Haematologica 2003 - Supplements

in MM cells. The apoptotic mechanism(s) mediating the anti-
MM effects of these novel agents is unclear. Our studies to date
have shown that all these agents share some common signals.
For example, a decrease in the mitochondrial transmembrane
potential, caspase-3 activation and Poly (ADP ribose) polymerase
(PARP) cleavage are universal events triggered in response to all
agents. However, these agents also induce differential and/or
additional upstream signaling cascades that lead to caspase-3 or
PARP cleavage. As an example for this diversity, here, we have
shown the delineation of 2ME2- or PS-341- versus Dex-induced
mitochondrial apoptotic signaling in MM cells.
Mitochondria harbor two key modulators of apoptosis,
cytochrome-c (cyto-c) and Smac (Second mitochondria-derived
activator of caspase) or DIABLO, which are released from
mitochondria into the cytosol during apoptosis. Both these
proteins activate caspase-9 via different mechanisms: cytosolic
cyto-c binds to Apaf-1 > Apaf-1 oligomerization > Capase-9
activation; cytosolic Smac binds to XIAP (an inhibitor of
apoptosis protein) and thereby eliminates its inhibitory effects on
caspase-9. In the context of MM, we showed that both 2ME2 and
PS-341, but not Dex, triggers the release of cyto-c; all these
agents activate caspase-9. IL-6, a growth factor for MM, blocks
Dex-, but not 2ME2 or PS-341, -induced apoptosis by preventing
the release of Smac. Irradiation and IMiDs also trigger the
release of cyto-c, suggesting that the cyto-c is essential for most
drug-induced apoptosis in MM. The lack of cyto-c signal in
response to Dex, coupled with the finding that IL-6 protects MM
cells against Dex-induced apoptosis, suggests that a combination
of Dex with the novel agents that restores an additional cyto-c
signal will enhance the anti-MM activity of Dex.
Figure Schema showing drug-induced signaling via
mitochondria.
The mechanism(s) mediating the release of cyto-c or Smac is
unclear. The c-Jun-NH2-terminal kinase (JNK) translocates to
mitochondria after genotoxic stress and inhibits the anti-apoptotic
function of proteins belonging to Bcl2 family members, thereby
allowing the release of mitochondrial apoptogenic proteins to
cytosol and subsequent activation of caspase cascades. Since
both 2ME2 and PS-341 induce cyto release, we next asked
whether 2ME2 or PS-341 affects JNK. Our results demonstrate
that 2ME2 or PS-341-induced apoptosis in MM cells is
associated with activation of JNK, translocation of JNK from
cytosol to mitochondria, and release of Smac from mitochondria
to cytosol. Blocking JNK either by dominant-negative mutant
(DN-JNK) or cotreatment with a specific JNK inhibitor
SP600125, abrogates both stress-induced release of Smac and
induction of apoptosis. These findings demonstrate that
activation of JNK is an obligatory event for the release of cyto-c
and Smac during 2ME2 or PS-341-induced apoptosis in MM
cells. Importantly, our prior studies have also shown that Dexinduced
apoptosis is not associated with activation of JNK.
Collectively, the cellular signaling data in MM cell have
important biologic and therapeutic implications. First, our results
showing that anti-MM drugs induce apoptosis via release of
mitochondrial Smac suggest that Smac agonists or active Smac
peptides may sensitize MM cells to various anti-MM agents.
Second, the observation that Dex-induced signaling lacks cyto-c
and JNK activation provides a rationale for combination of Dex
with novel agents that trigger both cyto-c release and JNK,
thereby allowing enhanced anti-MM activity.
P5.5
NOVEL RECEPTOR INTERACTIONS IN MYELOMA
CELLS AND DOWNSTREAM CONSEQUENCES
DF Jelinek (Presenter), DK Walters, JD French, BK Arendt,
and RC Tschumper
Dept. of Immunology and Tumor Biology Graduate Program, Mayo
Graduate and Medical Schools, Mayo Clinic, Rochester, MN 55905
USA. Tel: (507) 284-5617; FAX: (507) 266-0981; E-mail:
jelinek.diane@mayo.edu
Multiple myeloma (MM) is a universally fatal neoplastic disease
that results from the malignant transformation of plasma cells and
is characterized by the accumulation of these clonal plasma cells
in the bone marrow. Our current understanding of the
mechanisms that underlie transformation is far from complete,
however, it is clear that malignant plasma cells retain growth
potential whereas this property is lost during terminal
differentiation of normal plasma cells. Achieving a better
understanding of myeloma cell growth control has been
challenged by the striking heterogeneity that is often observed
between tumor cells obtained from different patients.
Differential alterations in growth factor and cytokine
responsiveness, which result in increased cell growth and/or
resistance to apoptosis, are frequently observed and are likely, at
least in part, to account for disease heterogeneity and the
accompanying challenge in treating this disease. Nevertheless,
growth factor/cytokine receptors remain attractive therapeutic
targets. However, for this strategy to be successful, it is
necessary to understand signaling molecules downstream of these
receptors. In this regard, there is a growing awareness of the
complications introduced by unexpected receptor interactions that
result in synergistic down-stream cellular effects, e.g., enhanced
growth or resistance to apoptosis of cancer cells. Thus, an added
biological challenge is introduced by the ability of some receptors
to act in a non-linear fashion and/or transactivate unrelated
receptors, which may provide an attractive mechanism by which
tumor cell growth factor/cytokine responses can be amplified
(reviewed in 1). Given that myeloma cells often acquire atypical
expression or overexpress one or more signaling receptors during
malignant transformation, it is becomes imperative to fully
understand non-linear signaling in myeloma cells.
Distinct receptors families are classically thought to function
independently, with potential interaction(s) only occurring
downstream between amplifying or competing signal
transduction intermediates. This paradigm has been challenged
in the last decade by growing evidence of receptor cross-talk, or
transactivation (reviewed in 1,2). In this regard, it has been
demonstrated that unrelated receptors can interact at the level of
the receptors themselves, either via a direct physical interaction
or via an associated kinase. Of particular interest, several reports
have recently demonstrated the ability of gp130, the signal-
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