Effect of new antimyeloma drugs
on bone microenvironment cells
Nicola Giuliani MD,PhD
"Ematologia e CTMO, Dipartimento di Medicina Interna e Scienze Biomediche"
University of Parma
13th International Myeloma Workshop, Paris 2011
New antimyeloma drugs
·
Targeting growth factors / receptors
· Targeting cell signaling pathway
AntiIL6 antibody
Farnesyl transferase inhibitor (Ras inhibitor)
·Sant7
·Tipifarnib or Zarnestra
VEGF receptor inhibitor
mTOR inhibitor
· PTK787/ZK
·Rapamycin
· GW654652
·CCI779
IGF1R inhibitor
Akt inhibitor
· AVE1642
·Perifosine
· GSK1838705AGSK1904529A
PKC inhibitor
FGFR tyrosine kinase inhibitor
· CHIR258
·Enzastaurin
Anti CD40 inhibitor
TRAIL receptor activator
·SNG40
·HGSETR
CS1
MAPK inhibitor
·Elotuzumab
·SCIO469
·
Targeting directly tumor cells
HSP90 inhibitors
· Multitarget drugs
· Geldanamicyne
Proteasome inhibitors: Bortezomib, Carfilzomib, NPI0052
· Tanespimycin
Thalidomide
Histone deacetylase inhibitor
IMiDS: Lenalidomide, Pomalidomide
·SAHA (vorinostat)
Arsenic trioxide
· LBH589 (panobinostat)
New drugs targeting MM cells and the microenvironment
E. Augment cytotoxicity
ThaI / Len.
IL2
C. Inhibit cytokines
CTL
IFN
PBMC
NK cells
ThaI / Len.
Bortezomib,
A. Induce growth arrest/apoptosis
ThaI / Len.
Bortezomib
B. Inhibit adhesion
IL6
ThaI / Len.
IGF1
Myeloma Cells
Bortezomib
VEGF
SDF1
IL1
VEGF
ThaI / Len.,
Bortezomib,
bFGF
Blood Vessels
Bone Marrow Stromal Cell
D. Antiangiogenesis
Key question:
Which are the effects of the new drugs on
bone microenvironment cells?
Prot
Pr easome
ot
inhibito
inhibit r
o s
r
IMiDs
HDAC inhibitors
Proteasome inhibitors block osteoclast formation
Zavrinski I et al.
Biochem Biophys Res
Commun 2005
Jimi E et al. JBiol Chem, 1998
Von Metzler et al.
Leukemia 2007
The new proteasome inhibitor NPI0052 (Salinosporamide A)
blocks RANKLinduced osteoclast formation
Ahn KS et al. Blood, 2007
Proteasome inhibitors stimulate bone formation in mice
Oyajobi et al. Br JHaematol, 2007
Garrett IR et al. J Clin Invest, 2003
BMPs stimulation by proteasome inhibitors
Garrett IR et al. J Clin Invest, 2003
Bortezomib increases osteogenic differentiation of hMSC
100
**
**
90
*
*
75
*
ld
60
45
nodules/fie
Bone 30
15
0
Giuliani et al. Blood 2007
Bortezomib increases the expression and activity of Runx2
and the osteogenic related markers
600
*
* p=0.05
ug
500
Runx2/CBFA1
270
/10
450)
OC
235
400
*
*
(OD
Col I
537
300
ation
ALP
475
activ
200
fa1
catenin
567
100
x2/Cb
GAPDH
209
Run
0
Giuliani et al. Blood 2007
Proteasome inhibitors activates TCF
trascriptional activity in osteoblasts and MSC
Quiang YW et al. Blood 2009
Bortezomib activates osteoblasts in vivo
Pre
Bortezomib
Post
Bortezomib
MM "responder"
MM "non responder"
Giuliani Net al. Blood 2007
Bortezomib stimulates bone alkaline phosphatase
level in MM patients
Zangari M et al. Br JHaematol, 2005
Heider U et al. Eur JHematol, 2006
Key question:
Which are the effects of the new drugs on
bone microenvironment cells?
