Targeting the Proapoptotic Factor Bcl-2 in Non-Hodgkin's Lymphoma
Targeting the Proapoptotic Factor Bcl-2 in Non-Hodgkin's Lymphoma
Bcl-2, a member of a family of
proteins primarily located between
the two layers of the
mitochondrial membrane, has been
identified as a logical therapeutic
target in a variety of human cancers,
given its role in regulating a major
apoptotic pathway, namely, the
mitochondria-mediated or intrinsic
pathway of caspase activation.[2,3]
Non-Hodgkin's lymphomas of B-cell
phenotype are among the malignancies
associated with a high level of
Bcl-2 expression, seen in greater than
90% of follicular or mantle cell histologies[
4,5] and in 50% of diffuse
No curative therapy exists for follicular
and mantle cell lymphomas,
and only 50% of all patients presenting
with diffuse large B-cell lymphoma
will be long-term failure-free
survivors. Therefore, strategies that
may reduce Bcl-2 expression in patients
with these diseases, which make
up almost two-thirds of all non-
Hodgkin's lymphomas, are being
evaluated for their potential to reduce
resistance to therapy and improve patient
Bcl-2 Expression and
Chemotherapy Drug Resistance
A number of research groups have
independently found that Bcl-2
overexpression is unequivocally associated
with (1) more advanced disease,
according to Ann Arbor criteria; (2) a
constellation of poor prognostic features,
as defined by the International
Prognostic Index; and (3) significantly
lower survival rates.[6-12] The studies
in diffuse large B-cell lymphoma
have implicated it as an independent
prognostic factor. For example, in a
British Columbia Cancer Agency
study of 116 patients with uniformly
staged and treated diffuse large B-cell
lymphoma, the 8-year overall, diseasefree,
and relapse-free survival rates
were significantly (P < .01) lower in
patients whose tumors expressed Bcl-
2 protein on initial biopsy: 34%, 32%,
and 25% vs 60%, 66%, and 59%, respectively,
for those with Bcl-2-negative
Does the expression of Bcl-2 have
a biologic impact on malignant cells?
In an elegant series of experiments, in
which lymphoma cells isolated from
Eμ-myc transgenic mice were
retrovirally transfected with the murine
bcl-2 gene, Schmitt and colleagues[
13] attempted to isolate the
effect of a single-gene change on tumor
behavior. Essentially, it was demonstrated
that upregulating Bcl-2 results
in greater cell viability and less
apoptosis but no difference in the actual
rate of proliferation (Figure 1),
confirming that the effects are specific
to the rate of cellular death. This
upregulation of Bcl-2 conferred relative
resistance to both cyclophosphamide
(Cytoxan, Neosar) and docetaxel
(Taxotere), with notable differences in
the rates of tumor-free survival between
animals bearing Bcl-2-positive
vs Bcl-2-negative lymphomas (Figure 2).
This direct experimental evidence,
taken together with the clinical data
amassed over the past decade, makes
it clear that strategies designed to decrease
Bcl-2 expression in lymphomas
and other malignancies have the potential
to alter outcomes significantly.
Oblimersen Sodium Antisense
Oligonucleotide Therapy in
Oblimersen sodium (Genasense),
formerly known as G3139, is an 18-mer
DNA oligonucleotide that is complementary
to, and thus selectively targets,
the first six codons of the bcl-2 messenger
RNA (mRNA) to decrease Bcl-
2 protein translation. The reverse orientation
of the nucleotides to the "sense"
strand of the RNA message led to the
antisense designation. A sulfa group,
added via a phosphorothioate linkage,
protects the molecule from degradation
by endonucleases and exonucleases,
allowing the formulation of a drug with
a favorable pharmacokinetic profile
when administered systemically.
The antisense molecules are believed
to function as "therapeutic
DNA" in that they bind to bcl-2 mRNA
in the cytoplasm to form a heteroduplex
by Watson-Crick base pairing,
thereby creating a biologically undesirable
complex.[14,15] Various endonucleases,
particularly RNase H, are
subsequently engaged to cleave the
mRNA portion off the DNA backbone,
thus eliminating the message while
leaving an intact antisense molecule
that, in theory, can proceed to target
additional bcl-2 mRNA. In this way,
the normal process by which the DNA
code is transcribed into a mRNA that
translocates into the cytoplasm, binds
a ribosome, and is translated into a
functional Bcl-2 protein is abrogated.
