DOI:10.1158/0008-5472.

CAN-07-5725

Aplidin, a Marine Organism−Derived Compound with Potent

Antimyeloma Activity In vitro and In vivo
Constantine S. Mitsiades, Enrique M. Ocio, Atanasio Pandiella, et al.

Cancer Res 2008;68:5216-5225. Published online July 1, 2008.

Updated Version Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-07-5725
Supplementary Access the most recent supplemental material at:
Material http://cancerres.aacrjournals.org/content/suppl/2008/06/30/68.13.5216.DC1.html

Cited Articles This article cites 40 articles, 25 of which you can access for free at:
http://cancerres.aacrjournals.org/content/68/13/5216.full.html#ref-list-1
Citing Articles This article has been cited by 1 HighWire-hosted articles. Access the articles at:
http://cancerres.aacrjournals.org/content/68/13/5216.full.html#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR
Subscriptions Publications Department at pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on April 12, 2011

Copyright © 2008 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-07-5725
Research Article
Aplidin, a Marine Organism–Derived Compound with Potent
Antimyeloma Activity In vitro and In vivo
1,2 3,4
Constantine S. Mitsiades, Enrique M. Ocio, Atanasio Pandiella, Patricia Maiso,
3,5 3 3 3
Consuelo Gajate, Mercedes Garayoa, David Vilanova, Juan Carlos Montero,
1,2 1,2 1,2 1,2
Nicholas Mitsiades, Ciaran J. McMullan, Nikhil C. Munshi, Teru Hideshima,
1,2 6,7 6,7 6,7
Dharminder Chauhan, Pablo Aviles, Gabriel Otero, Glynn Faircloth,
4 1,2 3
M. Victoria Mateos, Paul G. Richardson, Faustino Mollinedo,
3,4 1,2
Jesus F. San-Miguel, and Kenneth C. Anderson
1
Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute; 2Department of Medicine,
Harvard Medical School, Boston, Massachusetts; 3Centro de Investigación del Cáncer, Instituto de Biologı́a Molecular y Celular del
Cáncer, Universidad de Salamanca-CSIC, Salamanca; 4Departamento de Hematologı́a and 5Unidad de investigación,
Hospital Universitario de Salamanca, Spain; 6PharmaMar, Madrid, Spain; and 7PharmaMar, Inc., Cambridge, Massachusetts
Abstract
Despite recent progress in its treatment, multiple myeloma
(MM) remains incurable, thus necessitating identification of
novel anti-MM agents. We report that the marine-derived
cyclodepsipeptide Aplidin exhibits, at clinically achievable
concentrations, potent in vitro activity against primary MM
tumor cells and a broad spectrum of human MM cell lines,
including cells resistant to conventional (e.g., dexamethasone,
alkylating agents, and anthracyclines) or novel (e.g., thalido-
mide and bortezomib) anti-MM agents. Aplidin is active
against MM cells in the presence of proliferative/antiapoptotic
cytokines or bone marrow stromal cells and has additive or
synergistic effects with some of the established anti-MM
agents. Mechanistically, a short in vitro exposure to Aplidin
induces MM cell death, which involves activation of p38 and
c-jun NH2-terminal kinase signaling, Fas/CD95 translocation
to lipid rafts, and caspase activation. The anti-MM effect of
Aplidin is associated with suppression of a constellation of
proliferative/antiapoptotic genes (e.g., MYC, MYBL2, BUB1,
MCM2, MCM4, MCM5, and survivin) and up-regulation of
several potential regulators of apoptosis (including c-JUN,
TRAIL, CASP9, and Smac). Aplidin exhibited in vivo anti-MM
activity in a mouse xenograft model. The profile of the anti-
MM activity of Aplidin in our preclinical models provided
the framework for its clinical testing in MM, which has already
provided favorable preliminary results. [Cancer Res 2008;
68(13):5216–25]
Introduction
Although recent advances in the therapeutic management of
multiple myeloma (MM) have improved its prognosis (1), no
curative therapy currently exists for this plasma cell dyscrasia,
which is the second most commonly diagnosed hematologic
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
C.S. Mitsiades, E.M. Ocio, and A. Pandiella contributed equally to this work.
Requests for reprints: Constantine S. Mitsiades, Jerome Lipper Multiple Myeloma
Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard
Medical School, 44 Binney Street, Boston, MA 02115. Phone: 617-632-1962; Fax: 617-
812-7701; E-mail: Constantine_Mitsiades@dfci.harvard.edu.
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-07-5725
Cancer Res 2008; 68: (13). July 1, 2008 5216
Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
3 3
malignancy in the Western world (2). The identification of new
classes of agents with anti-MM activity, especially in patients who
relapse from or do not respond to available therapies, remains
an urgent priority. Within this context, we evaluated a series of
candidate antitumor agents derived from marine organisms. This
effort was based on the rationale that several established or
investigational anticancer drugs are derived from natural products
(3). These compounds are produced by various plants and other
organisms (mostly above sea level), and their antimitotic pro-
perties, which prompted the evaluation of their antitumor activity,
may serve the teleologic purpose of protecting from predators
or infections. Recently, increasing interest has been placed on
oceanic organisms, primarily invertebrates (e.g., sponges, tunicates,
bryozoans, and mollusks), as unique natural sources of a diverse
array of natural products with potent antimitotic and/or antitumor
properties, including compounds that have advanced to late-
stage clinical trials (4, 5). An intriguing hypothesis about marine
organism–derived compounds is that they may have evolved to be
more potent than similar compounds from above-water organisms
to compensate for the increased diffusion and thus rapidly
decreasing protective concentration gradient of these compounds
under water.
In this study, we characterize the anti-MM activity of one such
marine-derived compound, Aplidin, which is a cyclic depsipeptide
isolated from the Mediterranean tunicate Aplidium albicans (6). We
show that Aplidin, at clinically relevant concentrations, has a
potent activity against a broad panel of human MM cell lines and
primary MM tumor cells, including cells resistant to conventional
or novel anti-MM agents. Although cytokine- or cell adhesion–
mediated interactions with the bone marrow microenvironment
protect MM cells from conventional therapies (e.g., dexamethasone
or cytotoxic chemotherapy; refs. 7, 8), Aplidin is able to overcome
these protective effects in coculture models of MM cells with bone
marrow stromal cells. Moreover, Aplidin has additive or synergistic
effects with established anti-MM agents including bortezomib and
immunomodulatory agents. Mechanistically, in vitro exposure of
MM cells to clinically relevant doses of Aplidin for even a few hours
is sufficient to induce their cell death. This involves activation of
p38 signaling and is associated with suppression of a constellation
of proliferative/antiapoptotic genes and up-regulation of several
positive regulators of apoptosis. Aplidin also exhibited in vivo anti-
MM activity in a mouse xenograft model. The profile of the anti-
MM activity of Aplidin in preclinical in vitro and in vivo models,
www.aacrjournals.org
 DOI:10.1158/0008-5472.CAN-07-5725
coupled with its favorable safety profile in clinical trials for solid
tumors, provided the framework for clinical studies of this agent in
MM, where Aplidin has already shown encouraging preliminary
results.
Materials and Methods
MM cell lines, primary MM tumor cells, stromal cells, normal
hematopoietic cells, reagents, and antibodies. Detailed information on
the MM cell lines, primary MM tumor cells, stromal cells, normal
