KRN5500a spicamycin
spicamycin derivative,
derivative,
exerts anti-myeloma effects through
impairing both myeloma
myeloma cells and
osteoclasts
Hirokazu Miki, Shuji Ozaki, Osamu Tanaka, Shingen Nakamura,
Ayako Nakano, Kumiko Kagawa, Kyoko Takeuchi, Ken-ichiro Yata,
Masahiro Abe, and Toshio Matsumoto
Department of Medicine and Bioregulatory
Sciences, The University of Tokushima Graduate
School of Health Biosciences, Tokushima, Japan;
Division of Hematology and Division of Tr
T ansfusion
r
Medicine, Tokushima University Hospital,
Tokushima, Japan
Introduction
Multiple myeloma (MM)
(MM) is a malignant
malignant disorder characterized by
by
accumulation of neoplastic plasma cells in the bone marrow and by osteolytic
bone destruction.
MM remains incurable due to drug resistance mediated by interactions with
osteoclasts and stroma cells, and therefore, alternative approaches are
necessary to overcome drug
yg resistance and inhibit osteoclasts activity.
y
KRN5500 is a new derivative of spicamycin produced by Streptomyces
alanosinicus (Kyowa Hakko Kirin, Tokyo, Japan), which potently inhibits
protein synthesis and induces cell death in human tumor cell lines.
Phase I studies of KRN5500 in patients with solid tumors such as colon
cancer and gastric cancer showed acceptable toxicity
toxicity with
with Cmax
Cmax values
values of
1000-3000 nM.
Kamishohara M, et al. Cancer Chemother Pharmacol: 1996; 38: 495-98
Supko JG, et al. Clin. Cancer Res: 2003; 9: 5178-86
Yamamoto N, et al. Jpn J Clin Oncol: 2003; 33: 302-08
KRN5500
Tetradeca-dienoic acid
Glycine Spicamycin amino-nucleoside
Burger AM, et al. Clin Cancer Res 3:455, 1997
Objective
To clarify the effects of KRN5500 against
yg
MM cells and osteoclasts in vitro and in vivo.
KRN5500 inhibits the proliferation of myeloma cells
RPMI 8226
UTMC-2
100
100
50
50
0
0
0.1
1
10
100
1000
0.1
1
10
100
1000
KRN5500 (nM)
KRN5500 (nM)
KMS12-BM
INA-6
100
100
50
50
0
0
0.1
1
10
100
1000
0.1
1
10
100
1000
KRN5500 (nM)
KRN5500 (nM)
KRN5500 induces apoptosis in myeloma cells
Control
KRN5500
RPMI 8226
9%
24%
7%
13%
INA-6
13%
58%
16%
33%
Primary MM 1
18%
53%
10%
14%
PI
Primary MM 2
6%
21%
7%
16%
Annexin V-FITC
KRN5500 induces apoptosis in myeloma cells
KRN5500 (nM) L-PAM (µM)
RPMI 8226 cells
020
40
10
PARP
Cleaved
Caspase-8
Caspase-9
Caspase-3
Mcl-1
Bcl-2
Bcl-XL
Actin
KRN5500 induces cell cycle arrest in myeloma cells
RPMI 8226 cells
Control
KRN5500
sub G1
G1
3%
sub G1
G1
20%
G0/G1 53%
G0/G1
50%
S
17%
S
15%
G2/M
27%
G2/M
15%
KRN5500 induces apoptosis in osteoclasts
Control
KRN5500 (20
(20 nM)
TUNEL
Control
KRN5500 (100
(100 M)
n
TRAP
KRN5500 induces cell death in both myeloma cells
and osteoclasts
RPMI 8226 cells + primary osteoclasts
Control
Bortezomib (20 nM)
nM)
KRN5500 (20
(20 nM)
KRN 5500 inhibits MM cell growth in vivo (SC model)
Day -28 RPMI 8226 s.c. (5.0×
(5.0 106
×10 cells/mouse)
Day 0
randomized in two groups and started treatment
Control (saline: IP)
KRN5500 (5 mg/kg: IP)
1100
e
1000
m
Control (n
(n = 6)
900
KRN5500 (n = 6)
800
o
)
700
orvolu
600
(%
tum
500
ve
400
300
200
Relati
P< 0.001
100
0
Day 0
Day 7
Day 14
Day 21
KRN 5500 inhibits MM cell growth in vivo
(SCID-
(SCID rab model)
model)
day-70 implanted rabbit bones into SCID mice
day-28 intra-bone injection of INA-6 (1×106 cells/mouse)
day 0
randomized in two groups and started treatment
Control (saline: IP; thrice/week) KRN5500 (5 mg/kg: IP; thrice/week)
80
70
Control (n = 5)
60
KRN5500 (n = 5)
(ng/ml)
50
40
sIL-6R
30
20
P< 0.001
10
human
0
Day 0
Day 21
KRN5500 prevents bone destruction in vivo
(SCID-rab model)
Control
Control
Control (HE ×200)
Day 0
Day 21
osteoclasts
osteoclasts
KRN5500
KRN5500
KRN5500 (HE ×200)
Day 0
Day 21
The effects of KRN5500 against MM cells
KRN5500
Induction of apoptosis
pp
Induction of apoptosis
pp
Mcl-1
MIP-1
RANKL
Osteoblasts
Osteoclasts
DKK-1
Myeloma cells
sFRP-2
RANKL
BMP-2
Stromal cells
Abe M, et al. Blood 100: 2195, 2002
Abe M, et al. Blood 104: 2484, 2004
Oshima T, et al. Blood 106: 3160, 2005
Conclusion
KRN5500 exerts anti-
anti MM effects
ef
through
through
impairing both MM cells and osteoclasts
in vitro and in vivo.
