Haematologica 2003 - Supplements

human myeloma cell line (HMCL) to another. Actually, Akt
activation is highly dependent on either PTEN or CD45 phosphatase
expression. PTEN is a recently identified tumor suppressor gene that
is an important negative regulator controlling the Akt activation.
Indeed, out of 26 HMCL only 2 of them do not express PTEN and
have the strongest Akt activation in response to IGF-1. Among the 15
myeloma cell lines which has been analyzed for Akt activation, 3
expressed CD45 on 100% of cells (XG-1, XG2, MDN), 2 expressed
both CD45+ and CD45- subpopulations (U266 and JJN-3) and 9 are
completely negative for CD45 (LP-1, NCI-H926, OPM-2, ANBL-6,
BCN, NAN-1, RPMI-8226, L363, JIM-3 and Delta47). The Akt
response was significantly weaker for CD45+ or CD45+/- HMCL.
On the other hand, the highest Akt activation was restricted to CD45-
HMCL. Furthermore, the duration of Akt phosphorylation in
response to IGF-1 is more durable in CD45- myeloma cell lines
(>3hours) whereas a return to baseline level is reached as soon as 30
min in CD45+ HMCL. Finally, the growth of CD45- HMCL is
mainly or even totally controlled by the PI-3 Kinase pathway
whereas that of CD45+ HMCL is modestly controlled by it.
Altogether, the results suggest that CD45 as PTEN negatively
regulate IGF-1-dependent activation of PI-3 Kinase. Since we
recently demonstrated that patients lacking CD45 at diagnosis had a
bad prognosis, strategies that block IGF-1R signaling and
consequently Akt/PI-3Kinase pathway could be a priority in the
treatment of CD45- MM patients.
128
Two NF-κB activation pathways mediate CD40 signals
to promote primary myeloma cell survival
Eunice N. Hatada, Ruben Niesvizky, Sarah Meeus and
Selina Chen-Kiang
Weill Medical College of Cornell University, New York, NY10021, USA
Multiple myeloma cells represent plasma cell precursors which,
as a result of prior oncogenic transformation, fail to fully
differentiate to plasma cells and turn over by apoptosis. On this
basis, we hypothesize that the survival of MM cells in the bone
marrow is at least in part due to inappropriate retention of B cell
surface receptors. One candidate is CD40, which we have
recently demonstrated to be progressively reduced during normal
B cell terminal differentiation. CD40 signaling in MM cells has
been shown to correlate with IL-6 secretion, but its role in
myeloma clonogenic colony formation remains controversial.
To test our hypothesis, we have characterized the functional
consequence and the pathway of CD40 signaling in freshly
isolated bone marrow MM cells. Nearly all freshly isolated
CD138+ MM cells (20/21) expressed CD40 at varying levels, and
in 7 of them CD40 was expressed in the majority of cells (65-
100%). Stimulation with CD40L protected MM cells from
apoptosis ex vivo in 75% of the samples, and the degree of
protection correlated with CD40 expression. These results
support our hypothesis that most MM cells retain CD40
expression and that the primary consequence of CD40 signaling
is inhibition of apoptosis.
Next, we determined the signaling pathway(s) that mediate CD40
signals for MM cell survival. CD40 signaling in normal B cells leads
activation of various NF-B transcription complexes and the
downstream cell cycle and survival genes via the "classical" pathway,
which requires degradation of the inhibitor of NF-κBs (IκB) by the
ubiquitin-proteasome pathway. This "classical" pathway, notably the
transcriptional activating NF-κB1 p50/p65 NF-κB complex, was
activated in CD40 signaling in MM cells. Importantly, CD40
signaling also activated NF-B2 p52 in MM cells through the newly
discovered "alternative" pathway, which does not involve IκB
degradation. The two NF-B signaling pathways are crucial for the
survival of MM cells, because inhibition by proteasome inhibitors
dramatically accelerated MM cell apoptosis.
This study provides the first direct evidence that CD40 promotes
MM cell survival through activation of both classical and
alternative NF-κB signaling pathways. Our findings have
important implications for the mechanism of MM cell survival,
the timing of oncogenic transformation in MM pathogenesis, and
the use of proteasome inhibitors and possibly CD40 antagonists
for MM treatment. These will be discussed.
Supported by NIH grants (CA 80204, AR49436) and a
Specialized Center of Research for Myeloma grant by the
Leukemia and Lymphoma Society of America.
5.3 Drugs and signalling pathways
129
Selective receptor tyrosine kinase (RTK) inhibition
downregulates paracrine IL-6 in myeloma - stroma
crosstalk and induces apoptosis in t(4;14) myeloma
cells: therapeutic implications.
G. Bisping1, D. Wenning1, B. Dreyer1, M. Kropff1, F.
Hilberg2, G. Roth2, M. Stefanic2, W.E. Berdel1, J.
Kienast1
1Dept. of Medicine, Hematology and Oncology, University of
Münster, Germany, 2Boehringer Ingelheim, Austria, NCE
Pharmacology, Vienna, Austria
We have previously shown that basic fibroblast growth factor
(bFGF) and vascular endothelial growth factor (VEGF) derived
from MM cells stimulate bone marrow stromal cells (BMSC) to
produce and secrete interleukin-6 (IL-6), an important growth and
survival factor for human MM. In turn, IL-6 stimulates MM cells
to secrete bFGF and VEGF (Blood, 2002, Nov 27, epub ahead of
print, Blood,95:2630-2636; 2000).
In this study we investigated effects of BIBF1000 (125nM - 1µM), a
selective inhibitor of VEGF and FGF RTKs, on paracrine IL-6
secretion in non-contact MM-BMSC cocultures. IL-6 concentrations in
supernatants of serum-free 72hr-cocultures were significantly decreased
by BIBF1000. In detail, a 2.5–7.0-fold increase of stromal cell-derived
IL-6 by non-contact coincubation with KMS-11, U-266, RPMI-8226,
or MM patient cells, was abrogated by BIBF1000 to baseline levels of
unstimulated BMSC monocultures. BIBF1000 decreased IL-6 more
potently than anti-bFGF-, anti-VEGF-, or a combination of anti-bFGFand
anti-VEGF-antibodies. In summary, paracrine MM-BMSC circuits
can be completely blocked by BIBF1000 suggesting that selective
VEGF/FGF RTK inhibitors may be active in the treatment of MM, not
only through their anti-angiogenic activity, but also by downregulation
of paracrine IL-6 release.
Furthermore, we studied direct effects of BIBF1000 on
proliferation and apoptosis in MM cells. In KMS-11 and OPM-2,
but not in RPMI-8226, a significant inhibition of proliferation
occurred by addition of BIBF1000 (250nM – 1µM) as analyzed
by 3H-thymidine-uptake. In parallel, we observed a consistent 10
to 20% increase in apoptosis by Annexin V / PI staining in OPM-
2, KMS-18, and KMS-11 cell lines, all of which were shown to
carry the t(4;14) translocation and overexpress FGF receptor 3.
The proapoptotic activity was partially antagonized by exogenous
IL-6. BIBF1000 also caused a 5–18 fold upregulation of bcl-XLinhibiting
bmp-2 (bone morphogenetic protein-2) transcripts in
KMS-11 and KMS-18. No proapoptotic effects were observed
with RPMI-8226 and U-266.
Due to direct proapoptotic effects, FGF RTK inhibition appears
to be particularly promising in the treatment of MM with t(4;14).
S144