hematopoietic cells, reagents, and antibodies is included in Supplementary
data.
Evaluations of cell viability, cell proliferation, cell cycle profiles,
induction of apoptosis, and mitochondrial membrane potential.
Previously described protocols were used to evaluate MM cell viability
[with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric survival assay; refs. 9–13]; cell proliferation and cell
cycle profiles (with the CD38-CD138/propidium iodide double-staining
technique; ref. 14); induction of apoptosis (based on Annexin V-FITC/
propidium iodide staining of myelomatous plasma cells; ref. 13); and
changes in mitochondrial transmembrane potential (Dw m; using
3,3¶-dihexyloxacarbocyanine iodide [DiOC6(3)] staining of cells; ref. 13).
Detailed information on these protocols is included in Supplementary
data.
Oligonucleotide microarray analyses, Western blots, c-jun NH2-
terminal kinase in vitro kinase assays, and lipid raft isolation. Gene
expression profiling, Western blot analyses, c-jun NH2-terminal kinase (JNK)
in vitro kinase assays, and lipid raft isolation studies were done as in
previous studies (12, 15–18) and are described in detail in Supplementary
data.
Combinations of Aplidin with other anti-MM agents. The interac-
tion between Aplidin and other anti-MM agents (dexamethasone,
melphalan, doxorubicin, thalidomide, lenalidomide, and bortezomib) was
analyzed with the Calcusyn software (Biosoft). Data from cell viability
assays (MTT) were expressed as fraction of cells affected by the dose in
drug-treated cells compared with untreated cells (control). This program
is based on the Chou-Talalay method (19) according to the following
equation: CI = (D)1/(Dx)1 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are
the doses of drug 1 and drug 2 that have the same x effect when used
alone, and CI is the combination index. CI < 1.0 indicates synergism; CI ffi
1.0 indicates an additive effect; and CI > 1.0 corresponds to an
antagonistic effect. MM1S and U266-LR7 cells were treated for 72 h with
combinations of Aplidin and other anti-MM drugs at either a nonconstant
ratio ( for suboptimal doses of Aplidin) or a constant ratio combination
design, and MTT assays were done as previously described. CI values for
each drug combination were obtained from three different experiments
fulfilling experimental prerequisites of the Calcusyn program.
MM xenograft murine model. CB17-SCID mice (The Jackson
Laboratory) were s.c. inoculated into their right flank with 3  106
MM1S cells in 100 AL of RPMI 1640 and 100 AL of Matrigel basement
membrane matrix (Becton Dickinson). When tumors became palpable,
mice were randomly assigned into treatment and control groups. Aplidin
was administered via i.p. injection and mice in the control group
received an equal volume of vehicle (cremophor EL/ethanol/water
for injection, 15%:15%:70%). Caliper measurements of the tumor dia-
meters were done every other day and tumor volumes were calculated
according to the formula of the volume of an ellipse: V = 4/3p  (a/2) 
(b /2)2, where a and b correspond to the longest and shortest diameters
of the tumor, respectively. Animals were sacrificed, according to
institutional guidelines, when the diameter of their tumors reached
2 cm or when significant toxicity was observed. Differences in tumor
volumes between treated and control groups were evaluated using the
unpaired Student t test. Statistical significance was defined as P < 0.05.
Survival was estimated from the day of initiation of treatment, and
statistical differences were assessed by Kaplan-Meier curves with the
log-rank test.
www.aacrjournals.org 5217
Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
Antimyeloma Activity of Aplidin
Results
In vitro activity of Aplidin against MM cell lines. We first
evaluated, using MTT assays, the in vitro activity of Aplidin against
a large panel of MM cell lines including cells resistant to various
anti-MM agents. We observed that Aplidin exhibited potent in vitro
anti-MM activity (Fig. 1): The majority (18 of 19) of MM cell lines
treated with Aplidin at 10 nmol/L, a clinically achievable con-
centration (20), exhibited V30% viability compared with their
respective controls (Fig. 1A; Supplementary Fig. S1; Supplementary
Table S1). Aplidin-responsive cell lines included cells resistant to
conventional anti-MM agents [e.g., melphalan (RPMI-8226/LR5),
mitoxantrone (RPMI-8226/MR20), and doxorubicin (RPMI-8226/
Dox40)] as well as cells with low sensitivity to novel anti-MM
agents [e.g., immunomodulatory thalidomide derivatives (S6B45,
RPMI-8226/S); Fig. 1A]. Similarly, on comparison of the in vitro
effect of Aplidin on dexamethasone-sensitive MM1S and melpha-
lan-sensitive U266 cells versus the effect of the drug on the MM1R
and U266-LR7 isogenic sublines, which are resistant to dexameth-
asone and melphalan, respectively, we observed that Aplidin
potently suppressed MM cell viability in a dose-dependent manner
both in the drug-sensitive lines and in their drug-resistant sublines
(Fig. 1B). Consistent with the studies on other MM cell lines, the
IC50 values for these four cell lines were within the 1 to 10 nmol/L
range (4.1, 4.7, 5, and 8.2 nmol/L for MM1S, MM1R, U266, and
U266-LR7, respectively). Interestingly, this range of potency is
similar to that observed for bortezomib (data not shown), a potent
anti-MM agent used in the clinical practice (21, 22).
The effect of Aplidin on MM1S cells was time dependent:
Inhibition of cell viability progressively increased from day 1 to day
3 of treatment with any given Aplidin dose (Fig. 1C). Interestingly,
even a 24-hour exposure to Aplidin at concentrations as low as
5 nmol/L was sufficient to induce a >50% decrease in MM cell
viability. To address the question of whether constant exposure to
Aplidin was required for its anti-MM effect, we performed cell
death commitment experiments: MM cells were exposed to a
clinically relevant dose of Aplidin (40 nmol/L) for variable periods
of time (ranging from 0 to 10 hours) and then were washed to
remove the drug, followed by culture in drug-free media for 3 days.
At that time, cell viability was assessed by MTT and expressed as
percent cell viability compared with control cultures. These cell
death commitment assays evaluated whether a short exposure of
MM cells to Aplidin irreversibly commits them to undergo cell
death. In all five MM cell lines tested (JJN-3, S6B45, NCI-H929,
SKMM1, and KMS-18), even a 1-hour exposure to Aplidin was
sufficient to trigger, despite the subsequent washout of the drug, a
>50% decrease in cell viability (Fig. 1D). In fact, in four of five lines
tested (JJN-3, S6B45, SKMM1, and KMS-18), a 1-hour exposure to
Aplidin was sufficient to trigger a >70% decrease in MM cell
viability. These results supported the notion that, whereas up to 24
hours may elapse before the full effect of Aplidin-induced cell death
can be observed, a short exposure to the drug (1 hour) may be
sufficient to irreversibly commit MM cells to their death.
Effect of Aplidin on MM cells cultured with exogenous
interleukin 6, insulin-like growth factor I, and bone marrow
stromal cells. We next evaluated the effect of Aplidin on MM cell
proliferation induced by the stimulatory cytokines interleukin 6
(IL-6) and insulin-like growth factor I (IGF-I), as well as bone
marrow stromal cells (Fig. 2). MM1S cells were treated with
increasing doses of Aplidin in the presence or absence of IL-6
(1 nmol/L) or IGF-I (10 nmol/L). Neither of these growth factors
Cancer Res 2008; 68: (13). July 1, 2008
 DOI:10.1158/0008-5472.CAN-07-5725
Cancer Research

