Up-regulation of Cathepsin G by IMiDs
might contribute to the risk of
thromboembolic events in patients with
Multiple Myeloma
Suzanne Lentzsch MD, PhD
University of Pittsburgh
Background
· Treatment of MM patients with thalidomide and IMiDs (Lenalidomide
and CC-4047) is associated with a high incidence of TEEs (17-18%)
· Use of aspirin decreases the incidence of TEEs to 3% and is
recommended for "normal" risk patients
· Unclear why aspirin is effective in reducing TEEs in patients
receiving thalidomide or IMiDs
· Mechanism of
of development
development of
of TEEs
TEEs needs to
to be
be identified
identified to
to select
high risk patients and apply targeted therapies
CC-4047 induced up-regulation of serine
t
pro einases
RNA
m
s in hematopoietic
it
progen ors
Clone identification
Gene name
fold change
48hrs
72hrs
AT205653
Cathepsin G
3.4
7.6
AT206871
Elastase 2, neutrophil
4.5
11.8
AT207341
Proteinase 3
2.5
6.5
Oligonucleotide microarray analysis of human CD34+ cells treated with CC-4047 for
48 and 72
72 hours i
g ven in f l
o d
ld h
c ange in
i
compar son to
t
con
l
ro t
t
rea
t
men (DMSO)
Cathepsin G
· Cathepsin G, Neutrophil elastase 3 and Proteinase 3 are serine
proteinases of the chymotrypsin
chymotrypsin family
family present in
in the
the azurophilic
granules of human polymorphonuclear neutrophils (Attuci et al, Biochem
J 2001)
· Actit
tiva i
tion f
o
t
neu
h
rop il
hils ld
leads to translocati
tion f
o th
the
t
pro einases to th
the
cells surface
· Cathepsin G acts as a potent
pp
platelet
p
agg
g re
g gation
g
agonist
g
similar to
thrombin (La Rosa et al, Blood 1994; Si -Tahar et al, Biochem J 1996)
· Cathepsin G binds to cathepsin receptor on platelets (Herrman et al,
Thrombosis 2001)
· Neutrophil elastase 3 does not induce platelet aggregation by itself but
potently
py enhances Cathepsin
p
G dependent
p
platelet
p
agg
g re
g gation
g
(Renesto
(
et al, Blood 1993)
Methods
IV
In i
Vitro
· CD34+cells were cultured in IMDM media with SCF, IL-6 and IL-3 with
DMSO (control) and CC-4047 (drug)
In Vivo
· Blood samples were collected from 10 patients prior to treatment
(baseline), and at day 15 of each cycle of Lenalidomide treatment
· PMNs used for
·
Cathepsin G levels of cell culture supernatants and patient
samples by ELISA
·
Cathepsin G protein expression by western blot
·
Cathepsin G mRNA expression by q-RT-PCR
·
Platelet aggregation induced by supernatants from cell cultures
Cathepsin G-mRNA is up-regulated by
CC-4047 in CD34+ cells
6
l
CC-4047 (10M)
otr 5
n
Control (0.01%DMSO
(%
)
oc
**
4
veto
* p 0.05
3
*
** p 0.001
relati
2
crease
ind 1
olF
0
Day 6
Day 10
mRNA measured by q-
q RT-
RT PCR: fold induction by CC-4047 compared with control
mRNA was normalized with GAPDH expression, error bars indicate SD
Cathepsin G-protein is up-regulated by CC-4047 in
CD34+ cells
Western Blot of CD 34+ cells
Cathepsin G
- Actin
ELISA of
of supernatant
supernatant of CD34+
CD34 cultures
35
**
30
CC-4047 (10M)
**
25
/ml)
Control (0.01%DMSO)
g(n 20
G
**p 0.001
sin 15
ep
10
athC
5
0
Day 6
Day 10
Supernatant
p
of CD34+ cells cultured with CC-4047
induces platelet aggregation
20
18
CC-4047 (10M)
16
n
*
Control ( 0.01% DMSO)
14
atio
*
reg 12
g 10
* p0.05
ag
et 8
el
lat 6
p% 4
2
0
Supernatant from:
Day 6
Day 10
Supernatants 1:10 diluted from CD34+ cells cultured with CC-4047
were used for platelet aggregation studies
Cathepsin G levels of healthy volunteers (n=7)
measured by ELISA
50
45
SD
40 2.122611284
/ml)g
1 061305642
.
