Haematologica 2003 - Supplements

130
mTOR, a novel therapeutic target for multiple
myeloma?
F. Bakker, A. Wohlfarth, W. Baum, R. Burger, A. Guenther,
and M. Gramatzki
University of Erlangen-Nuremberg, Department of Medicine III,
Division of Hemato-/Oncology
Recently, it was reported that the phospatidyl inositol 3-kinase
(PI3-K) signaling pathway may play an important role for the
growth and survival of malignant plasma cells in multiple
myeloma (MM). The PI3-K and its downstream effector Akt,
activated by interleukin(IL)-6 or by insulin-like growth
factor(IGF)-1, were shown to mediate proliferative signals and to
inhibit dexamethasone induced apoptosis. In addition, it could be
shown that multiple downstream targets are phosphorylated in
MM cells upon activation of the PI-3K. Rapamycin is a potent
and highly selective inhibitor of the mammalian target of
rapamycin (mTOR), a downstream effector of PI3-K which
mediates activation of p70S6K and 4E-BP1 by Akt. Rapamycin is
a naturally occuring product isolated from Streptomyces
hygroscopicus, which has strong immunosuppressive properties.
In addition to its immunosuppressive properties, rapamycin was
found to inhibit the growth of normal and malignant B- and T-
lineage cells. In myeloma cells, rapamycin was shown to be able
to revert IL-6 and IGF-1 induced phosphorylation of p70S6K and
4E-BP1 as well as IL-6 and IGF-1 induced cell growth under
serum-free conditions. However, the overall effect of rapamycin
on myeloma cell growth was relatively modest in these
experiments. From other systems, it is known that serum is a
potent activator of the PI3-K signaling pathway. For example,
serum albumin was recently identified as the major plasma
component responsible for Akt activation in B-CLL cells (…).
Therefore, experiments conducted under serum-free conditions
may not reveal the relevance of PI3-K signaling for myeloma cell
growth and the potential role of PI3-K signaling pathway
inhibitors in the treatment of multiple myeloma. By Western Blot
analysis, we were able to show that fetal calf serum (FCS) is a far
more potent activator of the p70S6K than either IL-6 or IGF-1 in
all five human MM cell lines tested. Subsequently, we studied the
effect of rapamycin treatment on the growth of these MM cell
lines in the presence of FCS. Rapamycin was highly effective at
inhibiting the growth of all five MM cell lines tested, as
determined by [3H]-Thymidine incorporation (IC50 ranging from
0.24 to 1.8 ng/ml, growth inhibition at 100 ng/ml ranging from 74
to 97 %). Furthermore, we could show that this growth inhibition
is mainly mediated by a G1 cell-cycle arrest. Next, the in vivo
efficacy of rapamycin was evaluated in a SCID mouse xenograft
model for human plasmacytoma, based on the IL-6 dependent
human myeloma cell line INA-6. Starting on day 2 after tumor
inoculation, a total of 14 mice received an oral dose of 2 mg/kg
rapamycin 3 times per week for a total of two weeks. This dose
was comparable to the maximum tolerated dose determined in
studies with patients with advanced solid tumors. Treatment with
rapamycin significantly delayed tumor development and
prolonged survival of mice compared to untreated controls
(median survival: 108 days vs. 58 days). The in vitro and
preclinical in vivo efficacy of rapamycin indicates that inhibition
of mTOR may provide an interesting treatment option for patients
with malignant plasma cell tumors.
131
Inhibition of Protein Geranylgeranylation Induces
Apoptosis in Myeloma Cells by Reducing Mcl-1 Protein
Levels
Niels W.C.J. van de Donk1, Marloes M.J. Kamphuis1,
Berris van Kessel2, Henk M. Lokhorst2 and Andries C.
Bloem1
Departments of Immunology1 and Hematology2, University
Medical Center Utrecht, Utrecht, The Netherlands
Prenylation is a class of lipid modification involving covalent
attachment of hydrophobic isoprenoid molecules to target
proteins including small GTP-binding proteins such as Ras and
members of the Rac and Rho families. Farnesyl transferase
(FTase) and geranylgeranyl transferase (GGTase) catalyze the
transfer and binding of farnesyl and geranylgeranyl moieties from
farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate
(GGPP), respectively. Prenylation is essential for membrane
attachment and the subsequent participation of prenylated
proteins in diverse signaling pathways regulating growth and
survival. Lovastatin inhibits 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase, which is the rate-limiting
enzyme of the mevalonate pathway. This pathway leads to
formation of cholesterol and isoprenoids like FPP and GGPP.
Treatment of plasma cell lines and ex vivo purified tumor cells
from myeloma patients with lovastatin resulted in the induction
of apoptosis. Co-treatment of cells with lovastatin and
mevalonate or GGPP, but not FPP, abrogated lovastatin-induced
apoptosis. Furthermore, induction of apoptosis and reduction of
cell viability by inhibition of FTase were less pronounced when
compared to inhibition of GGTase I. This implies that
geranylgeranylation of proteins is critical for the regulation of
survival of myeloma cells. Apoptosis triggered by inhibition of
geranylgeranylation was associated with reduction of Mcl-1
protein expression, which, in turn, resulted in the collapse of the
mitochondrial transmembrane potential, cytochrome c release
from mitochondria into the cytosol, and stimulation of caspase-3
activity. Lovastatin enhanced the cytotoxic effects of
dexamethasone and doxorubicin in a synergistic fashion. This
synergism may be due to the lovastatin-mediated Mcl-1
downregulation. These results show that geranylgeranylation of
proteins is a key event in the regulation of myeloma tumor cell
survival, probably through the regulation of Mcl-1 expression.
Based on these findings, we started a Phase I/II trial to evaluate
the combination of dose-escalating lovastatin and CHOP or VAD
chemotherapy in refractory non-Hodgkin’s lymphoma and
multiple myeloma patients. At the moment, 5 patients have been
included in this trial.
132
Targeting the IGF-I Receptor Survival Pathways in
Multiple Myeloma Using Selective IGF-I Receptor
Tyrosine Kinase Inhibitors
T. Strömberg, L. Dimberg, O. Larsson, M. Axelsson, K.
Nilsson, H. Jernberg-Wiklund
Department of Genetics and Pathology, Rudbeck Laboratory,
Uppsala, Sweden; Cancer Center Karolinska, Karolinska Institute,
Stockholm, Sweden
The insulin-like growth factor I receptor (IGF-IR) is strongly
suggested to play a key role in malignant transformation. In
tumor cells from multiple myeloma (MM), IGF-I has been
established as an important survival factor, and we and others
have previously shown that a blocking anti-IGF-IR antibody
(IR3) augments apoptosis induced by serum-starvation, Fas-
S145