Microsoft PowerPoint - Session21.pptx

Centrosomal Clustering
A Novel Therapeutic Target for Multiple Myeloma
Marc S. Raab
CCU Molecular Hematology/Oncolo
gy
gy,
gy German Cancer Research Center &
Myeloma Research Program, Department of Medicine,
University of Heidelberg, Germany
Jerome Lipper Myeloma Center
Dana-Fb
Farber Cancer I
t
ns it
tit t
u e, B
t
os on, MA
MA, USA
USA

Introduction
·
Centrosomes are small intracellular organelles that function as
the microtubule-organizing centers of eukaryotic cells
·
Supernumerary centrosomes have been described in virtually
all human malignancies, highly associated with chromosomal
instability and the clinical aggressiveness of tumor cells -
including MM (Maxwell et al., 2004&2005; Chng et al., 2006&2008)
·
Supernumerary centrosomes lead to multipolar spindles,
aberrant mitoses, chromosome missegregation, and cell death
·
Therefore, malignant cells need to specifically cluster multiple
multiple
centrosomes into two spindle poles to ensure bipolar mitosis
and to regain secondary karyotype stability
·
Aim: To identify therapeutic molecules
molecules that
that disrupt
disrupt
centrosomal clustering in malignant cells, thereby inducing
multipolar mitoses followed by apoptotic cell death

Introduction
Most tumor cells that harbor multiple centrosomes still undergo bipolar divisions
allowing for secondary karyotype restabilization and survival
Apoptosis
Ml
Multi
l
po ar
Neoplasm
Bipolar
Clustering
Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint , Kops et al., PNAS 2004
Spindle multipolarity is prevented by centrosomal clustering, Quintyne et al., Science 2005

Screening of fungal extracts
Phenotype-based screen of a fungal small
molecule library for centrosomal clustering
inhibitors using a model cell line SCC114
GFP--Tubulin SCC114
phenotype
identification
in collaboration with Thomas Ostenfeld Larsen
(Center for Microbial Biotechnology, Kopenhagen)
Cancer Res, 2007

Griseofulvin is an inhibitor of centrosomal clustering
Mock
fulvino
Grise
bipolar
4:00 h
9:20 h
9:40 h
10:00 h
11:20 h
Griseofulvin specifically
induces apoptotic death
in cells with spindle
multipolar
6:20 h
7:40 h
12:00 h
12:40 h
13:00 h
multipolarity
Cancer Res, 2007

Griseofulvin analogs improving potency
O
O O
O
O
O O
O O
OH
O
N
O
O
O
O
O
O
O
Cl
Cl
Cl
1
Griseofulvin
GF15
GF 61
120
GF
GF
100
100
GF15
100
96
94
GF15
GF61
GF61
]
77
80
%[
69
ität
58
ar
60
55
53
53
olp
46
ltip
40
36
35
Mu
21
21
20
12
10
7
10
9
8
9
88
7
9
66
9
8
8
6
5
0
0
100
75
50
35
20
10
5
1
0.75
0.5
0.25
0.1
0.05
0.01
0.001
Konzentration [µM]
Testing of numerous derivatives of Griseofulvin improved potency by ~ 2log
>
= GF15

GF15 inhibits cell survival in tumor cell lines
Myeloma
Myeloma resist.
Leukemia
Solid tumors
Non-malignant
tl
controls
0
10
20
30
40
50
60
02
10
0
30
40
50M GF15
Treatment of tumor cell lines with GF15 induced cell death (IC50) in a
concentration-dependent manner

GF15 - specificity
PHA
sensitive
resistant
3
PBMCs
) 60
ntrol)o
y(%
co
50
of 2
40
fold
30
(48h;
emultipolarit
1
20
Spindl 10
iablilityV
0
0
0
5
10
30
50 M

-+
-
+
GF15
GF15 does not affect growth and survival of activated PBMCs
GF15 does not induce multipolar spindles in resistant cells

MM cells are highly sensitive to GF15
OPM2 48h
48h
MM1R
RPMI 48h
100
Dox40
NCI 48h
LR5
OPM1 48h
PAT 1
KMS12BM
80
control)
KMS12PE
of
KMS11
60
%
KMS18
l(48h; 40
20
Surviva
0
00.5
1
2
3
4
5
10M GF15
00.5
1
2
3
4
5
10M
GF15 inhibits survival and induces
G2/M arrest in MM cells
GF15
Mock