35
30 0.925224808
levels(n 25 1.102941176
G 20 0.925224808
15 1.27356677
10
Cathepsin
0.636783385
5 2.122611284
0 2.122611284
2.783778601
1
2
3
4
5
6
7
EDTA blood: Cathepsin G mean 33.3 ng/mL
Na Citrate blood: Cathepsin G mean 37.9 ng/mL
Patients characteristics
Patient
Age/Gender
Stage
Ig
History of DVT
DVT on
Treatment
1.
59 M
II
IgAk
PE on Coumadin
None
2.
58 M
III
IgAk
None, Lovenox
None
prophylaxis
3.
70M
II
IgAk
None
None
4.
47M
III
IgGk
DVT while on
None
Thalidomide,
placed on
Coumadin
5.
75M
III
IgGk
DVT while on
None
Thali
hal domide, on
on
Coumadin
6.
48M
III
IgAk
None, on aspirin
None
during study
7.
61F
III
IgGk
None, on aspirin
None
during study
8.
58F
III
IgGk
None
None
9.
80M
II
IgGk
None, on aspirin
None
during study
10.
63 M
III
IgAk
None, on aspirin
None
during study
Increase of Cathepsin G-
G mRNA levels with
with
the length of Lenalidomide treatment
q-RT-PCR of PBMC
G
25
nsi
*
20
* p <0.001
ep ls
cath
15
eve
in l
Baseline 1
A
Cycle 2 1.70
se N 10
Cycle 3 4.79
crea mR 5
Cycle 4 20 72
.
nild 0
Fo
Baseline
Cycle 2
Cycle 3
Cycle 4
·mRNA fold induction compared to baseline (pretreatment)
·level of mRNA was normalized with GAPDH expression
Increase of Cathepsin G with the length of
Lenalidomide treatment
treatment
250
ELISA of blood plasma
*
l)
* p <0.05
m/ 200
gn
150
vels(
Baseline 53 ng/mL
leG 100
Cycle
y
2 77.5 ng/mL
nsi
Cycle 3 129.2 ng/mL
ep
50
ath
Cycle 4 145.5 ng/mL
C
0
Baseline
Cycle
y
2
Cycle
y
3
Cycle
y
4
3
e1 2.5
cyclto 2
ve
1.5
Baseline 1
elati
er
Cycle 2 1.45 fold
g
1
an
Cycle 3 2 4
. 3
43
hc 0.5
ld
Baseline
Cycle 2
Cycle 3
Cycle 4
Cycle 4 2.73
oF 0
Results
· Cathepsin G RNA of CD34+ cells treated with CC-4047 was significantly
up-regulated in olig
ggonucleotide gene arrays and q-RT-PCR
· Cathepsin G measured by ELISA in supernatants of CD34+ cells cultured
with CC-4047 was significantly increased
· Platelet aggregation was significantly enhanced by supernatants from
CD34+ cells treated with CC-4047
· MM patients had higher baseline Cathepsin G levels than healthy
volunteers
· Continuous significant increase of
of the
the Cathepsin G mRNA
mRNA levels over the
course of treatment (up to 20.7 fold during cycle 4)
· Cathepsin G significantly increased from a baseline mean of 53 ng/ml to
145 5
.
/
ng l
m d i
ur ng
l
cyc e 4
Conclusions I
·Cathepsin
p
G levels are higher
g
in MM patients
p
·IMiDs up-regulate the potent platelet activator
Cathepsin G in vitro and in vivo
·Increased Cathepsin G might contribute to the
dl
devel
t
opmen
f
o TEEs inp ti
a
t
en s r
ii
eceiving IMiD
treatment
·Our results might explain why aspirin is effective in
reducing TEEs in patients receiving IMiDs
Conclusions II
·Inhibition of Cathepsin G might be a potential
therapeutic target for preventing TEEs induced by
IMiD treatment
·Further studies are needed
needed to
·determine if Cathepsin G helps to predict TEEs and
to select high risk patients
·apply targeted
targeted therapies
therapies
ACKNOWLEDGMENTS
Research Team
Clinical Team
Rekha Pal
Pal
Patricia Schaefer
Markus Mapara
Ryan Kennedy
Martin Janz
Rene Mansour
G. David Roodman