Figure 1. Activity of Aplidin on MM cell lines. A, dose-response matrix of human MM cell lines to Aplidin in vitro (0–100 nmol/L for 72 h). The viability of Aplidin-treated
MM cells (expressed for each cell line as the percent of its respective control) is visualized in color format according to their values on a linear scale (0–100%).
B, dose effect of Aplidin after 48 h of treatment in MM1S, MM-1R, U266, and U266-LR7 cell lines using the MTT uptake assay (as described in Materials and Methods).
The mean cell viability values are presented for each condition as percent of the respective control of each cell line. A similar pattern of decrease in cell viability
in a dose-dependent manner was observed for the four cell lines tested. C, time course analysis of Aplidin-induced death of MM1S cells. D, results of cell death
commitment experiments of five MM cell lines. MM cells were cultured in the presence of Aplidin (40 nmol/L) for 0 to 10 h and, after washout of the drug, were cultured
for an additional 72 h in the presence of drug-free media to assess the shortest duration of exposure to Aplidin that could commit MM cells to eventual death.
A 1-h exposure to Aplidin was sufficient, despite the subsequent absence of drug in the culture medium, to trigger a significant decrease in viability in the five MM cell
lines tested.

protected against Aplidin-induced growth inhibition (Fig. 2B and
C). Binding of MM cells to patient bone marrow stromal cells
triggered increased MM cell proliferation, which was attenuated by
Aplidin (Fig. 2A). Even low doses of Aplidin (e.g., 10 nmol/L) display
a potency similar to that of higher concentrations of bortezomib
(e.g., 50 nmol/L). Importantly, Aplidin did not affect the viability of
bone marrow stromal cells, as determined by MTT assays (data not
shown).
Efficacy of Aplidin against freshly isolated cells from MM
patients. The activity of Aplidin against MM plasma cells freshly
isolated cells from 16 patients (8 newly diagnosed and 8 with
relapsed/refractory disease) was next analyzed using a multi-
parametric cytometry protocol. This test allows for separate
analysis of the induction of cell death in the myelomatous plasma
cell population versus other bone marrow cells. Thirteen of 16
primary MM cell samples tested in vitro were sensitive to the drug.
Two of the three nonresponsive MM primary cell samples were
isolated from newly diagnosed patients and the third one was
from a patient with refractory disease. Interestingly, of the 13
cases sensitive to Aplidin in vitro, 7 patients had shown clinical
resistance to several treatments including alkylating agents and
high-dose dexamethasone (all of them), autologous stem cell
transplantation (5 cases), bortezomib (3 cases), and thalidomide-
based regimens (2 cases) (Supplementary Table S2). Aplidin
(100 nmol/L) induced cell death in >80% of MM plasma cells from
8 of 16 patients, and between 60% to 80% in 5 more patients. The
average cytotoxicity of Aplidin (100 nmol/L) in sensitive patients
was 83% (SD, 14.4; 95% confidence interval, 74.1–91.6). Even at a
10-fold lower dose of Aplidin (10 nmol/L), the average cytotoxic
activity on MM plasma cells remained close to 60%. Supplementary
Fig. S2A illustrates the effect of Aplidin on MM plasma cells, as well
as on normal lymphoid and myeloid cells, from four representative
patient cases. Interestingly, whereas Aplidin had a pronounced
effect on the viability of MM cells, minimal toxicity was observed
on normal lymphocytes and myeloid cells from the same patient
samples (Supplementary Table S2; Supplementary Fig. S2A). Sup-
plementary Fig. S2B depicts the results from the analysis of a
representative primary MM patient sample, which exhibited a
dose-dependent induction of cell death in MM plasma cells. Again,
only minimal toxicity was observed on normal mononuclear and
myeloid cells, even at high doses of Aplidin.
Evaluation of combinations of Aplidin with other anti-MM
agents. Clinical experience in the therapeutic management of
MM patients supports the concept that drug combinations can
induce higher response rates compared with single-agent treat-
ment (23–26). We therefore evaluated the effect of combinations
of Aplidin with other anti-MM drugs on the viability of MM1S
and U266-LR7 cells. Specifically, Aplidin was combined with con-
ventional agents used in MM treatment (such as dexamethasone,
doxorubicin, or melphalan), as well as with recently developed
drugs (bortezomib, lenalidomide, or thalidomide). In these ex-
periments, both nonconstant ratio ( for suboptimal doses of
drugs) and constant ratio experimental designs were used. The
effects of single and combined treatments after 72 hours were

