Bcl-2 Antisense Therapy in Multiple Myeloma
Bcl-2 Antisense Therapy in Multiple Myeloma
Multiple myeloma (MM) remains
incurable with conventional
with a median survival between 2 and
3 years. MM is typically chemosensitive
earlier in its clinical course but
is ultimately characterized by the development
of multidrug resistance in
almost all patients. There is growing
evidence that the apoptosis-regulating
Bcl-2 protein may play an important
role in the development of multidrug
resistance in patients with MM. Bcl-2
antisense therapy may therefore provide
a new therapeutic approach to
reverse this resistance and to potentiate
antitumor effects of chemotherapy
in the treatment of MM.
Bcl-2 Protein and Multidrug
Resistance in Multiple Myeloma
Bcl-2 is a conserved, ubiquitous
protein associated with the inner mitochondrial
membranes. It exhibits a
regulatory role in apoptosis by blocking
the release of cytochrome c.
Most human myeloma cell lines and
samples obtained from patients with
MM overexpress Bcl-2 protein.[5,6]
Various preclinical studies have
demonstrated that the Bcl-2 protein
plays an important role in mediating resistance
of MM cells to apoptosis induced
by dexamethasone and cytotoxic
agents.[7-13] Tian and Gazitt initially
demonstrated that the extent of
dexamethasone-induced apoptosis in
MM cell lines in vitro was inversely
correlated with intracellular levels of
Bcl-2. Subsequently, these investigators
low Bcl-2-expressing MM cell
lines with a Bcl-2-inducible gene construct
expressed under the control of
a lac repressor operon. Activation
of the inducible gene resulted in increased
intracellular levels of Bcl-2,
enhanced cell growth, and decreased
spontaneous apoptosis, with concomitant
increased resistance to dexamethasone.
Conversely, inactivation of the
inducible gene restored sensitivity to
Collectively, these seminal studies
demonstrated a potential role of Bcl-2
in apoptosis regulation of MM cell
lines as well as development of resistance
apoptosis in these cells. This provides
the basis for the hypothesis that Bcl-2
antisense therapy might decrease Bcl-
2 protein production and thereby facilitate
apoptosis in malignant myeloma
Preclinical Studies With
Oblimersen sodium (Genasense,
previously known as G3139) is a Bcl-
2 antisense oligonucleotide designed
to specifically bind to the bcl-2 messenger
RNA. It binds to the first
six codons of the human bcl-2 mRNA,
forming a heterodimer. This doublestranded
mRNA is perceived as aberrant
and is subsequently degraded in
the cell's cytoplasm. The result is a
decreased production of the Bcl-2 protein
by the ribosome.
Recent preclinical studies have
evaluated the potential of oblimersen
to decrease Bcl-2 protein in MM cells
and to sensitize or reverse resistance
of MM cells to agents active against
MM. Results from these studies indicate
that oblimersen is taken up by myeloma
cells and can decrease Bcl-2
protein production. Thus, the sensitivity
of myeloma cells to therapeutic
agents commonly used in the treatment
of MM, such as dexamethasone
and doxorubicin, is enhanced, providing
a rationale for conducting clinical
trials using oblimersen in patients with
Liu and Gazitt reported the effects
of pretreatment with oblimersen
on dexamethasone-, paclitaxel-, and adenovirus
p53-induced apoptosis and
intracellular levels of Bcl-2 in myeloma
cells expressing varying levels of Bcl-
2. Multiple myeloma cells were treated
with oblimersen (10 μg/mL) for 3 days,
followed by exposure to dexamethasone,
paclitaxel, or adenovirus p53 for
up to 2 additional days. In myeloma
cells expressing relatively low levels of
Bcl-2, oblimersen exposure resulted in
substantial apoptosis with a concomitant
decrease of Bcl-2 protein. This decrease
in Bcl-2 protein levels and the
increased apoptotic events were timeand
apoptosis was noted to be mediated
through activation of caspase-9 and
caspase-3 and by the release of cytochrome
c into the cytosol.
