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Review in translational hematology
Oncogenes as molecular targets in lymphoma
Ali Hachem and Ronald B. Gartenhaus
Bcl-2 family
The activation of oncogenes is essential to the development of
lymphoid malignancies. Frequently, these oncogenes are involved in relaying extracellular messages to the nucleus by
means of signaling pathways, causing changes in the cell
transcriptional patterns. The altered function of those molecules
associated with lymphomagenesis presents unique opportunities
to specifically disrupt those signaling pathways critical to
maintaining the malignant phenotype. Distinct oncogenic signaling cascades have been specifically associated with the different
subtypes of non-Hodgkin lymphoma. Many of the effectors
involved in these pathways may be essential for signaling
function, thus offering an expansive array of candidate targets.
Targeted therapeutic approaches are more likely to be specific
and associated with fewer deleterious side effects. They are also
expected to be highly active. Diverse and novel strategies have
been used to target the products of these oncogenes in nonHodgkin lymphoma; several have already reached the clinic
with promising preliminary results.
The non-Hodgkin lymphomas (NHLs) are clonal malignant
expansion of B or T cells that are at various stages of maturation.
Multiple classification schemes have been employed for these
diseases. Initially, these were based on the morphologic appearance of the neoplastic cell. However, as our understanding of the
immunology, cytogenetics, and molecular biology of the lymphomas broadened, these classification systems changed in order to
refine diagnoses and to provide a clinical perspective. Similarly,
treatment of the NHLs evolved to include targeted therapies
directed against specific signaling pathways critical to
lymphomagenesis.
Human cells possess genes that are homologous to retroviral
oncogenes. These normal cellular genes, referred to as protooncogenes, are involved in the regulation of proliferation, cell
cycle progression, and apoptosis. These proto-oncogenes have
the potential for becoming activated (oncogenes) by a variety of
genetic mechanisms including amplification, point mutations,
and chromosomal translocations. Frequently, these oncogenes
are involved in relaying extracellular messages to the nucleus by
means of signaling pathways, causing changes in the cell
transcriptional patterns. Any of the effectors involved in these
pathways may become oncogenic if overexpressed or inappropriately activated, leading to deregulation of cell growth. Therefore, intercepting involved signaling cascades at discrete points
holds therapeutic promise. Distinct oncogenes have been specifically associated with the different subtypes of NHL. This review
highlights promising targeted approaches to interfering with
oncogenic signaling pathways in NHL.
The Bcl-2 (B-cell CLL/lymphoma 2) protein is a member of a large
family of proteins involved in the regulation of programmed cell
death, primarily the intrinsic pathway. These proteins determine the
cellular decision to live or die by their ability to modulate
mitochondrial function. While some members, properly called
antiapoptotic, preserve mitochondrial integrity, other proapoptotic
members promote the release of cytochrome c from the intermembrane space of mitochondria (IMS). Following its release in the
cytosol, cytochrome c interacts with Apaf-1 and caspase-9, forming
an apoptosome, which activates caspase-9, which then activates
caspase-3, the effector protease leading to the cleavage of target
proteins and DNA fragmentation.1,2 All members in the Bcl-2
family contain one or more conserved Bcl-2 homology (BH)
domains but have considerable variation in overall structure and
function. The antiapoptotic members, which include Bcl-2 and
Bcl-XL, share sequence homology in 4 BH domains designated
BH1, BH2, BH3, and BH4. The proapoptotic members are divided
into 2 groups, the multidomain Bax and Bak, which contain BH1 to
BH3 domains, and the BH3-only proteins, including Bad, Bim,
Bik, and Bid, which only have the BH3 domain. While Bcl-2 and
Bcl-XL are integral membrane proteins that are predominantly
located in the outer mitochondrial membrane (OMM), the multidomain proapoptotic and the BH3-only proteins are primarily localized in the cytosol and are translocated to the mitochondria
following the activation of a death signal. The proposed model
through which these proteins interact to determine the cell fate is
intriguing and suggests that the BH3 segment of the entire
BH3-only protein acts as a death ligand and hence can be exploited
to develop synthetic peptide capable of inducing tumor cell death.2-4
Under normal conditions, Bax predominantly exists as a
monomeric protein within the cytosol or is loosely attached to
membranes. In response to an apoptotic stimulus, Bax translocates
to the mitochondria where it deeply inserts into the OMM,
undergoing a conformational change that induces its homooligomerization into large complexes, and facilitates release of cytochrome
c, a process that is mediated by the BH3-only members and
antagonized by Bcl-2 and Bcl-XL. The best understood example is
that of Bid, where the generation of a signal through the death
receptor (surface Fas and tumor necrosis factor [TNF] receptor)
results in the cleavage of BH3-only protein Bid by caspase-8. The
truncated version, tBid, then interacts with Bax or Bak and
facilitates its translocation to OMM and oligomerization. The
antiapoptotic property of Bcl-2 and Bcl-XL is mainly derived from
their ability to sequester the BH3-only molecules and to interact
From the University of Maryland Greenebaum Cancer Center, Baltimore, MD.