Cancer Res 2008; 68: (13). July 1, 2008 5218 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-07-5725
evaluated by MTT assays, and data were analyzed with the
Calcusyn program.
As shown in Fig. 3A, Aplidin increased the anti-MM effect
of dexamethasone, melphalan, lenalidomide, thalidomide, and
bortezomib. Analysis with the Calcusyn program indicated that
Aplidin exhibited a synergistic effect when combined with lenali-
domide or thalidomide (Fig. 3B); Aplidin combinations with dexa-
methasone or melphalan were additive; combinations of Aplidin
and bortezomib presented an additive effect that was even
moderately synergistic when low doses of both drugs were used.
No synergism was observed in combinations with doxorubicin
(data not shown). In experiments with U266-LR7 cells (resistant
to melphalan), combinations with dexamethasone or melphalan
were not amenable to evaluation with the Calcusyn program
because the prerequisite of a dose-effect relationship for each of
these drugs as single agents was not fulfilled. When Aplidin was
Figure 2. Effect of Aplidin on MM cells stimulated with proliferative cytokines or
cocultured with bone marrow stromal cells: MM1S cells were treated for 48 h
with the indicated concentrations of Aplidin in the presence or absence of bone
marrow stromal cells (BMSC ) derived from a MM patient (A), IL-6 (B),
or IGF-I (C ). DNA synthesis was determined by measuring bromodeoxyuridine
(BrdUrd ) incorporation during the last 8 h of 48-h cultures. Columns, mean of
quadruplicates; bars, SD.
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
Antimyeloma Activity of Aplidin
combined with lenalidomide or thalidomide (Fig. 3C), a synergistic
effect on U266-LR7 cells was again observed, although less
pronounced than that against MM1S cells. The effect of Aplidin
and bortezomib on U266-LR7 cells remained moderately synergis-
tic at lower doses and additive at higher doses. Conversely, in
U266-LR7 cells, the Aplidin plus doxorubicin combination
remained antagonistic.
Effects of Aplidin on the cell cycle and viability of MM cells.
Treatment with 10 nmol/L Aplidin induced a modest increase in
the percentage of MM1S cells in the G2-M phase (Supplementary
Fig. S3A). A similar increase in the percent of cells in G2-M was
observed after treatment of MM1R cells with 1 nmol/L Aplidin:
Percent of cells in G2-M was 19% in control cells versus 29% in 1
nmol/L Aplidin–treated cells (data not shown). Higher doses
of Aplidin (100 nmol/L) triggered a clear sub-G0 accumulation
(data not shown). The latter fact, together with the increased
Annexin V staining obtained in patient plasma cells, indicates
that Aplidin does not merely block the proliferation of MM cells
but also actively triggers cell death. This hypothesis is also
supported by a time-dependent increase in Annexin V staining of
MM1S cells treated with Aplidin (Supplementary Fig. S3B). In
addition, Aplidin induced DNA laddering in MM1S cells (data not
shown).
To gain insights into the mechanism(s) of action of Aplidin on
MM cells, we performed gene expression profiling, as well as
biochemical and cell biological analyses, with emphasis on the
pathways involved in the regulation of apoptosis. Gene expression
profiling identified 805 transcripts that were significantly up-
regulated or down-regulated by z2-fold with Aplidin treatment.
The classification of these genes according to functional categories
indicated that 9.8% were involved in apoptosis/responses to stress,
22.4% in the control of cell cycle/proliferation, 5.8% were kinases
that participate in cell cycle and survival signaling, and 1%
participated in adhesion (Supplementary Fig. S3C). These results
suggest that genes involved in cell cycle and cell death regulation
may participate in the mechanism of action of Aplidin. Indeed,
microarray data showed that Aplidin down-regulated several genes
required for cell proliferation and/or cell survival (MYBL2, BUB1,
BUB1B, cyclin B1, cyclin E1, CENPF, MCM2, MCM4, MCM5, and
survivin; Supplementary Fig. S3). In addition, Aplidin up-regulated
the transcript levels of potential regulators of apoptosis, such as
GADD45A, GADD45B, TRAIL, CASP9, CASP6, CIDEC, and Smac
(Supplementary Fig. S3D). Of interest, we also observed an increase
in the expression of c-JUN and a decrease in the expression of MYC
(Fig. 4; Supplementary Figs. S4–S8). Previous data from our group
and others have shown that activation of the pathway and/or up-
regulation of c-JUN expression is associated with the apoptosis
induced by novel drugs such as bortezomib (11). Furthermore,
extensive work in MM and other neoplasias has indicated that myc
is an important oncogenic protein and a putative therapeutic
target (as reviewed in ref. 27). A complete list of transcripts modu-
lated by Aplidin treatment is included as a Supplementary table,
and schematic representations of functional pathways involving
Aplidin-modulated transcripts are depicted in Supplementary
figures.
Drug-induced apoptotic cell death is frequently linked to the
release from mitochondria to the cytosol of proteins, which then
partner with other cytosol-resident proteins to activate caspases.
Loss of mitochondrial membrane potential (Dw m) often reflects an
increase in mitochondrial outer membrane permeability. Treat-
ment of MM1S cells with Aplidin caused a decrease in Dw m,
5219 Cancer Res 2008; 68: (13). July 1, 2008
 DOI:10.1158/0008-5472.CAN-07-5725
Cancer Research

indicative of an increase in mitochondrial outer membrane Bcl-2 family member Mcl-1 (Fig. 4B). No changes in Bcl-2
permeability (Fig. 4A). Bcl-2 family members act as important expression were induced by Aplidin, whereas a small increase in
regulators of mitochondrial outer membrane permeability, and Bcl-X was noticed (Fig. 4B). Apoptosis triggered by Aplidin was
Western blot analyses indicated that Aplidin down-regulated the further confirmed by cleavage of poly(ADP-ribose) polymerase,

Figure 3. Evaluation of combinations of Aplidin with other

anti-MM agents. A, Aplidin (1.25 nmol/L) was combined with
dexamethasone, bortezomib, melphalan, lenalidomide, or
thalidomide; after 48 h, MTT assays were done on MM1S cells.
B, calculated CIs of different double combinations for the
MM1S cell line. A fraction affected-CI plot simulation is
provided when a constant ratio design is used. CR, constant
ratio; NCR, nonconstant ratio. C, CIs observed for the
U266-LR7 cell line. CIs of <0.3, 0.3 to 0.7, 0.7 to 0.85, 0.85 to
0.90, 9.0 to 1.10, and >1.10 indicate strong synergism,
synergism, moderate synergism, slight synergism, additive
effect, and antagonism, respectively.

Cancer Res 2008; 68: (13). July 1, 2008 5220 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-07-5725
Antimyeloma Activity of Aplidin

Figure 4. Analysis of the pathways involved in

Aplidin-induced apoptosis. A, MM1S cells were
treated with Aplidin (100 nmol/L), and Dw m analyses
done with [DiOC6(3)low] by flow cytometry.
B, MM1S cells were treated with Aplidin (100 nmol/L)
for the indicated times, and expressions of
poly(ADP-ribose) polymerase (PARP ), caspase-3,
caspase-7, caspase-8, caspase-9, Bcl-2, Bcl-X,
Mcl-1, and myc proteins were analyzed by Western
blotting of cell extracts with specific antibodies.
C, effect of the caspase inhibitors z-VAD-fmk,
z-IETD-fmk, and z-LEHD-fmk on Aplidin-induced cell
death. MM1S cells were plated and pretreated,
where indicated, with caspase inhibitors (50 Amol/L)
for 60 min. Aplidin (100 nmol/L) was added to the
corresponding samples and the experiment was
continued for 6 and 12 h. Annexin V staining was
carried out as described above.