This unique mechanism of action provides
a valuable
valuable therapeutic option to improve
the prognosis in patients with MM.
Acknowledgments
We thank Asuka Oda and Hiroe Amou for their
excellent technical assistance
assistance.
KRN 5500 was a gift from Ky
gyowa Hakko Kirin Co.,
Ltd. (Tokyo, Japan).
70
201010-12
SpicamycinKRN5500
1)
2)
3)
1)1)2)1) 1)1)
1)1) 1)3)
1) 1)
1) 1
Structure of spicamycin analogue KRN5500 (NSC 650426),
6-[4-deoxy-4-(tetradeca-2(E)
(),4(E)
()-dienoylgly
ygycyl)amino-L-gly
gycero
--L-manno-hepatapyransoyl]amino-9H-purine, SPA.
All spicamycins contain the common spicamycin amino-nucleoside,
linked through gl
gg ycine to different fatty acid moieties.
MM cell lines such as RPMI 8226, UTMC-2, KMS12-BM, INA-6 were
incubated with various concentrations of KRN5500 for 3 days.
WST-8 cell proliferation assay showed marked inhibition of cell
growth in MM cell lines (IC50 = 3-40 nM).
Rabbit bones were implanted into SCID mice, then INA-6 cells were
injected into rabbit bones (
j(SCID-rab model). Serum levels of human
sIL-6R were measured at day 0, and mice were randomized into two
groups. Mice were treated with either saline or KRN5500 thrice a week
for 3 weeks. KRN5500 inhibits MM cell growth in the bone marrow as
assessed by serum levels of human sIL-6R derived from INA-6 at day
21 (52.8 ±16.5 ng/ml vs 11.6 ± 2.8 ng/ml, p < 0.001).
MM cell lines and primary MM cells were incubated with KRN5500
(100 nM)
() for 48 hours, and stained with Annexin V-FITC and
propidium iodide (PI). Cells were analyzed by flow cytometry to
determine the percentage of cells displaying Annexin V staining
(early apoptosis) or both Annexin V and PI staining (late apoptosis).
Treatment with KRN 5500 induced early and late apoptosis in MM
cells.
RPMI 8226 cells were treated with KRN5500 or L-PAM for 24 hours.
Western
Western blot
blot analysis confirmed
confirmed cleavage
cleavage of poly (ADP
(ADP-ribose)
polymerase (PARP), activation of caspase-8, -9, and -3, and
marked reduction of Mcl-1.
SCID-rab/INA-6 mice were killed at day 21, and the implanted rabbit bones
were examined radiologically
gy and histologicall
g
y.
y
Tumor growth and bone destruction was found in the control mice but not
in the KRN5500-treated mice. KRN5500 also inhibited activation of
osteoclasts in the bones as shown in HE staining.
RPMI 8226 cells were treated with KRN5500 (40 nM) for 24 hours, and
analyzed for
for cell cycles. Flow
Flow cytometric analysis showed
showed an increase
in sub G1 cell population and G1 arrest.
Osteoclasts were induced from primary bone marrow samples of MM
patients. Osteoclasts or
or stromal
stromal cells were treated with KRN5500 for
for 48
hours.
KRN5500 induced apoptosis in primary osteoclasts as assessed by TUNEL
staining. More than 90% of osteoclasts were
were killed
killed even
even at
at low
low
concentration of KRN5500 (20 nM).
TRAP staining showed that KRN5500 induced apoptosis in osteoclasts but
not stromal cells.
RPMI 8226 cells were co-cultured with osteoclasts and treated with
bortezomib or KRN5500 for 48 hours.
Bortezomib induced cell death in MM cells but not osteoclasts;
however, KRN5500 mediated strong cytotoxicity to both MM cells and
osteoclasts.
Upper panels: inverted microscopy findings
Lower panels: TRAP staining of residual osteoclasts.
SCID mice were subcutaneously inoculated with RPMI 8226 cells, then
treated with either saline or KRN5500 thrice a week for 3 weeks. KRN5500
inhibits the tumor growth compared with control mice (relative tumor
volume, 232 ± 54% vs 950 ± 422%, p < 0.001).