Blood First Edition Paper, March 31, 2005; DOI 10.1182/blood-2004-12-4621.
Supported in part through funding from the Veterans Administration Merit
Review Award (R.B.G.).
Reprints: Ronald B. Gartenhaus, University of Maryland Greenebaum Cancer
Center, 9-011 BRB, 655 West Baltimore St, Baltimore MD 21201; e-mail:
[email protected].
Submitted December 14, 2004; accepted March 27, 2005. Prepublished online as
© 2005 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
Role of Bcl-2 in lymphomagenesis
1911
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1912
HACHEM and GARTENHAUS
with different components of the mitochondrial membrane promoting its stability. However, the recent work by Letai and colleagues2,3 established that there are 2 classes of BH3-only proteins
involved in the execution of the apoptotic signal. By using BH3
peptidic sequences derived from representative BH3-only molecules, the authors described BH3 domains from Bid and Bim
capable of activating mutidomain proapoptotic Bak and Bax and
described BH3 domains from Bad and Bik that are incapable of
inducing Bak oligomerization but effectively bind to antiapoptotic
Bcl-2, displacing sequestered BID and BIM and making them
available to activate Bak or Bax. There are several proposed
mechanisms through which Bax, by means of its ability to interact
with membrane lipids and protein channels, promotes the release of
cytochrome c from the IMS. One mechanism, denoted as the
mitochondrial permeability transition (MPT), involves an increase
in the permeability of inner mitochondrial membrane (IMM) with
secondary depolarization of the IMM and swelling of the matrix
causing the rupture of the OMM. MPT is believed to be a
consequence of the opening of a complex pore system designated
as the permeability transition pore (PTP), which includes the
voltage-dependent anion channel (VDAC) in the OMM and the
adenine nucleotide translocator (ANT) in the IMM. Other mechanisms include the formation of de novo protein channels in the
OMM from Bax oligomers, Bax regulation of VDAC activity
without disruption of the ultrastructure or volume homeostasis of
mitochondria, and the formation of lipidic channels by Bax induction
of membrane instability without rupture.4
Enhanced Bcl-2 expression results most commonly from t(14;
18)(q32;q21) translocation that juxtaposes the Bcl-2 locus with the
immunoglobulin heavy chain (IgH) enhancer.1,5-8 t(14;18)(q32;
q21) is present in 70% to 90% of follicular lymphoma (FL) cases.1,8
Bcl-2 can be overexpressed in several B-cell malignancies through
mechanisms other than t(14;18) and may confer a worse prognosis.9-12 The overexpression of Bcl-2 and/or Bcl-XL is believed to
contribute to both progression of cancer cells and their resistance to
chemotherapeutic drugs and radiation therapy.13-16 While Bcl-2 is
important for the development of a B-cell malignancy, it is
probably as important for the continuation of the malignant clone.
The different aberrations accrued during tumorigenesis, like cell
cycle dysregulation, angiogenesis, and invasion, are very likely to
produce apoptotic signals. This can be viewed as an intrinsic
checkpoint mechanism that is triggered by these breaches to
terminate the abnormal cell. An apoptotic defect, like overexpression of Bcl-2, will be then necessary to oppose cell death signals.
From a therapeutic point of view, eliminating Bcl-2 will remove the
constraints on these inherent death signals and lead to cell demise.
This was explored by Letai and colleagues, who generated mice
expressing a conditional Bcl-2 gene and a constitutive c-Myc that
develop lymphoblastic leukemia They demonstrated that Bcl-2 was
required for the B-cell malignancy maintenance, because elimination of Bcl-2 resulted in rapid loss of the leukemic cells and
prolonged survival of the mice. They also showed that while BAD
BH3 did not affect mitochondria from nonmalignant pro-B lymphocytes, it resulted in release of cytochrome c from the lymphoblastic
leukemia cells, which is consistent with the theory that Bcl-2 must
be sequestering a large amount of continuously generated and
activated BIM.2
Targeting Bcl-2 with ASOs
Disruption of these antiapoptotic signals became feasible with the
advent of antisense oligonucleotides (ASOs). ASOs are short sequences of single-stranded DNA that are complementary to the
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
mRNA of interest. The binding of ASO to mRNA on a base-pairing
basis inhibits translation and also leads to cleavage of the mRNA by
RNase H.8,17-20 The result is a reduction in the encoded protein pool.