caspase-3, caspase-7, caspase-8, and caspase-9, with generation
of active low M r cleaved fragments (Fig. 4B). To investigate the
importance of caspases in cell death triggered by Aplidin, we
evaluated the ability of the pan-caspase inhibitor z-VAD-fmk,
the caspase-8 inhibitor z-IETD-fmk, and the caspase-9 inhibitor
z-LEHD-fmk to protect against Aplidin-induced apoptosis. As
shown in Fig. 4C, preincubation with z-VAD-fmk attenuated
Aplidin-induced cell death, whereas no effect was observed with
caspase-8 and caspase-9 inhibitors, suggesting that only the
blockade of both the intrinsic and extrinsic pathways can partially
block Aplidin-induced apoptosis.
Aplidin-induced translocation of Fas/CD95 death receptor
into lipid rafts. A novel mechanism regulating Fas/CD95 death
receptor activation in apoptosis involves its translocation into lipid
rafts (16). In this regard, Aplidin-induced apoptosis in leukemic
cells involves translocation of Fas/CD95 in lipid rafts (28), and
recruitment of death receptors in lipid rafts has recently been
shown to be a crucial event in the anti-MM activity of the synthetic
alkyl-lysophospholipid analogues edelfosine and perifosine (18).
Therefore, we analyzed whether Aplidin induced translocation of
Fas/CD95 death receptor in lipid rafts by isolating these membrane
microdomains from untreated and drug-treated MM cells by taking
advantage of their insolubility in Triton X-100 lysis buffer at 4jC,
followed by discontinuous sucrose gradient centrifugation. GM1-
containing lipid rafts, at the upper part ( fractions 3–5) of the
sucrose gradient (Fig. 5A), were identified using cholera toxin B
subunit conjugated to horseradish peroxidase. We observed that
Fas/CD95 was located in the soluble fractions ( fractions 9–12) of
the sucrose gradient and not in the detergent-insoluble lipid
raft region in untreated MM144 cells, indicating that Fas/CD95
is excluded from the lipid rafts in untreated MM144 cells, in
agreement with previous studies (18). Importantly, Aplidin treat-
ment induced a partial recruitment of Fas/CD95 into the lipid raft
region ( fractions 3–5) of the sucrose gradient (Fig. 5A). Conversely,
disruption of lipid rafts by methyl-h-cyclodextrin, which interferes
with protein association with lipid rafts by cholesterol depletion
(16), partially inhibited Aplidin-induced cell death in MM144 cells
(Fig. 5B). These results suggest that Aplidin-induced cell death
in MM cells involves, at least in part, Fas/CD95 translocation
into lipid rafts.

www.aacrjournals.org 5221 Cancer Res 2008; 68: (13). July 1, 2008

Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-07-5725
Cancer Research

The role of the JNK and p38 apoptotic pathways in the
mechanism of action of Aplidin. To further explore the
mechanism of action of Aplidin on MM cells, we analyzed the
expression and activation status of JNK, p38, extracellular signal–
regulated kinase (Erk)-1/2, and Erk5 in Aplidin-treated MM cells.
Aplidin (100 nmol/L) induced activation of JNK, which was already
detectable by 15 minutes of Aplidin treatment and reached a
maximum at 1 to 3 hours (Fig. 5C). Activation of JNK was also
observed in MM1R cells (data not shown). Aplidin also rapidly
increased phosphorylation of p38. On the other hand, no significant
effect on the Erk1/2 or Erk5 pathways was observed at times up to
6 hours of treatment. At later times, Aplidin decreased Erk1/2 and
Erk5 expression. To further characterize the biological consequen-
ces of the activation of the JNK and p38 signaling pathways, cells
were treated with or without inhibitors for p38 kinase (SB203580;
25 Amol/L) and JNK (SP600125; 25 Amol/L) before Aplidin treat-
ment. Both the p38 inhibitor SB203580 and JNK inhibitor SP600125
partially rescued MM1S cells from Aplidin-induced cell death
(Fig. 5D), implicating p38 and JNK activation in the mechanism of
action of this drug. Comparison with bortezomib indicated
differences in the sensitivity to mitogen-activated protein kinase
(MAPK) inhibitors because the p38 inhibitor (SB203580) was
more efficient in preventing the action of Aplidin (Fig. 5D). In
fact, inhibition of the p38 route had a small effect on bortezomib-
induced cell death (Fig. 5D), in agreement with the poor
p38-stimulating activity of bortezomib (Fig. 5C).
Aplidin exhibits in vivo anti-MM activity in a xenograft-
plasmocytoma murine model. In view of the potent in vitro
activity of Aplidin against MM cell lines and freshly isolated
primary MM patient cells, we next studied the in vivo anti-MM
effect of Aplidin in a human plasmocytoma model in immunode-
ficient mice. To address the possibility that the side effect profile of
Aplidin is strain dependent, we first conducted a pilot study to
explore the potential toxicity of this drug in CB17-SCID mice. For
this purpose, 20 mice were injected i.p. with different doses of
Aplidin and were serially monitored for the development of any
significant toxicity. Maximum tolerated dose for this strain was
established at a total weekly dose of 700 Ag/kg.
A separate cohort of 16 mice were treated with a total weekly
dose of 700 Ag/kg Aplidin at two different schedules of treatment:
nine mice received Aplidin 100 Ag/kg i.p. daily and seven were given
Aplidin 140 Ag/kg i.p. 5 days a week. Nine mice served as control
cohort and received i.p. injections of the vehicle alone. The toxicity
profile of Aplidin was quite favorable, evidenced by a body weight
loss of only 6% to 8% in the treated groups as compared with the
control group. Only one mouse in the 140 Ag/kg cohort had to be
euthanized (on day 26 after initiation of treatment) due to a
significant loss in body weight (>20%). Aplidin treatment decreased
tumor growth in both cohorts of treated mice, and this effect was
greater in the group receiving 140 Ag/kg of Aplidin 5 days a week
(Fig. 6A). The differences in tumor volumes between the vehicle and
the 140 Ag/kg cohort were statistically significant from day 9 of
treatment (P = 0.01) until the end of the experiment. With 100 Ag/kg
treatment, the tumor volume also decreased compared with the
controls, but differences did not reach statistical significance
(P = 0.08). Kaplan-Meier survival analysis revealed a significant
prolongation of survival for the Aplidin-treated groups compared
with the vehicle-treated controls (log-rank P = 0.02; Fig. 6B).

Figure 5. Effects of Aplidin treatment on Fas/CD95 translocation into lipid rafts and on JNK and p38 signaling. A, translocation of Fas/CD95 into lipid rafts in
Aplidin-treated MM cells. Untreated MM144 cells (Control ) and MM144 cells treated with 10 nmol/L Aplidin for 15 h (Aplidin ) were lysed in 1% Triton X-100 and
fractionated by centrifugation on a discontinuous sucrose density gradient. An equal volume of each collected fraction was subjected to SDS-PAGE before analysis of
Fas/CD95 with a specific antibody. Location of GM1-containing lipid rafts (fractions 3–5) was determined using cholera toxin B subunit conjugated to horseradish
peroxidase. Representative of three experiments. B, MM144 cells were untreated (Control ) or pretreated with methyl-h-cyclodextrin (MCD ) and then incubated with
10 nmol/L Aplidin (APL ) for 24 h and examined for the percentage of apoptotic cells by flow cytometry. Columns, mean of three independent determinations;
bars, SE. C, MM1S cells were treated for the indicated times with 100 nmol/L Aplidin (left ) or 25 nmol/L bortezomib (right ) and lysed. One part of the cell extracts
were used for the analyses of JNK activity using glutathione S-transferase-c-Jun. Another part of the extracts were used to detect p-Erk1/2, Erk1/2, p-p38, and
poly(ADP-ribose) polymerase by Western blots. Erk5 was analyzed by Western blotting of anti-Erk5 immunoprecipitates. Shown are the results of an experiment that
was repeated twice. D, MM1S cells were pretreated for 60 min with inhibitors for p38 kinase (SB203580) and JNK (SP600125) and subsequently treated with
Aplidin for 6 and 12 h. Induction of cell death in each condition was assessed by Annexin V staining.