MM cells are highly sensitive to GF15
GF15 (µM), 24h
0
0.5
1
3
5
blot:
MCL-1
k
BCL-2
Moc
tubulin
Eg5/centrin
g
-tubulin
DAPI
merge
g
GAPDH
Casp-8
CF
Casp-9
GF15
CF
Casp-3
CF
GF15 specifically induces multipolar
PARP
multipolar
CF
spindles and apoptotic death in MM cells

GF15 and the microenvironment
)
)4
4 0
BMSCs
0
BMSCs
60
MM.1S
20
RPMI 8226
MM.1S +BMSCs
50
RPMI +BMSCs
(cpmx1
(cpmx1 15
40
30
ation
ation 10
a 20
5
10
Prolifer 0
Prolifer 0
01
5
10M GF15
01
5
10M GF15
Controls
VEGF/FN
IGF-1
2.5
2
ntrol) 2
oc
GF15 overcomes the
h
growt
1.5
of
advantage conferred by BMSCs
1
(fold 1
and abrogates cytokine-
tion
induced MM cell migration
migration
0.5
Migra
0
0
0
5
00
13
5
0
1
3
5 M GF15

In vivo activity
GF15 OPM2
GF61 p.o.
1600
2500
1400
2000
) 1200
3
1000
(mm
1500
150
e
Control
)
800
lum
T1
m
o
Control
v
T2
cm 1000
600
(
T1
mor
TU
Tu
400
500
200
0
0
1
3
5
8
10131517
0
1
3
5
8
10
131517
19
-500
Days of Tx
days of Tx
30.0
30
30.
30
30
30
25.0
25
30
GF15 and its orally available
20.0
)
20
25
g
analog GF61 significantly
(
15.0
th
15
g
20
gi
decrease tumor growth
growth in
in a
we
10.0
10
15
xenograft mouse model
5.
5
10 0
0.
0 0
0
0.
1
3
5
7
9
11
13
15
17
19
21
0
7
14
21
28
010
28
35
51 42
5
49 56
20
days
56 day
0
7
14
21
28
35
42 49 56 da
0
7
14
21
28
35
42 49 56

Summary
·
Aim: To evaluate centrosomal clustering as a novel therapeutic
target in MM and other malignancies
·
Identification of griseofulvin as a clustering inhibitor in a
phenotype-
phenotype screen of fungal extracts
·
GF specifically induces spindle multipolarity and cell death in
cells with supernumerary centrosomes
·
Analoges of GF show ~2log improved potency (GF15, GF61)
·
GF15 specifically induces multipolar spindles, G2/M arrest and
apoptotic death in MM cells
·
GF15 overcomes the growth advantage conferred by BMSCs and
abrogates cytokine-induced MM cell migration
·
GF15 and its
its orally available analog
analog GF61
GF61 significantly
significantly decrease
decrease
tumor growth in a xenograft mouse model of human MM
Identification of intracellular target, continue small molecule
screen, optimize
,p
GF analogs
g , develop for clinical
application

Thank you!
Danish Technical University
Natural Products Research Group
Thomas O. Larsen
Danish Technical University
Department of Chemistry
Mads H. Clausen
Mads H. Ronnest
University of Heidelberg
Myeloma Program
Kai Neben
Dirk Hose
Hartmut Goldschmidt
Goldschmidt
German Cancer Research Center
CCU Molecular Hematology/Oncology
EMBL Heidelberg
Blanka Rebacz
Chemical Biology Core Facility
Dana-Farber Cancer Institute
Phillip Riffel
Joe Lewis
Harvard Medical School
Brigitte Schreiter
Sonja
Sonja He
H n
e nemann
n
Iris Breitkreutz
Breitkreutz
Joachim Czaja
Beth Israel Deaconess
Patrick J. Hayden
Alwin Kraemer
Medical Center
Klaus Podar
Nikhil Munshi
Johannes Fruehauf
and all lab members!
Paul Richardson
Thu Nguyen
Kenneth C. Anderson