Proteasome inhibitors
IMiDs
HDAC inhibitors
Thalidomide and IMiDs inhibit osteoclast
formation and activity
Anderson G. et al. Blood 2006
· Immunomodulatory drugs (IMIDs®), such as Lenalidomide (LEN)
and the new more potent Pomalidomide (POM), inhibit osteoclast
formation directly through the block of osteoclast maturation.13
Lenalidomide
P omalidomide
· Which is the potential effects of the IMiD® LEN and POM on the
mechanisms involved in MMinduced osteoclast formation ?
1 Breitkreutz I et. al. Leukemia. 2008; 2Anderson G et al. Blood. 2006; 3Lacy MQ, et al. Am J Hematol. 2010
IMiD® compounds effect on RANKL and OPG production
by BMSC/osteoprogenitor cells
1.2
RANKL
1
einot 0.8
l prta 0.6
to
0.4
/mg
pg
0.2
0
BMSC
LEN 2M
LEN 100M
POM 2M
POM 100M
300
OPG
250
200
einot
150
l prta 100
to
/mg
50
pg
0
BMSC
LEN 2M
LEN 100M
POM 2M
POM 100M
IMiD® compounds blunt RANKL upregulation in coculture
einot
*
l pr
*
tio
*
ta
ra
to
**
**
/mg
**
pg
BMSC
BMSC+JJN3
LEN 100M
POM 2M
POM 100M
BMSC
BMSC
BMSC+JJN3
BMSC+JJN3
LEN
LEN 100
100
M
M
POM
POM 2
2
M
M
POM 100M
RANKL
RANKL/OPG
einot
l pr
tio
ta
ra
to
**
/mg
**
pg
**
**
MSC
BMSC
BMSC+JJN3
BMSC+XG1
LEN 2M
POM 2M
BMSC
BMSC
BMSC+JJN3
BMSC+XG1
LEN
LEN100
2 M
M
POM
POM
22
M
M
The proosteoclastogenic property of the CM of
BMSC/MM cells was reduced in the presence of IMiDs®
Vehicle
LEN 2M
POM 2M
Effect of IMiD® compounds on the secretion of the pro
osteoclastogenic cytokines by MM cells: CCL3/MIP1
JJN3
ein
U266
ot
l prta
to
/mg
pg
CON
LEN 100 µM
POM 100 µM
Effect of IMiD® compounds on osteoclastogenic cytokine secretion
by activated peripheral blood mononuclear cells (PBMCs)
RANKL/OPG
IL6
con
con
IL3
CCL3/MIP1
con
con
Adhesion molecules were significantly modulated
at gene level by IMiD® compounds in MM cells
By microarray analysis (Affymetrix ® Genechips U133 Plus 2.0 ) we found:
· 71 and 214 genes were significantly modulated in hBMSC by LEN and POM, respectively, including
those belonging to focal adhesion, cell cycle, BMP2, TGF and IL6 signaling.
· 40 and 83 genes were significantly modulated by LEN and POM, respectively, in HMCLs (JJN3).
Downregulation of ITGA4 (CD49d), ITGA8 and ICAM2 (CD102)
Fold change LEN vs Fold change POM
Probe Set
Gene Title
Gene symbol
CON
vs CON
213416_at
integrin, alpha 4
ITGA4
0.43
0.49
214265_s_at
Integrin, alpha 8
ITGA8
0.33
0.23
Intercellular adhesion
213620_s_at
ICAM2
0.42
0.48
molecule 2
205692_s_at
CD38 molecule
CD38
1.59
2.36
IMiD® compounds reduce VLA4 (CD49d) expression by
MM cells at a wide range of concentrations
LEN 10 pM
LEN 10nM
LEN 100nM
LEN 2µM
LEN 100µM
POM 10pM
POM 10nM
POM 100nM
POM 2µM
POM 100µM
Control JJN3
CD49d
Treated JJN3
IMiD® compounds reduce CD102 expression by MM cells
LEN 10pM
LEN 10nM
LEN 100nM
LEN 1µM
LEN 2µM
POM 10pM
POM 10nM
POM 100nM
POM 1µM
POM 2µM
Control JJN3
CD102
Treated JJN3
ITGA8 is a target of IMiD® compounds in cells
ITGA8
ChangeldFo
JJN3
LEN
LEN POM POM
CD138+
LEN POM
2M 100M 2M 100M
2M 2M
SUMMARY
·IMiD® compounds blunt RANKL overexpression by BMSC in coculture
with MM cells normalizing RANKL/OPG ratio.
