Fusion Genes as

Potential Therapeutic

Targets

Olaf Heidenreich

32nd Cancer Cytogenetics

Group Meeting 2010

Improvement of Survival

St. Jude‘s Children‘s Hospital

Drugs for Chemotherapy

Cytarabine

Dexamethasone

Vincristine

Daunorubicin

Etoposide

Cyclophosphamide

Drugs in Cancer Therapy

Rene Bernards

Paul Ehrlich (1854 – 1915)

"Wir müssen lernen, magische Kugeln zu gießen,

die gleichsam wie Zauberkugeln des

Freischützen - nur die Krankheitserreger treffen."

Current and Ideal Treatment

Chromosomal Aberrations in AML and ALL

Look (1997)

Science 278, 1059 ff.

t(8;21)(q22;q22)

Incidence 8x10-7 – 1.5x10-6

80 new presentations/a

40 relapses/a

Breakpoint conserved

mainly AML-M2

RUNX1/RUNX1T1

(AML1/MTG8, AML1/ETO)

Transcriptional repressor

RHD

50-178

TAFH

NHR1

270-360

NHR2

485-551

CCR

NHR3

592-634

MYND

NHR4

663-699

DNA binding

C/EBPa

CBFb

MTGs

HDAC1, 3

SIN3A

HDAC1, 3

SMRT

N-CoRE proteins

MLL/AF4 (MLL/AFF1)

P-CTD

RNA Pol II

H3K79me2

LEDGF

MLLN AF4C

MT

AT

LAF4

AF5

AF9

ENL pTEFb

DOT1L

MEN1

siRNA-mediated Suppression of Leukaemic Fusion Genes

RISC

AML1/MTG8

AML1

Mo

ck

siA

ML

1/M

TG

8

siC

TL

2

Kasumi-1 SEM

Mo

ck

siM

LL

/AF

4

siC

TL

1

MLL/AF4

GAPDH

Heidenreich (2004)

Curr. Pharm. Biotech. 5, 349 ff.Heidenreich et al. (2003)

Blood 101, 3157 ff.

Thomas et al. (2005)

Blood 106, 3559 ff.

Thomas et al. (2005)

Blood 106, 3559 ff.

0

25

50

75

100

125

Mo

ck

siC

TL

1

siM

LL

/AF

4

siA

ML

1/M

TG

8

siC

TL

2

% c

olo

nie

s

siRNA

SEM Kasumi-1

siRNA Specificity

Delivery of Antileukaemic siRNAs ex vivo

Leukaemic

self-renewal

Regulation of Leukaemic Stemness

MLLN AF4C

MT

AT

AML1N

MTG8C

RHD

TAFH

Telomerase

Leukaemic Fusion Proteins Regulate TERT

Maria Thomas

Andreas Gessner

Patricia Garrido Castro

Lars Büchler

Leukaemic Fusion Proteins

Regulate Stem Cell Markers

6_7.016

FL1-H10

010

110

210

310

4

0

40

80

120

160

no_7.014

FL1-H10

010

110

210

310

4

0

40

80

120

160

1_7.015

FL1-H10

010

110

210

310

4

0

40

80

120

160

Mock siAML1/MTG8 siCTL2

CD34

Co

un

ts

Thomas et al. (2005)

Blood 106, 3559 ff.

siMLL/AF4siCTL1

Mock

Martinez et al. (2004)

BMC Cancer 4; 44

Leukaemic Fusion Proteins Regulate ANGPT1

Patricia Garrido Castro

Leukaemic

self-renewal

Regulation of Leukaemic Stemness

MLLN AF4C

MT

AT

AML1N

MTG8C

RHD

TAFH

TERT

ANGPT1

CD

34

CD

133

Targeting Fusion Protein-mediated

Leukaemogenesis

X

XX

AML1/MTG8 Impairs Drosophila Crystal Cell Formation

Pro-hemocytes

Crystal cells

progenitors

Plasmatocytes

progenitors

Plasmatocytes

AML1 MTG8+

Crystal cells

differentiation blocked

Increase of the number

of crystal cells progenitors

Dani Osman

Toulouse

AML1 MTG8 RNAi [NIG Japan]

UAS

Hairpin

dsRNA

1500 genes: 10% of Drosophila genome

2500 dsRNA transgenic lines screened

Dani Osman

Toulouse

Screen for Suppressors of AML1/MTG8-induced Lethality

Effect of CalpB knock-down on crystal cells number

Nu

mb

er

of

Lz+

cells p

er

larv

ae

Osman et al., 2009

PNAS 106, 12043 ff.

Calpains

Merck Calbiochem

Calpain Functions

Cleavage of cytoskeletal components such

as Talin

GSK3 and b-Catenin (both activating!)

XIAP (proapoptotic)

IkB (antiapoptotic, pro-proliferative)

Androgen receptor (androgen

independence)

Nitric Oxide Synthase (HSP90-modulated)

Calpain Inhibition Impairs Leukaemic Clonogenicity

0

20

40

60

80

100

120

140

160

DMSO ALLN 10µM

ALLN 30µM

Inh III 10µM

Inh III 30µM

Colo

nie

s

(% o

f co

ntr

ol)

Kasumi-1

0

0.2

0.4

0.6

0.8

1

1.2

DMSO ALLN Inh IIIR

el.

co

lon

y n

um

ber

CFU-GEMM

CFU-GM

CFU-M

CFU-G

B/CFU-E

Osman et al., 2009

PNAS 106, 12043 ff.

Targeting Fusion Protein-mediated

Leukaemogenesis

X

How to achieve specificity and efficacy?

Tumour specifity

Target

specificity

Tissue

specificity

Efficacy

Tissue

enrichment

siRNA Delivery

Uptake of siRNA ImmunoliposomesM

erg

eC

y3-s

iRN

A

Cell

tracker

Jurkat + IL Kasumi-1 + L Kasumi-1 + IL SKNO-1 + L SKNO-1 + IL

Rothdiener et al., 2010

J. Contr. Release, in press

Liposomal siRNA Delivery

Rothdiener et al., 2010

J. Contr. Release, in press

Targeted siRNA Delivery

Cytostatic

drugs

Thomas et al. (2006)

Acta Pharm. Sin. 27, 273 ff.

Thanks toDani Osman, Lucas Waltzer, Marc HaenlinCentre de Biologie du Développement, Toulouse

Gerhard Heil & Jürgen Krauter

Michaela Scherr & Matthias EderHämatologie, Hämostaseologie und Onkologie

Medizinische Hochschule Hannover

Johann GreilKinderheilkunde III

Universitätsklinikum Heidelberg

Jenny Dunne & Bryan YoungMedical Oncology

Barts and the London School of Medicine

Markus ManzIstituto di Ricerca in Biomedicina

Bellinzona

Philipp Hadwiger & Hans-Peter VornlocherAlnylam Europe, Kulmbach

Mike Batey

Lars Buechler

Patricia Garrido Castro

Vicky Forster

Andreas Geßner

Elda Latif

Svetlana Mysina

Frida Ponthan

Anja Scholz

James Allan

Josef VormoorNICR, Newcastle University

Natalia Martinez Soria

Meriem Nouaimi-Bachmann

Heidemarie Riehle

Maria ThomasAbteilung Molekularbiologie & Proteomzentrum

Eberhard Karls Universität Tübingen

Miriam Rether

Roland KontermannInstitut für Zellbiologie und Immunologie

Universität Stuttgart

JGW Patterson Foundation