Can Bcl-2 actually be demonstrably
downregulated in this way? Working
with tumor cell lines, numerous
groups, including our own with lymphoid
tumors, have shown specific
downregulation of Bcl-2 protein using
oblimersen. Murine SCID/human
tumor xenograft models have allowed
study in vivo that at least mimics the
clinical therapeutic situation. In solidtumor
studies that examined human
melanoma-bearing mice in a subcutaneous
model, Jansen and colleagues[
17], from the University of
Vienna, showed that systemically delivered
oblimersen is clearly capable
of downregulating Bcl-2 and improving
sensitivity to a cytotoxic agent,
In studies in our laboratory,
oblimersen alone, somewhat to our
surprise, completely eliminated
DoHH2 (a transformed follicular lymphoma
cell line associated with highlevel
Bcl-2 expression) in a fraction
of animals with systemic disease. Reverse
polarity oligonucleotides used as
controls had no effect, a proof in principle
that such widespread disease
could be targeted by oblimersen.
Guinness and coworkers, from
the Yale Comprehensive Cancer
Center, examined the effect of
oblimersen as a single-agent modality
in a human/SCID model of Epstein-
Barr virus-associated posttransplant
lymphoproliferative disease. In two
separate in vivo experiments, the administration
of oblimersen (10 mg/m2/
d) for 12 days led to a significantly
longer disease-free survival than that
seen in the control group (P < .001),
with no evidence of disease in most
oblimersen-treated animals at the time
However, given what is known about
the role of Bcl-2 in preventing the engagement
of the cell's apoptotic program,
it stood to reason on theoretical
grounds that Bcl-2 blockade in the face
of the genotoxic insult associated with
the types of therapy customarily given
in clinical practice might yield the greatest
benefit (Figure 3). Most chemotherapeutic
agents are known to cause DNA
damage, which is sensed via p53 and
other mechanisms, in turn, forcing
malignant cells toward a critical decision
point-to activate either repair or
cell death programs. A reduction in Bcl-
2 expression at this critical moment
could shift the balance in favor of proceeding
down the apoptotic pathway.
Combining Oblimersen With
In our own studies at the British Columbia Cancer Agency, oblimersen given as a single agent was shown to improve the median survival and the 90-day survival rate in the SCID xenograft model of DoHH2. At all dose levels evaluated, ranging from 2.5 mg/kg every other day to 12.5 mg/ kg/d, oblimersen-treated animals had a highly significant (P < .000001) survival advantage over the untreated and irrelevant oligonucleotide control groups. Cyclophosphamide, given as a single agent at a dose of 35 mg/kg on days 4, 8, and 12, exerted modest effects, improving median survival to 47 days from 33 days in the control group, but with no cured animals seen (Figure 4). When oblimersen was combined with cyclophosphamide in a variety of doses and schedules, the vast majority of animals were cured. Perhaps the most striking finding was that adding a 28-day course of oblimersen (2.5 or 5 mg/kg every other day, a total of 14 doses) to a completely ineffective single-agent cyclophosphamide dose (15 mg/kg on days 4, 8, and 12) dramatically increased the 90-day survival rate from 0% to 50%. The specificity (use of a number of control oligonucleotides), consistency (across a large number of animal cohorts studied), and durability (cured animals were human Bcl-2 negative by polymerase chain reaction at necropsy) of this effect provided the scientific rationale for the design of subsequent clinical trials. In both preclinical and clinical settings, there has been interest in combining oblimersen with other agents, including the CD20-targeted monoclonal antibody rituximab (Rituxan). In an in vitro study by Auer et al, exposing primary chronic lymphocytic leukemia (CLL) cells to the combination of oblimersen plus rituximab dramatically increased the degree of apoptosis in a synergistic, dose-depen- dent (for rituximab) manner. Lacy and collaborators from Yale University reported that combining oblimersen with rituximab in a human/SCID model of Epstein-Barr virus-associated posttransplant lymphoproliferative disease increases cure rates and significantly prolongs survival compared with either agent alone. When combining the results of two identical experiments, the tumorfree survival rates were 79% (11/14) for the combination of oblimersen/ rituximab, vs 7% (1/14) and 15% (2/13) for single-agent rituximab and oblimersen, respectively. Expanding on that work, this research team showed that oblimersen enhances the in vitro and in vivo cytotoxicity of etoposide in this model. Oblimersen with etoposide was associated with a significantly prolonged median survival of > 150 days, vs 98 days for oblimersen alone and 86 days for etoposide alone (Figure 5). As one further example of the synergistic interaction between oblimersen and other targeted therapies, Cotter and colleagues from Barts and the London School of Medicine found that the combination of oblimersen plus alemtuzumab (Campath) exerts more proapoptotic activity than oblimersen alone in CLL and non-Hodgkin's lymphoma cell lines. Collectively, in vitro and in vivo preclinical data support that oblimersen has a direct effect on downregulating Bcl-2 and that this effect has considerable therapeutic potential in treating lymphomas. Clinical Studies of Single-Agent and Combination Therapy
To date, clinical data on oblimersen in lymphomas are available from one completed phase I trial of single-agent therapy and two ongoing phase II trials of rituximab-containing combination regimens (Table 1).[23-27] Lymphomas are arguably the most logical target for developing oblimersen; however, until recently, clinical research efforts in hematologic malignancies have been slow to develop relative to solid tumors.