In another experiment with high
Bcl-2-expressing myeloma cell lines
(ARH-77, U266), oblimersen pretreatment
followed by exposure to dexamethasone
or paclitaxel resulted in a
substantial increase in the percentage of
myeloma cells undergoing apoptosis
when compared with the effects
obtained with each agent with prior
exposure to oblimersen. The increased
apoptosis was associated with a decrease
in Bcl-2 protein. Similar results
were obtained when freshly isolated
myeloma cells from patients were used.
In another study, van de Donk et
al also evaluated the effects of decreased
Bcl-2 protein in ex vivo purified
malignant plasma cells from patients
with MM. Following incubation
of the cells with oblimersen, but not with
solvent or the sense oligonucleotides, a
substantial reduction (> 75%) of bcl-2
mRNA levels occurred after 2 and 4
days of exposure as measured by realtime
polymerase chain reaction. Exposure
of the cells to oblimersen resulted
in a sequence-specific reduction of
Bcl-2 protein within 4 days in 10 of
11 patient samples. Significant enhancement
of dexamethasone- or
doxorubicin-induced apoptosis and
cytotoxicity was also noted in these
experiments (Figures 1 and 2).
O'Connor et al were the first
to report on the beneficial antitumor
effects of oblimersen when combined
with the biologic agent bortezomib
(Velcade) in non-Hodgkin's lymphoma
(NHL) and MM cell lines.
These authors recently shared the preliminary
results of in vitro and in vivo
experiments with oblimersen and
bortezomib with established MM
(U266, RPMI 8226, KMS-11) and
NHL (Raji, SKI-DLCL-1, DOHH2)
cell lines. These seminal experiments
showed that oblimersen enhances the
antiproliferative effect of bortezomib
in MM cell lines. The augmented
antiproliferative response of oblimersen
was particularly evident in the
In a xenograft experiment with
SKI-DLCL-1 comparing oblimersen
alone, bortezomib alone, and oblimersen
prior to bortezomib administration,
mice receiving either agent
alone showed 20% to 25% inhibition
of tumor growth, whereas animals receiving
the combination experienced
a reduction in tumor growth of approximately
50% at 24 days. This
effect of oblimersen was noted to be
sequence-specific. Based on these experiments,
future studies are proposed
to explore the role of oblimersen in
optimizing the use of bortezomib in
patients with B-cell malignant disorders.
Clinical Studies of Oblimersen
Based on the potential role of antiapoptotic
Bcl-2 protein in MM and the
encouraging preclinical studies with
oblimersen, several clinical trials in patients
with relapsed or refractory MM
were initiated. These clinical studies are
designed to evaluate the potential role
of oblimersen to enhance sensitivity
or reverse resistance to standard MM
therapies, including dexamethasone,
and the combination of vincristine,
doxorubicin (Adriamycin), and
Phase II Trials
Badros et al[19,20] are conducting a phase I/II clinical trial evaluating the administration of oblimersen followed by dexamethasone/thalidomide in patients with relapsed or refractory MM. This study has been active as of May 2004, with a target accrual of up to 46 patients. To be eligible, patients may have received no more than four prior chemotherapy regimens. Oblimersen (7 mg/kg/d) is administered by continuous intravenous infusion on days 1 to 7 (the first three patients received 5 mg/kg/d), with dexamethasone (40 mg/d) given on days 4 to 7 and thalidomide (100 to 400 mg/d) starting on day 4. Treatment cycles are repeated every 3 weeks. After three induction cycles, responding patients continue oblimersen on a 5-week cycle with dexamethasone (20 mg/d) for 4 days and thalidomide at the tolerated dose for up to 1 year. Preliminary data have been reported for the first 18 patients treated. The median age was 58 years (range: 47 to 74), and 11 patients were male. Patients had received a median of 3 prior regimens (range: 2 to 4 regimens), including prior autologous (n = 15) or allogeneic (n = 1) stem cell transplantation. Eight patients had received thalidomide previously, for a median duration of 6.5 months (range: 2 to 10 months), with 6 of these patients demonstrating disease progression while receiving thalidomide. Ten patients had complex karyotype. Of the enrolled patients, 16 have completed the induction phase. Two patients had a