Post on 22-Mar-2018
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