Cancer Res 2008; 68: (13). July 1, 2008 5222 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
 DOI:10.1158/0008-5472.CAN-07-5725
Figure 6. In vivo anti-MM effect of Aplidin. CB17-SCID mice were inoculated
with 3  106 MM1S cells s.c. in the right flank. Treatment was started when
tumors became palpable. Mice were randomly assigned into three groups
(with equal average tumor volumes before initiation of treatments): vehicle
(n = 9), Aplidin 140 Ag/kg/d i.p.  5 d/wk (n = 7), and Aplidin 100 Ag/kg/d
i.p.  7 d/wk (n = 9). Caliper measurements of tumor diameters were done
every other day, and tumor volumes were estimated with the formula
V = 4/3p  (a/2)  (b/2)2, where a and b correspond to the longest and shortest
diameters, respectively. Aplidin significantly inhibited the increase in tumor
growth of the MM xenograft (A; P = 0.01) and increased survival (B ; P = 0.02)
compared with control mice. A, bars, SE.
Discussion
The results of this preclinical study suggest a favorable profile of
the in vitro and in vivo anti-MM activities of Aplidin. First, this
compound exhibits potent in vitro activity against both MM cell
lines and primary MM tumor cells, including cell lines resistant to
established anti-MM treatments. Furthermore, Aplidin was able to
induce cytotoxicity in myelomatous plasma cells obtained from
patients with clinical resistance to most of the current treatments
for the clinical management of MM, including combination
chemotherapy regimens, autologous stem cell transplant, as well
as novel agents such as bortezomib or thalidomide and their
combinations. Interestingly, among the primary tumor samples
tested with Aplidin, one of the most sensitive ones (patient sample
no. 3) had shown clinical refractoriness to all of these treatments.
These data suggest a different mechanism of action of Aplidin than
other antimyeloma agents and suggest that it may overcome
resistance generated against currently available therapies for MM.
Moreover, our studies also show that the potency of Aplidin is high
and comparable to that of bortezomib, a most potent anti-MM
agent in vitro. Additional favorable aspects of Aplidin include the
sparing of normal hematopoietic cells at concentrations that are
www.aacrjournals.org 5223
Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
Antimyeloma Activity of Aplidin
still active against MM cells; the ability to overcome the protective
effect of growth factors, such as IL-6 or IGF-I, on MM cells; and the
additive (bortezomib and melphalan) or synergistic (dexametha-
sone, thalidomide, and lenalidomide) effects when Aplidin is
combined with other anti-MM agents. Another positive feature of
the anti-MM activity of Aplidin is that even a brief exposure of
MM cells to this agent is capable of committing them to undergo
cell death. Indeed, a 1-hour incubation with clinically relevant
concentrations of Aplidin, based on pharmacokinetic studies from
clinical trials in solid tumors (20), was capable of irreversibly
initiating the process of MM cell death in vitro. Finally, this anti-
MM activity of Aplidin in MM cells was confirmed in an in vivo
setting, as evidenced by a delay in tumor growth and prolongation
of survival in Aplidin-treated MM-bearing mice. Importantly, the
toxicity profile of Aplidin in this model was favorable, as evidenced
by the lack of major side effects.
Cell cycle studies indicated that low doses of Aplidin caused G2-
M accumulation. In addition, molecular profiling studies indicated
that Aplidin down-regulated several genes required for cell cycle
progression. In addition to the cell cycle effects, Aplidin triggered
increased Annexin V staining, decreased Dw m, and DNA laddering.
It is important to emphasize the prompt increase in Annexin V
staining, which is detectable early, after 3 hours of treatment. Due
to this rapid proapoptotic action of Aplidin, we selected early times
of exposure to Aplidin (4 hours) to identify genes involved in the
triggering of cell death. These gene expression analyses identified
several genes, including c-JUN and c-MYC, that control cell cycle
and apoptotic phenomena. This apoptotic cell death was also
supported by the induction of poly(ADP-ribose) polymerase pro-
cessing as well as caspase-3, caspase-7, caspase-8, and caspase-9
cleavage induced by Aplidin. This treatment also induced a sub-
stantial decrease in Mcl-1, an important regulator of MM survival.
Finally, studies with inhibitors of the different caspases confirmed
that Aplidin induced cell death via the caspase machinery.
However, because the inhibitory effect of these caspase inhibitors
was only partial, it remains possible that caspase-independent
mechanisms may also contribute to apoptotic cell death triggered
by Aplidin.
Our results offer new insights into the mechanism of action of
Aplidin on tumor cells. Here we found that Aplidin induces
apoptosis in MM cells, at least in part, through the translocation of
the death receptor Fas/CD95 into lipid rafts. In addition, our data
suggest that this is not the only mechanism of action of Aplidin.
The difficulty in delineating a singular molecular target for Aplidin
results, in part, from its complex molecular structure, which
includes many potential biologically active moieties, and the
pleiotropicity of its molecular sequelae. Both of these factors,
which often apply as well for other similarly identified natural
products, can confound our ability to pinpoint specific candidate
molecule(s) that might physically interact with Aplidin and
function as exclusive mediator(s) of its antitumor effects. Our
studies, however, do provide insight into the pathways involved in
the anti-MM activity of this compound. The rapid commitment of
MM cells to undergo cell death on exposure to Aplidin and the
early activation of JNK signaling are similar to the effects of the
proteasome inhibitor bortezomib. However, whereas Aplidin also
activates p38, bortezomib treatment is enhanced by p38 inhibition
(29). Therefore, although equimolar concentrations of Aplidin and
bortezomib have similar potency in rapidly killing MM cells in vitro,
they have distinct differences in their molecular effects (11). This
apparent dichotomy may be related to the ability of the p38 MAPK
Cancer Res 2008; 68: (13). July 1, 2008
 DOI:10.1158/0008-5472.CAN-07-5725
Cancer Research

