Haematologica 2003 - Supplements

5. Signal transduction pathways and
cytokine networks
P5.1
OVERVIEW OF MULTIPLE MYELOMA SIGNAL
TRANSDUCTION PATHWAYS
Anderson K
Cytokines in the BM microenvironment mediate growth of MM
cells [interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1),
vascular endothelial growth factor (VEGF), tumor necrosis
factor- (TNF-); tumor cell survival or resistance to apoptosis
(IL-6, IGF-1, IL-21); and migration [VEGF, stromal cell-derived
factor-1 (SDF-1)] Adhesion of tumor cells to BM stromal
cells further upregulates transcription and secretion these
cytokines in BM stromal cells and/or MM cells, thereby
promoting autocrine and paracrine tumor cell growth and
survival. Moreover, cytokines can modulate adhesion of MM
cells in BM. For example, TNFα in the BM milieu induces NFκB
dependent upregulation of cell surface adhesion molecules
[ICAM-1, vascular cell adhesion molecule-1 (VCAM-1)] on both
MM cells and BM stromal cells, with related increased binding as
well as induction of transcription and secretion of cytokines (IL-
6, VEGF) in BM stromal cells. The delineation of signaling
cascades mediating proliferation, survival, and migration of MM
cells in the BM milieu both enhances understanding of
pathogenesis and provides the framework for identification and
validation of novel molecular targets. Proliferation of MM cells
triggered by cytokines (IL-6, IGF-1, VEGF, TNFα, SDF-1α, IL-
21) is mediated primarily via the Raf/mitogen activated protein
kinase kinase (MEK)/p42/44 mitogen activated protein kinase
(MAPK) signaling cascade. Cytokine-induced survival or
resistance to apoptosis in MM cells is mediated via Janus kinase
(JAK)/signal transducers and activators of transcription
3(STAT3) (IL-6), as well as the phosphatidylinositol 3-kinase
(PI3-K)/Akt (IL-6, IGF-1, TNFα, SDF-1α) pathways. In
contrast, MM cell migration induced by cytokines (VEGF) is
mediated via a protein kinase C (PKC) dependent, p42/44MAPKindependent,
pathway. Importantly, the BM microenvironment
also confers drug resistance via two mechanisms. First, MM cell
binding to fibronectin confers cell adhesion mediated drug
resistance (CAM-DR) associated with induction of p27 and G1
growth arrest. Second, cytokines in the BM milieu induce
JAK/STAT and PI3-K/Akt signaling which mediates resistance to
conventional and novel therapies. Specifically, DNA damaging
agents, irradiation (IR), Fas, TNF-related apoptosis-inducing
ligand (TRAIL), as well as Thalidomide (Thal) and its
immunomodulatory derivatives (IMiDs) activate caspase 8 and
arsenic trioxide (As 2 O 3 ) activate caspase 9; and proteasome
inhibitor PS-341 activates both caspases 8 and 9. In all cases
downstream death signaling is mediated via activation of caspase
3, poly ADP ribose polymerase (PARP) cleavage, and apoptosis.
Apoptosis triggered by Dex, commonly used clinically to treat
MM, is associated with activation of related adhesion focal
tyrosine kinase (RAFTK) as well as release of second
mitochondria activator of caspase (Smac), but not cytochrome c,
from mitochondria. IL-6 confers drug resistance via activation of
JAK/STAT signaling and upregulation of Bcl-xL and Mcl-1
expression. In addition, IL-6 activates SHP2 phosphatase, which
blocks Dex induced activation of RAFTK and apoptosis. Both
IL-6 and IGF-1 inhibit drug-induced MM cell apoptosis via PI3-
K/Akt signaling and NF-κB activation, with downstream
induction of intracellular inhibitors of apoptosis (IAPs) including
FLICE inhibitory protein (FLIP), survivin, cIAP-2, A1/BFL-1
and XIAP. These studies therefore both define mechanisms of
tumor cell adhesion and cytokine mediated anti-apoptotic
sequelae in the BM milieu, and identify potential novel
therapeutic targets.
P5.2
GENETIC DEREGULATION EFFECTS ON SIGNALING
Alan Lichtenstein, Joseph Gera, Yijiang Shi, Fuyuhiko
Tamanoi and Brian Van Ness
Oncogenic K-ras or N-ras mutations are some of the most
common genetic defects in myeloma cells, occurring in up to 40-
50% of patients in selected series. The associations of these
mutations with stage III disease, disease progression and plasma
cell leukemia suggest they impart an aggressive phenotype to
their myeloma clones. Constitutive activity of mutant ras
proteins results in deregulated downstream signaling through
several cascades. Use of the IL-6-dependent ANBL-6 myeloma
cell line has allowed us to identify these cascades. When ANBL-
6 cells are stably transfected with oncogenic N-ras or K-ras
genes, they become IL-6-independent. In addition, expression of
oncogenic ras in these cells results in the following constitutively
upregulated signal pathways: 1) MEK/ERK; 2) PI3-kinase/AKT;
3) mTOR/p70S6kinase, and; 4) NF-kB. As these pathways are
known to promote myeloma cell growth, they may be
contributing to the ras-dependent proliferation and anti-apoptotic
signals in the ANBL-6 model..
Although ras mutations certainly appear to provide a growth
advantage and aggressive behavior in myeloma cells, they may
also conferr a sensitivity to therapy targeted to ras or the above
described upregulated downstream pathways. For example, in
model systems, hyperactive ras is known to induce an apoptosissensitive
phenotype via its ability to upregulate expression of
caspases and accelerate/stimulate release of cytocheomre C from
mitochondria. Thus, mutant ras-containing myeloma cells may
be either specifically deficient in those apoptotic effects of
mutant ras or additional ras-dependent downstream pathways
may protect against this ras signature of enhanced apoptosis.
Investigations in this arena with the goal of re-instituting the
apoptosis signature would have therapeutic potential.
A second strategy for targeting oncogenic ras is to prevent its
farnesylation with newly developed farnesyl transferase inhibitors
(FTIs). Ras proteins must be processed by farnesylation or
geranylgeranylation in order to be properly localized to the cell
membrane. Promising work on use of FTIs in patients will be
presented later in this symposium. However, there are theoretical
problems with this therapeutic concept. For example, although
H-ras is very sensitive to inactivation by FTIs, K-ras and N-ras
are much less so, owing to their greater avidity for farnesyl
transferase itself, the target of FTIs, or their great facility for
geranylgeranylation in the face of farnesyltransferase inhibition.
In fact, using the same ANBL-6 model transfected with
oncogenic ras, we found a strong apoptotic effect of FTIs in the
absence of any effect on ras processing, suggesting a mechanism
of action independent of effects on ras itself.
In a third approach, we have targeted the upregulated AKT
activation in mutant ras-containing myeloma cells. Our prior
work with myeloma cells containing heightened AKT activation
due to PTEN mutations indicated that upregulated AKT activity
was associated with hypersensitivity to mTOR inhibitors such as
rapamycin and its newer analog, CCI-779. This influence of
AKT on sensitivity to mTOR inhibitors has been seen in other
tumor models as well. MTOR inhibitors induce cytostasis by
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