·Direct and indirect inhibitory effect of POM and LEN on OC formation at
apoptotic and subapoptotic concentrations reached in vivo in MM
patients.
·IMiD® compounds inhibit VLA4 (CD49d) expression by MM cells in a
dose dependent manner they and reduce the expression of other
adhesion molecules including ICAM2 (CD102) and ITAG8.
· IMiD® compounds inhibit MMinduced osteoclast formation through the
inhibition of RANKL/OPG ratio targeting the expression of adhesion
molecules by MM cells.
Lenalidomide treatment reduces RANKL/OPG
ratio serum level in MM patients
Breitkeutz et al. Leukemia 2008
Key question:
Which are the effects of the new drugs on
bone microenvironment cells?
Proteasome inhibitors
IMiDs
HDA
HD C
A
C inhibit
o
inhibit r
o s
r
In vitro and in vivo effects of HDAC inhibitors on bone cells
McGeeLawrence ME et al. Gene 2011
HDAC inhibitor SAHA (Vorinostat) causes bone loss
by inhibiting immature osteoblasts
McGeeLawrence ME et al. Bone 2011
HDAC inhibitors in combination with Bortezomib show a
positive effect on MM bone disease in mice
Deleu S et al. Cancer Res 2009
CONCLUSIONS
·The new class of antimyeloma drugs such as
proteasome inhibitors, IMiDs and HDAC inhibitors
show a significant effects on both osteoclasts and
osteoblasts with potential impact on bone remodeling
process and MM bone disease in MM patients
·Osteoclasts and osteoblasts are targets of the new
antimyeloma drugs whose effect may be due in part
to their action on the bone microenvironment cells.
THANKS TO
EMATOLOGIA E CTMO, PARMA
Vittorio Rizzoli
LABORATORY STAFF
FONDAZIONE IRCCS POLICLINICO, MILANO
Marina Bolzoni
Luca Agnelli
Paola Storti
Katia Todoerti
Sabrina Bonomini
Antonino Neri
Gabriella Sammarelli
CLINICAL STAFF
Benedetta Dalla Palma
IMiD® compounds have not effect on osteogenic differentation
Munemasa S et al. In J Oncol 2008
De Matteo M et al. Leuk Res 2010
Pathophysiology of osteoclast activation in multiple myeloma (MM)
BMSC/Preosteoblast
VLA4/VCAM1
MM cell
T lymphocytes
IL7
OPG
/ RANKL
Th17
IL6
IL6, OPN
CCL3/MIP1
BAFF,APRIL
RANKL
CCL20/MIP3
RANKL
CCR6
RANK
IFN
Bone destruction
Osteoclast
progenitor
Osteoclast
Giuliani N et al Blood 2001 and Blood 2002; Giuliani N et al. Cancer Res 2008; Choi SJ et al. J Clin Invest. 2001; Abe M et al. Leukemia 2006; Noonan K et al. Blood 2010
Pathophysiology of Osteoblast inhibition in MM
MM cell
MM cell
IL7, IL3
VLA4
VLA4
VCAM1
Mesenchymal/stro
VCAM1
mal cell
COL
ALP
Runx2/Cbfa1
DKK1, SFRP2, SFRP3
OC
WNT?
OPN
IL3
Immature
COL ++
ALP ++
Osteoblast
OPN +
OC +
T cell
Mature
Osteoblast
Tian E et al. NEJM, 2003
Ehrlich LA et al Blood, 2005
Giuliani N et al. Cancer Res, 2007
Giuliani N et al Blood, 2005