pathway to respond to different stimuli with diverse biological
effects, which can also depend on the specific cellular context (30).
For example, p38 can trigger apoptosis in some cells but inhibit its
induction in other pathophysiologic settings (30). It has been
proposed (as reviewed in ref. 31) that such stress-activated MAPK
pathways may function as a double-edged sword: They may protect
normal cells against environmental stress and malignant cells
against forms of stress inherent to the neoplastic transformation
(e.g., oncogenic ‘‘addiction,’’ drug treatments); conversely, they may
also trigger apoptosis if the stress levels exceed a threshold (31),
which in turn is influenced by the particular context in which the
tumor cells are placed. We hypothesize that p38 activation, in the
context of Aplidin treatment, is complemented by other molecular
events (also triggered by the compound) that reinforce the pro-
apoptotic role of p38. Such complementary molecular events
include the suppression of myc (an important regulator of pro-
liferative/antiapoptotic responses for MM cells; ref. 27) or anti-
apoptotic molecules such as survivin (32, 33) and/or the increased
expression of proapoptotic modulators such as TRAIL (9, 32, 34, 35)
and Smac (36, 37).
This favorable preclinical profile of the anti-MM activity of
Aplidin provided the rationale for the initiation of a clinical trial of
this agent in patients with advanced MM (38). Preliminary results
of this ongoing clinical study indicate that Aplidin has promising
clinical anti-MM activity in heavily pretreated MM patients
refractory to several conventional and novel anti-MM agents (e.g.,
bortezomib and/or lenalidomide; ref. 38). A high proportion of
patients in this trial had received prior high-dose therapy and
autologous stem cell transplant (60%), thalidomide (58%), and/or
bortezomib (48%). Among 26 evaluable patients, 2 (8%) achieved
partial response, 3 (12%) showed minor response, and 8 (31%) had
stable disease. Due to the enhanced anti-MM activity observed
with the in vitro combination of these agents, high-dose
dexamethasone was added to Aplidin in patients not achieving
response, and preliminary results of the clinical combination
regimen are also very encouraging. Importantly, in the clinical trials
Aplidin did not cause significant toxicities that are present in
established anti-MM agents, such as neuropathy or myelosuppres-
sion (38). Furthermore, preliminary data indicate that its
combination with dexamethasone is not associated with the
thromboembolic complications observed with the combinations of
glucocorticoids with thalidomide or lenalidomide (39, 40).
In conclusion, our preclinical data on the potent anti-MM
activity of single-agent Aplidin and its potential in vitro synergistic/
additive effect with other anti-MM agents, coupled with promising
clinical data on the safety profile and objective clinical responses in
heavily pretreated refractory patients, indicate that this novel
compound (alone or in combination with other agents) may
contribute to improve the outcome of MM patients.
Disclosure of Potential Conflicts of Interest
C.S. Mitsiades: Honoraria, Pharmion and Millenium. A. Pandiella: Commercial
research grant, PharmaMar. P. Aviles: Employment, PharmaMar. G. Faircloth:
Employment, PharmaMar. P.G. Richardson: Speakers bureau/honoraria, Millennium
and Celgene. J.F. San-Miguel: Commercial research support, PharmaMar. K.C.
Anderson: Commercial research support, Millennium and Celgene; speakers bureau/
honoraria, Novartis, Millennium, and Celgene. C.S. Mitsiades and N. Mitsiades are
inventors of a patent, co-owned by Dana-Farber and PharmaMar, that relates to the
present work. The other authors disclosed no potential conflicts of interest.
Acknowledgments
Received 10/2/2007; revised 3/21/2008; accepted 4/11/2008.
Grant support: The Multiple Myeloma Research Foundation (C.S. Mitsiades,
N. Mitsiades, and J.F. San-Miguel), The Lauri Strauss Leukemia Foundation (C.S.
Mitsiades and N. Mitsiades), The International Waldenstrom’s Macroglobulinemia
Foundation (C.S. Mitsiades), The Fund to Cure Myeloma (K.C. Anderson), The
Fulbright Commission (C.J. McMullan), Doris Duke Distinguished Clinical Research
Scientist Award (K.C. Anderson), Fondo de Investigación Sanitaria and European
Commission grants FIS-FEDER 04/0843 (F. Mollinedo) and 06/0813 (C. Gajate),
Ministerio de Educación y Ciencia grant SAF2005-04293 (F. Mollinedo), Fundación de
Investigación Médica Mutua Madrileña (F. Mollinedo), and Fundación ‘‘la Caixa’’ grant
BM05-30-0 (F. Mollinedo). C. Gajate is supported by the Ramón y Cajal Program from
the Ministerio de Educación y Ciencia of Spain; E.M. Ocio is supported by Carlos III-
Fondo de Investigación Sanitaria-Ministerio de Ciencia y Tecnologı́a grant CM04/0001;
This work was supported by a grant from the Ministry of Education and Science of
Spain (BFU2006-01813/BMC), and by an Institutional Cancer Center Network program
from the ISCIII. The Cancer Research Institute receives support from the European
Community through the regional development funding program (FEDER). P. Maiso
was supported by the FIS-FEDER through projects to J.F. San-Miguel, and a Spanish
Myeloma Network Program (G03/136). M. Garayoa is supported by ‘‘Instituto de Salud
Carlos III-Fondo de Investigación Sanitaria-Ministerio de Ciencia y Tecnologı́a’’ grant
CP 05/0279; C.S. Mitsiades is a Special Fellow of the Leukemia and Lymphoma Society
and is supported by the ‘‘Dunkin Donuts Rising Stars’’ Program at the Dana-Farber
Cancer Institute.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Joseph M. Negri, Gary Fanourakis, Supriya Rao, Despoina Sykoutri,
Montserrat Martin, and Laura San Segundo for technical assistance.
C.S. Mitsiades, E.M. Ocio, and A. Pandiella designed research, performed research,
analyzed data, and wrote the paper; P. Maiso, C. Gajate, M. Garayoa, and F. Mollinedo
designed research, performed research, analyzed data, and contributed to the writing
of the paper; N. Mitsiades, C.J. McMullan, J-C. Montero, D. Vilanova, and M.V. Mateos
designed research, performed research, and analyzed data; T. Hideshima and D.
Chauhan contributed research tools; N.C. Munshi, G. Otero, P. Aviles, and
G. Faircloth contributed research tools and analyzed data; P.G. Richardson, J.F. San-
Miguel, and K.C. Anderson designed research, analyzed data, and contributed to the
writing of the paper.

References
1. Richardson PG, Hideshima T, Mitsiades C, Anderson
KC. The emerging role of novel therapies for the
treatment of relapsed myeloma. J Natl Compr Canc
Netw 2007;5:149–62.
2. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ.
Cancer statistics, 2007. CA Cancer J Clin 2007;57:43–66.
3. Newman DJ, Cragg GM. Natural products as sources
of new drugs over the last 25 years. J Nat Prod 2007;70:
461–77.
4. Rinehart KL. Antitumor compounds from tunicates.
Med Res Rev 2000;20:1–27.
5. O’Hanlon LH. Scientists are searching the seas for
cancer drugs. J Natl Cancer Inst 2006;98:662–3.
6. Urdiales JL, Morata P, Nunez De Castro I, Sanchez-
Jimenez F. Antiproliferative effect of dehydrodidemnin
B (DDB), a depsipeptide isolated from Mediterranean
tunicates. Cancer Lett 1996;102:31–7.
7. Chauhan D, Uchiyama H, Urashima M, Yamamoto K,
Anderson KC. Regulation of interleukin 6 in multiple
myeloma and bone marrow stromal cells. Stem Cells
1995;13 Suppl 2:35–9.
8. Hideshima T, Richardson P, Chauhan D, et al. The
proteasome inhibitor PS-341 inhibits growth, induces
apoptosis, and overcomes drug resistance in human
multiple myeloma cells. Cancer Res 2001;61:3071–6.
9. Mitsiades CS, Treon SP, Mitsiades N, et al. TRAIL/
Apo2L ligand selectively induces apoptosis and over-
comes drug resistance in multiple myeloma: therapeutic
applications. Blood 2001;98:795–804.
10. Mitsiades N, Mitsiades CS, Richardson PG, et al. The
proteasome inhibitor PS-341 potentiates sensitivity of
multiple myeloma cells to conventional chemothera-
peutic agents: therapeutic applications. Blood 2003;101:
2377–80.
11. Mitsiades N, Mitsiades CS, Poulaki V, et al. Molecular
sequelae of proteasome inhibition in human multiple
myeloma cells. Proc Natl Acad Sci U S A 2002;99:14374–9.
12. Carvajal-Vergara X, Tabera S, Montero JC, et al.
Multifunctional role of Erk5 in multiple myeloma. Blood
2005;105:4492–9.
13. Maiso P, Carvajal-Vergara X, Ocio EM, et al. The
histone deacetylase inhibitor LBH589 is a potent
antimyeloma agent that overcomes drug resistance.
Cancer Res 2006;66:5781–9.
14. Orfao A, Garcia-Sanz R, Lopez-Berges MC, et al. A
new method for the analysis of plasma cell DNA content
in multiple myeloma samples using a CD38/propidium
iodide double staining technique. Cytometry 1994;17:
332–9.