Additionally, there is evidence to support that ASOs have nonantisense
effects including immune stimulation and enhancement of natural
killer (NK) cell activity.8,21,22 Substituting the phosphodiester linkage of
the bases with phosphorothioates conferred ASOs nuclease resistance.
G3139, a prototype ASO targeting Bcl-2, down-regulated both mRNA
and protein expression in Bcl-2–overexpressing lymphoma cell lines.
After demonstrating therapeutic activity in tumor xenograft models,
G3139 entered phase 1 clinical trials, both alone and in combination
with chemotherapeutic agents.8,20,23,24 As a single agent in heavily
pretreated patients with relapsed NHL expressing Bcl-2, G3139 resulted
in responses and disease stability that correlated in most of these patients
with down-regulation of Bcl-2.25,26 Combining G3139 with chemotherapeutic drugs is based on both in vitro as well as vivo experiments
showing that targeting Bcl-2 with antisense therapy can promote
apoptosis following cellular damage induced by chemotherapy.27-32
A phase 1 trial in relapsed FL using combination cyclophosphamide and
G3139, in addition to other preclinical and clinical evidence, suggests
that G3139 synergizes with many cytotoxic and biologic agents against
a variety of hematologic malignancies including NHLs.8,33 Similar
synergism may be achieved by the simultaneous disruption of the
antiapoptotic (Bcl-2) and proliferation (mitogen-activated protein kinase
kinase/mitogen-activated protein kinase [MEK/MAPK]) pathways to
promote apoptosis and improve the cytotoxicity of conventional chemotherapeutic agents.34
Targeting Bcl-XL
There has also been interest in targeting another antiapoptotic molecule,
Bcl-XL, with ASOs. Bcl-XL and proapoptotic Bcl-XS are products of
different splicing of the same Bcl-X pre-mRNA. As a result, earlier ASOs
that targeted Bcl-XL also possessed activity against the proapoptotic
molecule Bcl-XS. Interestingly, these oligonucleotides were still able to
induce apoptosis in several cancer cells.20,35-37 Subsequently, investigators
looked for newer generations ofASOs that shift the splicing of the mRNA
into producing Bcl-XS. Several were described and tested in cancer cells
with promising results.20,38,39 Following the evidence that Bcl-2 and
Bcl-XL have different roles, despite being closely related,13,40-43 a Bcl-2/
Bcl-XL–bispecific ASO was designed with ability to induce in vitro
apoptosis in tumor cells with diverse tissue origins.44-47
Low-molecular-weight compounds
The use of low-molecular-weight compounds to inhibit Bcl-2 and
Bcl-XL function is a novel method with several potential therapeutic advantages. Small molecules inhibitors have the benefit of being
more stable with better bioavailability as compared with other
biologic and peptide inhibitors. They also have the ability to
penetrate the central nervous system blood brain barrier.13 The
search for small-molecule inhibitors of Bcl-2 and Bcl-XL became
possible with the understanding of the detailed atomic structure of
both the proapoptotic and antiapoptotic members. Both Bcl-2 and
Bcl-XL bind, through their BH3 binding pockets, to the BH3
domains on proapoptotic members, an essential step in the
execution of the antiapoptotic signal. The BH3 domains of these
proapoptotic members are also required for implementing the
apoptotic signal.13,48 Based on these findings, nonpeptide small
molecules can be designed to compete with the proapoptotic
members for binding to the BH3 binding pocket and subsequently
disrupting the function of the antiapoptotic molecules. The first of
these small molecules, HA14-1, was described by Wang et al.49
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
HA14-1 was shown to bind to the BH3 binding site on Bcl-2
protein and inhibit its interaction with Bak. At an inhibitory
concentration of 50% (IC50) of 9 ␮M, it was able to induce
apoptosis in human myeloid leukemia cells overexpressing Bcl2.13,49 Afterward, several more small-molecule inhibitors of Bcl-XL
and Bcl-2 were described and shown to bind to the BH3 binding
site. An excellent review by Wang et al describes several of these
small-molecule inhibitors of Bcl-2 and Bcl-XL and illustrates the
various approaches entailed in discovering them.13 The work by
Letai and colleagues describing 2 functionally different BH3-only
proteins opens the door on several therapeutic potentials. Bad-like
peptidomimetics or small molecules can be used to bind Bcl-2 and
displace Bid. Because an apoptotic signal is needed to activate the
BH3-only proteins, such agents may be harmless to normal tissues
but lethal to transformed cells where inherent apoptotic signals are