- Phase I Trial of Single-Agent Oblimersen in Heavily Pretreated Lymphoma-In 1997 and 2000, Waters, Webb, and colleagues[26,27] from the United Kingdom published the results of a phase I trial of oblimersen in 21 patients with various types of previously treated non- Hodgkin's lymphoma, all of whom had documented Bcl-2 protein expression by tumor biopsy. Oblimersen was delivered as a 14-day continuous subcutaneous infusion with eight doses, ranging from 4.6 to 195.8 mg/m2/d. Three patients received a second cycle. Most patients had indolent lymphomas (nine with follicular and eight with small lymphocytic histology) and had received a median of four chemotherapy regimens. Toxicities generally were mild to modest, with no significant grade 3/4 toxicity up to 110 mg/m2/d. Reversible grade 2/3 thrombocytope- icity in three of five patients treated at the maximum tolerated dose of 147 mg/m2/d, which is equivalent to 4 mg/kg/d. In one patient treated at the maximum tolerated dose, who had an unexpectedly high plasma level of oblimersen, dose-limiting toxicity consisted of grade 3 fever in conjunction with grade 3 hypotension (it resolved rapidly and did not recur on retreatment at a lower dose). At doses exceeding 37 mg/m2/d, 10 patients developed transient grade 3/4 lymphopenia; of note, most of these patients had preexisting lymphopenia, and any worsening that occurred during oblimersen therapy was deemed clinically insignificant. Grade 1/2 hyperglycemia in the nonfasting state was transiently seen in 19 patients, possibly associated with the sulfur molecule that is part of the phosphorothioate backbone of oblimersen. One patient with node-positive follicular lymphoma with bone marrow involvement achieved a complete response of longer than 3 years' duration. Additionally, in this heavily pretreated population, there were two minor responses and nine patients with stable disease. Disease-related symptoms improved in 6 of 10 patients (60%) who were symptomatic prior to treatment, and Bcl-2 protein expression decreased in 7 of 16 (44%) assessable biopsy samples. After a median follow-up of nearly 1 year, median overall and disease progression-free survival times were 13.4 and 3.6 months, respectively. The feasibility, tolerability, and activity of oblimersen in this study have prompted investigators to initiate phase II trials of oblimersen as a component of cytotoxic therapy for both relapsed and untreated non-Hodgkin's lymphoma.
- Phase I/II Trial of Oblimersen Plus Rituximab-Cyclophosphamide/ Doxorubicin/Vincristine/Prednisone in Mantle Cell Lymphoma-Preclinical work within our own group, characterizing mantle cell lymphoma cell lines and exposing them to various agents in vitro and in murine xenograft models, indicates that both rituximab and oblimersen are active agents with potentially additive/synergistic effects.[ 28,29] Leonard and colleagues[ 23,24] designed a multicenter phase I/II study of oblimersen in combination with rituximab-cyclophosphamide/ doxorubicin HCl/vincristine (Oncovin)/prednisone (R-CHOP) in mantle cell lymphoma, a disease with a median survival of only 3 to 4 years associated with overexpression of both Bcl-1 and Bcl-2, the latter of which has been somewhat underappreciated. Treatment was initiated with oblimersen (3 to 5 mg/kg/d) administered as a 7-day continuous infusion every 21 days for up to six cycles or until disease progression; treating relapsed/ refractory patients with an additional six cycles was permitted. In chemotherapy-naive patients failing to achieve a complete response after six cycles of oblimersen alone, subsequent therapy consisted of oblimersen (3 mg/m2/d) on days 1 to 7 plus R-CHOP on day 5 for up to six cycles. Results for the first 45 enrolled patients, 27 with relapsed/refractory and 18 with chemotherapy-naive disease, have been reported in abstract form (Table 2). All six cycles of initial oblimersen therapy were completed without evidence of disease progression in seven relapsed/refractory and two chemotherapy-naive patients. Of 18 patients with relapsed/refractory disease treated at the 3-mg/kg/d dose (determined to be the maximum tolerated dose for cycle 1), 1 patient achieved a complete response and 7 patients had stable disease. No objective responses to single-agent oblimersen were seen among the 13 evaluable patients in the chemotherapy- naive subset. However, six of eight evaluable patients had a complete response (n = 2) or partial response (n = 4) on completion of oblimersen plus R-CHOP, an objective response rate of 75%. Based on preliminary safety analysis, combination therapy has been well tolerated, and adding oblimersen to R-CHOP did not appear to increase the expected toxicity of this regimen.
