMGA2

cDNABC052158

Complementary sites of let-7a

1 3 4 52 6 7

Introduction of truncated cDNA clone of Hmga2 into pPGKPuro

( )

5’-UTR 3’-UTRcoding

XhoI ClaI

Coding5’-UTR PGK-PAPGK-PROM

Specific primer pair

SalISalI

XhoI

ClaI

SalI

SalI

Replace puro with the truncated cDNA of Hmga2

Cleaved recombinant with SalI for injection

Homologous recombination for conditional truncation of Hmga2 3’-UTR

loxP

QuickTimeý DzTIFFÅiàèkǻǵÅj êLí£ÉvÉçÉOÉâÉÄ

ǙDZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ ÇÅB

Exon 1 2 3 4 5

wt Hmga2

QuickTimeý DzTIFFÅiàèkǻǵÅj êLí£ÉvÉçÉOÉâÉÄ

ǙDZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ ÇÅB

IRES/NEO-lox-Hmga2

loxP

IRES/NEO

0

0.2

0.4

0.6

0.8

1

1.2

1.4

BM Spleen Thymus Liver

TG (n=3 or 4)

WT (n=3)

qRT-PCR

Western Blotting

Actin

HMGA2

TG WTSpleen

TG WTThymus

TG WTLiver

TG WTBM

Expression of Hmga2 in Truncated-Hmga2+ TG mouse

Re

lativ

e e

xpre

ssio

n

P<0.05 P=0.0509

P<0.01

P<0.02

42 kDa

17 kDa

Peripheral blood count

• Age- and sex-matched 19 truncated Hmga2+ TG and wild-type BL6 mice

Competitive repopulation assay

Truncated-Hmga2+

(CD45.2)PEP

(CD45.1)

Donors

1. 50% vs 50%2. 10% vs 90%3. 100% vs 0%

vs

5 x 106 cells

Recipients PEP

Total 1000 radInjection

(BM transplantation; BMT)

Observe chimerisms of PB cells 6 weeks and 10 to 12 weeks after BMT

Prepare BM cells

0

10

20

30

40

50

606w (n=2)

12w (n=3)

Proportions of donor-derived cells after competitive repopulation assay

10% Hmga290% Recipient

50% Hmga250% Recipient

%C

D4

5.2+

ce

lls

Granulocytes Monocytes B cellsT cells

100% Hmga2

Granulocytes Monocytes B cellsT cells

Granulocytes Monocytes B cellsT cells

Truncated-Hmga2+/-

X Piga-

with 1% GPI-

60% GPI- etc.

Truncated-Hmga2+/-Piga-Piga- (without truncated Hmga2)

Breed truncated Hmga2+ mice with Piga- mice to see effect of Hmga2 on growth advantage of PNH

hematopoietic cells

• Time courses of changes in proportion of GPI- cells• Blood cell count• Competitive repopulation assay• Spontaneous or ENU-induced leukemogenesis

2 mice 1 mouse(need more breeding)

AcknowledgementsAcknowledgementsWashington University St. Louis:Kazu IkedaJeffrey HickenMarek JasinskyPeter Keller Bing Han Ike PantazopoulosShashikant KulkarniPhilip J. Mason Division of HematologyMorey BlinderJoshua Fields

Bone Marrow Transplant TeamJohn DiPersioPeter Westervelt

Hereditary Cancer CoreChissie KampJennifer Ivanovich

Division of RheumatologyCelia Fang John Atkinson

Dept. PathologyRichard BurackSusan Treese

Center for Clinical StudiesParticipating Medical CentersForTRIUMPHSEPHEREDEMBRACE

All patients with PNH for participating