Haematologica 2003 - Supplements

368
PHARMACOGENOMIC STUDIES OF BORTEZOMIB
(VELCADETM) TREATMENT IN LATE STAGE
MULTIPLE MYELOMA
G.Mulligan, B. Bryant, J.Stec, S. Kim, M.Morrissey,
A.Damokosh, A.Singh, A.Bolt, A.Schmitt, J.Metivier,
J.Larsen-Gallup, D.Esseltine, J.Adams, D.Schenkein, A.
Boral, J. Brown, G. Linette, J. Ross, and the SUMMIT
Myeloma Study Group*
Millennium Pharmaceuticals, Inc. Cambridge, MA
The clinical development of the proteasome inhibitor, bortezomib
(VELCADETM) for the treatment of late stage multiple myeloma
has included a transcriptional profiling-based pharmacogenomics
strategy to identify genomic predictors of response and further
elucidate the mechanism of action of the drug. Through a
collaborative effort with the Phase II SUMMIT clinical trial sites,
patient bone marrow aspirates were subjected to a rapid negative
selection enrichment protocol to purify myeloma cells before being
frozen and shipped for centralized pharmacogenomics assessment.
Sixty-two percent of 202 patients agreed to the optional expression
profiling analysis. The intrinsic gene expression patterns of these
samples were determined on Affymetrix U-133 oligonucleotide
microarrays prior to treatment with bortezomib. Multiple
bioinformatic marker selection algorithms with supervised learning
were used to identify genes expression patterns that characterized
the differences between responders and non-responders to
bortezomib using the Blade response criteria. Predictive accuracy
as determined by 5 fold cross validation was significant (71-78%,
range) in multiple models. These pharmacogenomic studies of late
stage multiple myeloma patients is continuing with the evaluation
of new samples obtained from patients in the Phase III clinical trial
designed to further discover new markers and validate the current
predictive gene sets.
369
Effects of PS-341 and PS-1145 on cytokine-mediated
proliferation and anti-apoptosis in myeloma cells.
Unn-Merete Fagerli, Vadim Baykov, Torstein Baade Ro,
Anders Waage, Anders Sundan and Magne Borset.
Norwegian University of Science and Technology, Dept. of Cancer
Research and Molecular Medicine; Dept. of Oncology and Section
of Haematology, Trondheim University Hospital, Trondheim,
Norway.
Several cytokines have been implicated in the pathogenesis of
multiple myeloma (MM). Interleukin (IL)-6 is an important
growth and anti-apoptotic factor for MM cells. However, other
cytokines may substitute for IL-6, for example tumor necrosis
factor (TNF), IL-10, IL-15, insulin-like growth factor (IGF)-1 or
IL-21. For instance, in the human myeloma cell line OH-2,
combinations of TNF with IL-10, IL-15 or IL-21 give synergistic
effects on proliferation and a stronger response than IL-6 alone.
We examined in vitro effects of the proteasome inhibitor PS-341
and the IkB kinase inhibitor PS-1145 on the cytokine-dependent
myeloma cell lines OH-2 and IH-1 as well as on the cytokineindependent
cell line RPMI 8226. We wanted to see if the two
substances discriminated between MM cells stimulated to grow by
different cytokines and combinations of different cytokines. We
examined proliferation by 3H-thymidine incorporation and
induction of apoptosis by Annexin V-FITC/propidium iodide flow
cytometry.
PS-341 inhibited proliferation and induced apoptosis in all cell
lines, irrespective of which cytokine stimulated the growth. In the
proliferation assay ED50 was 1.3 nM.
PS-1145 inhibited TNF-induced proliferation of OH-2 cells by
more than 50% in a dose-dependent manner, whereas growth
stimulated by other cytokines was inhibited less than 25%. In the
apoptosis assay, PS-1145 totally inhibited the anti-apoptotic
effect of TNF in OH-2 cells, whereas little or no inhibition was
seen against anti-apoptosis mediated by IL-6 or IL-21. When
growth of OH-2 cells was supported by a combination of TNF
and other cytokines, proliferation or anti-apoptosis was inhibited
to the level seen in controls without TNF. Because of the striking
synergy between TNF and other cytokines in OH-2 cells, the
effect of PS-1145 was most pronounced under these conditions,
both in absolute and in relative sense (e.g. more than 60%
reduction of proliferation stimulated by TNF and IL-21).
Conversely, in the IH-1 cell line, which is not growth-stimulated
by TNF, PS-1145 gave a less than 10% reduction in proliferation
of cells stimulated with a combination of TNF and IL-6.
In the cytokine-independent cell line RPMI-8226, PS-1145 was
pro-apoptotic only in the presence of TNF, showing that TNF
became cytotoxic when added together with PS-1145.
In conclusion, PS-341 induces apoptosis irrespective of which
cytokine is promoting growth of the cells, suggesting that its proapoptotic
effect is not linked to any specific signaling pathway. In
contrast, PS-1145 seems to be specific for TNF-mediated growth,
and in fact, TNF itself becomes cytotoxic in the presence of PS-
1145. The anti-proliferative effect of PS-1145 is most
conspicuous when TNF is combined with other growthpromoting
cytokines. We have earlier suggested that TNF might
be more important to myeloma growth as a synergistic stimulant
than its effect as a single agent suggests. Inhibition of TNFinduced
and NF-kB-mediated proliferation and anti-apoptosis is
promising, suggesting a place for PS-1145 or other agents
targeting NF-kB in treatment of MM.
370
Proteasome inhibitors induce growth inhibition and
apoptosis in human myeloma cells irrespective of
chromosome 13 deletion
Ivana Zavrski, Christian Jakob, Ulrike Heider, Claudia
Fleissner, Jan Eucker, Kurt Possinger, Orhan Sezer
Department of Hematology and Oncology, Universitätsklinikum
Charité, Berlin, Germany.
The proteasome is a ATP ubiquitin-dependent protease which
plays a crucial role in degrading cytosolic and nuclear proteins,
which are strongly involved in basic cellular functions, like the
cell cycle regulation, transcription of growth factors and
apoptosis. The inhibition of the ubiquitin-proteasome pathway
leads to an uncoordinated expression and degradation of several
short living proteins required for appropriate cell cycle
progression and survival in malignant cells and activates
apoptosis. In this study, we investigated the effects of cellpermeable
proteasome inhibitors MG-132, MG-262, PSI and
lactacystin on multiple myeloma cell lines OPM-2, U266,
RPMI8226 and freshly isolated plasma cells with or without
deletion of chromosome 13 from patients with multiple myeloma
or plasma cell leukemia, and CD34+ human hematopoietic stem
cells. The effects of proteasome inhibitors on cell cycle
progression, cell growth and apoptosis were determined.
Using the MTT-assay, we found PSI with the half maximal
cytotoxicity (IC50) of 5.7 nM to be the most potent proteasome
inhibitor among those tested, followed by MG-262 (31 nM), MG-
132 (177 nM) and lactacystin (>1 µm). The growth inhibition
occurred despite of deletion of chromosome 13, there was no
significant difference for IC50 between myeloma cells with and
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