complete response, two others had a near-complete response, and five patients had a partial response. The overall response rate of 75% is notable in a group of patients with advanced disease who had failed multiple prior therapies. At a median follow-up of 4 months (range: 1.5 to 8.5 months), 1 responding patient had relapsed and 11 continued on study therapy. Bone marrow with adequate plasma cells was available for 11 patients; in this study no significant decrease in Bcl-2 protein was observed at days 4 to 7 or day 28 of oblimersen infusion compared with baseline. In addition, there were no detectable differences between responders and nonresponders with respect to Bcl-2 protein. The combination of oblimersen, dexamethasone, and thalidomide was well-tolerated. Oblimersen toxicities included reversible increases in serum creatinine (to > 2 mg/dL) in 10 patients, which required oblimersen dose reduction to 3 to 5 mg/kg/d, and thrombocytopenia in 6 patients. No grade 4 toxicities occurred, and the majority of toxicities were reversible. In another phase II trial, the same concept of restoring chemosensitivity in patients refractory to standard chemotherapy by administration of oblimersen to decrease Bcl-2 protein production in combination with chemotherapy is being evaluated. Ten patients with refractory MM, including eight patients whose disease was refractory to VAD, were treated with oblimersen (7 mg/kg/ d) by continuous intravenous infusion for 7 days in combination with VAD. All patients were heavily pretreated and had received a median of 4 previous chemotherapy regimens (range: 2 to 6). Four patients had a partial response, and three patients had a minor response. Median disease progression-free survival was approximately 6 months (range: 2 to 7+ months), and median overall survival had not been reached. Oblimersen decreased the amount of Bcl-2 protein in peripheral blood myeloma cells, T cells, B cells, and monocytes. The oblimersen/VAD combination was feasible and well tolerated. The median number of treatment cycles was 2.5. Baseline Bcl-2 protein in bone marrow, Ki-67 growth fraction, and the presence of deletion of chromosome 13 were not predictive of response. These preliminary results suggest that oblimersen may help to overcome chemotherapy resistance and restore sensitivity of myeloma tumor cells to VAD chemotherapy. Phase III Trial
A randomized phase III trial of dexamethasone with or without oblimersen in patients with relapsed or refractory MM (GMY302) recently completed accrual (n = 220). Patients could have had up to six prior therapies. In a 4-week induction period, oral dexamethasone (40 mg) was given every day for 4 days of weeks 1, 2, and 3 to both cohorts. Patients who were randomized to the study arm received oblimersen (7 mg/kg/d) as a continuous infusion for 7 days on weeks 1 and 3, with dexamethasone initiated on day 4 of the oblimersen infusion. Subsequent cycles in both cohorts incorporated only one 4-day dexamethasone pulse every 3 weeks. The primary end point is time to disease progression. Results of this study are expected to be presented at the Annual Session of the American Society of Hematology (ASH) in 2004. Conclusion Bcl-2 protein confers a clinically relevant chemoresistant phenotype on many types of cancer cells, including multiple myeloma, making it a relevant target for MM therapy. Preclinical studies indicate that oblimersen decreases Bcl-2 protein in MM cell lines as well as in ex vivo MM cells from patients and enhances the cytotoxic potential of dexamethasone, doxorubicin, and bortezomib. Preliminary data from phase II clinical trials in patients with refractory or relapsed MM are encouraging. Clinically oblimersen appears to enhance the cytotoxic potential of antimelanoma therapies and is noted to have an acceptable safety profile in these patients; the results of a fully accrued phase III trial comparing dexamethasone plus oblimersen to dexamethasone alone in patients with refractory or relapsed disease are awaited with considerable interest. Results from these ongoing studies will determine the role of oblimersen in the treatment of patients with MM. Future studies will focus on combining oblimersen with other novel biologic agents such as bortezomib.
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