Cancer Res 2008; 68: (13). July 1, 2008 5224 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research

15. Li C, Wong WH. Model-based analysis of oligonucle-
otide arrays: expression index computation and outlier
detection. Proc Natl Acad Sci U S A 2001;98:31–6.
16. Gajate C, Mollinedo F. The antitumor ether lipid
ET-18-OCH(3) induces apoptosis through translocation
and capping of Fas/CD95 into membrane rafts in
human leukemic cells. Blood 2001;98:3860–3.
17. Cheng PC, Dykstra ML, Mitchell RN, Pierce SK. A role
for lipid rafts in B cell antigen receptor signaling and
antigen targeting. J Exp Med 1999;190:1549–60.
18. Gajate C, Mollinedo F. Edelfosine and perifosine
induce selective apoptosis in multiple myeloma by
recruitment of death receptors and downstream signal-
ing molecules into lipid rafts. Blood 2007;109:711–9.
19. Chou TC, Talalay P. Quantitative analysis of dose-
effect relationships: the combined effects of multiple
drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:
27–55.
20. Maroun JA, Belanger K, Seymour L, et al. Phase I
study of Aplidine in a dailyx5 one-hour infusion every
3 weeks in patients with solid tumors refractory to
standard therapy. A National Cancer Institute of Canada
Clinical Trials Group study: NCIC CTG IND 115. Ann
Oncol 2006;17:1371–8.
21. Richardson PG, Sonneveld P, Schuster MW, et al.
Bortezomib or high-dose dexamethasone for relapsed
multiple myeloma. N Engl J Med 2005;352:2487–98.
22. Richardson PG, Mitsiades C, Hideshima T, Anderson
KC. Bortezomib: proteasome inhibition as an effective
anticancer therapy. Annu Rev Med 2006;57:33–47.
23. Richardson PG, Jagannath S, Avigan DE, et al.
Lenalidomide plus bortezomib (Rev-Vel) in relapsed
www.aacrjournals.org
Downloaded from cancerres.aacrjournals.org on April 12, 2011
Copyright © 2008 American Association for Cancer Research
DOI:10.1158/0008-5472.CAN-07-5725
and/or refractory multiple myeloma (MM): final results
of a multicenter phase 1 trial. Blood 2006;108:124A–A.
24. Orlowski RZ. Bortezomib and its role in the
management of patients with multiple myeloma. Expert
Rev Anticancer Ther 2004;4:171–9.
25. Rajkumar SV, Hayman SR, Lacy MQ, et al. Combina-
tion therapy with lenalidomide plus dexamethasone
(Rev/Dex) for newly diagnosed myeloma. Blood 2005;
106:4050–3.
26. Rajkumar SV, Blood E, Vesole D, Fonseca R, Greipp
PR. Phase III clinical trial of thalidomide plus dexa-
methasone compared with dexamethasone alone in
newly diagnosed multiple myeloma: a clinical trial
coordinated by the Eastern Cooperative Oncology
Group. J Clin Oncol 2006;24:431–6.
27. Bergsagel PL, Kuehl WM. Molecular pathogenesis
and a consequent classification of multiple myeloma.
J Clin Oncol 2005;23:6333–8.
28. Gajate C, Mollinedo F. Cytoskeleton-mediated death
receptor and ligand concentration in lipid rafts forms
apoptosis-promoting clusters in cancer chemotherapy.
J Biol Chem 2005;280:11641–7.
29. Hideshima T, Akiyama M, Hayashi T, et al. Targeting
p38 MAPK inhibits multiple myeloma cell growth in the
bone marrow milieu. Blood 2003;101:703–5.
30. Bradham C, McClay DR. p38 MAPK in development
and cancer. Cell Cycle 2006;5:824–8.
31. Dhillon AS, Hagan S, Rath O, Kolch W. MAP
kinase signalling pathways in cancer. Oncogene 2007;
26:3279–90.
32. Mitsiades N, Mitsiades CS, Poulaki V, et al. Biologic
sequelae of nuclear factor-nB blockade in multiple
5225
Antimyeloma Activity of Aplidin
myeloma: therapeutic applications. Blood 2002;99:
4079–86.
33. Romagnoli M, Trichet V, David C, et al. Significant
impact of survivin on myeloma cell growth. Leukemia
2007;21:1070–8.
34. Mitsiades N, Mitsiades CS, Poulaki V, et al. Apoptotic
signaling induced by immunomodulatory thalidomide
analogs in human multiple myeloma cells: therapeutic
implications. Blood 2002;99:4525–30.
35. Mitsiades N, Mitsiades CS, Poulaki V, Anderson KC,
Treon SP. Intracellular regulation of tumor necrosis
factor-related apoptosis-inducing ligand-induced apo-
ptosis in human multiple myeloma cells. Blood 2002;99:
2162–71.
36. Chauhan D, Li G, Hideshima T, et al. JNK-dependent
release of mitochondrial protein, Smac, during apopto-
sis in multiple myeloma (MM) cells. J Biol Chem 2003;
278:17593–6.
37. Chauhan D, Neri P, Velankar M, et al. Targeting
mitochondrial factor Smac/DIABLO as therapy for
multiple myeloma (MM). Blood 2007;109:1220–7.
38. Mateos MV, Cibeira MT, Blade J, et al. Phase II trial
with plitidepsin (Aplidin) alone and in combination
with dexamethasone (dex) in patients with relapsed/
refractory multiple myeloma: preliminary results. Hae-
matologica 2007;92:153–4.
39. Rajkumar SV, Blood E. Lenalidomide and venous
thrombosis in multiple myeloma. N Engl J Med 2006;354:
2079–80.
40. Knight R, DeLap RJ, Zeldis JB. Lenalidomide and
venous thrombosis in multiple myeloma. N Engl J Med
2006;354:2079–80.
Cancer Res 2008; 68: (13). July 1, 2008