- Phase I/II Trial of Oblimersen Plus Rituximab in B-Cell Non-Hodgkin's Lymphoma-With oblimersen and rituximab demonstrating synergy against lymphoma in vitro and at least additive effects in xenografts, Pro and colleagues[25,30] from the University of Texas M.D. Anderson Cancer Center initiated a phase II trial of this combination in refractory/ recurrent B-cell non-Hodgkin's lymphoma. Oblimersen (3 mg/kg/d) is given as a 7-day continuous intravenous infusion every 2 weeks (on days 1 to 7, 15 to 21, and 29 to 35) in conjunction with six doses of once-weekly rituximab (375 mg/m2) on days 3, 8, 15, 22, 29, and 36. In the absence of disease progression or unacceptable toxicity, one additional course was permitted. Most of the first 16 enrolled patients had indolent histologies, the most common being follicular lymphoma (n = 8), and 14 patients had previously received rituximab. In the 11 evaluable patients, four responses were seen, for a preliminary response rate of 36%. One complete response was seen in a patient with stage IV mucosa-associated lymphoid tissue lymphoma with bone marrow involvement. All but one of the responders had a history of rituximab exposure. Accrual into this trial is ongoing as of March 2004, with a target enrollment of 120 patients.
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17. Jansen B, Schlagbauer-Wadl H, Brown BD, et al: Bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice. Nat Med 4:232-234, 1998.
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19. Auer RL, Corbo M, Fegan CD, et al: Bcl- 2 antisense (Genasense™) induces apoptosis and potentiates activity of both cytotoxic chemotherapy and rituximab in primary CLL cells (abstract 3358). Blood 98(suppl 1):808, 2001.
20. Loomis R, Carbone R, Reiss M, et al: Bcl-2 antisense (G3139, Genasense) enhances the in vitro and in vivo response of Epstein- Barr virus-associated lymphoproliferative disease to rituximab. Clin Cancer Res 9:1931- 1939, 2003.
21. Lacy J, Loomis R: Bcl-2 antisense (G3139, oblimersen) and rituximab enhance chemosensitivity of EBV-associated lymphoproliferative disease in vitro and in vivo (abstract 863). Proc Am Soc Clin Oncol 22:215, 2003.
22. Cotter FE, Auer R, Corbo M, et al: Oblimersen sodium (G3139) sensitizes malignant B-cells to alemtuzumab (Ab) induced apoptosis (abstract 910). Proc Am Soc Clin Oncol 22:227, 2003.
23. Leonard JP, Coleman M, Vose J, et al: Phase II study of oblimersen sodium (G3139) alone and with R-CHOP in mantle cell lymphoma (MCL) (abstract 2276). Proc Am Soc Clin Oncol 22:566, 2003.
24. Leonard JP, Hainsworth J, Bernstein S, et al: Genasense (oblimersen sodium, G3139) is active and well-tolerated both alone and with R-CHOP in mantle cell lymphoma (MCL) (abstract 490). Blood 102:143, 2003.
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29. Williams ME, Bebb DG, Tucker C, et al: Mantle cell lymphoma: In vitro and murine preclinical models utilizing five human cell lines (abstract 1378). Blood 100(11 pt 1):355, 2002.
30. Pro B: Phase II study of oblimersen and rituximab in patients with recurrent B-cell non-Hodgkin’s lymphoma. Protocol MDA-ID-02148, NCI-5808. Available at:http://cancer.gov/clinicaltrials/view_ c l i n i c a l t r i a l s . a s p x ? v e r s i o n = healthprofessional&cd rid=270426& protocolsearchid=842445. Accessed March 26, 2004.