generated but blocked, for example, by excess Bcl-2 that sequesters
BID.2,3 Walensky and colleagues designed a hydrocarbon-stapled
BH3 helix that mimics Bid BH3 that activated apoptosis in vitro
and inhibited leukemia cells in vivo. The use of ␣, ␣-disubstituted
nonnatural amino acids containing olefinic side chains to tie amino
acids within the BH3 peptide improved the stability and cell
permeability of the mimics while preserving the helical structure,
critical for biologic activity, and specificity.50 Moreover, a novel
approach to inhibit Bcl-2 was recently reported by Cohen-Saidon
and colleagues. Using a semisynthetic human phage-display library, they generated a single-chain Fv antibody (scFv) against
Bcl-2. Attaching a basic domain of Tat to the antibody allowed the
single-chain antibody complex to penetrate into several types of
living cells. The anti–Bcl-2–scFv–Tat was specific and was directed to the BH1 domain of Bcl-2. The single-chain antibody was
also shown to decrease the level of Bcl-2, depolarize the mitochondrial membrane, and induce cell apoptosis in 2 types of mast cells
and in a human breast cancer cell line. This is a potentially promising
approach that allows the construction of highly specific single-chain
antibodies that are smaller than regular antibodies and thus easier to
introduce into cells.51
Cyclin D1
Bcl-1 proto-oncogene, also called CCND1 and PRAD1, has been
implicated in the pathogenesis of NHLs. Its gene product, cyclin
D1, plays a critical role in cell transition from the G to S phase in
response to mitogens. Cyclin D1 is 1 of at least 11 different
mammalian cyclins that are involved in the regulation of cell
cycle.52-54 Overexpression of cyclin D1 has been shown to shorten
the G1 phase and reduce the dependency of the cell on mitogens.55,56 The prototypical NHL with deregulation of the Bcl-1
oncogene is mantle cell lymphoma (MCL).
The development of MCL involves the acquisition of specific
cytogenetic abnormalities at the different phases of the disease.
t(11;14)(q13;q32) juxtaposes the cyclin D1 (Bcl-1, CCND1,
PRAD1) locus on chromosome 11 and the immunoglobulin heavy
chain IGH locus on chromosome 14. The translocation results in
overexpression of cyclin D1 with secondary cell cycle deregulation.52,57,58 Depending on which method is used, t(11;14)(q13;q32)
is identified in 50% to 70% of cases of MCL. However, cyclin D1
RNA and/or protein are detected in 90% to 100% of cases,
suggesting that alternative mechanisms including point mutations
may also result in increased expression.59-64
ONCOGENES AS MOLECULAR TARGETS IN LYMPHOMA
1913
Cell-cycle control
Cyclin D1, in association with cyclin-dependent kinase-4 (CDK4)
and CDK6, will induce the cell to enter the S (DNA synthesis)
phase by phosphorylating the retinoblastoma tumor-suppressing
protein (pRB), which binds to transcription factors, including
E2F.52,58 The process begins with phosphorylation of cyclin
D1–bound CDK4 by CDK-activating kinase (CAK).58,65,66 The
resultant active cyclin D–CDK4 complex phosphorylates pRB,
releasing E2F and other DNA binding proteins that are necessary
for transcriptional activation of genes required for cell transition
into the S phase.52,58,67,68 Several CDK inhibitors have entered
clinical trials, with others still undergoing preclinical testing. These
agents fall into 2 categories depending on their mechanism of CDK
inhibition. Flavopiridol, R-roscovitine (CYC202), and BMS387032 directly bind to the CDK catalytic subunit, while 7-hydroxystaurosporine (UCN-01) modulates the upstream regulatory
pathways needed for CDK activation.69
CDK inhibitors
Flavopiridol, a semisynthetic flavone derivative, is a protein kinase
inhibitor with demonstrated activity against a number of protein
kinases including cyclin D1 and CDK4. The drug exhibits in vitro
activity against a variety of cycling as well as noncycling tumor
cells including lymphomas.8 The antitumor effects of flavopiridol
include cell cycle arrest, induction of apoptosis, activation of
caspase-3, decreased expression of p53 protein, and downregulation of MCL-1.8 Flavopiridol is the first CDK inhibitor to be
tested in patients with MCL. Its efficacy was limited in both the
Dana-Farber Cancer Institute and National Cancer Institute (NCI)–
Canada trials but was also schedule dependent. In the NCI-Canada
trial, flavopiridol was administered at a dose of 50 mg/m2/d by
intravenous bolus for 3 consecutive days every 21 days. Although
no complete responses (CRs) were seen, 3 patients had a partial
response (11%), and 20 patients had stable disease (71%). At the
Dana-Farber Cancer Institute, patients received flavopiridol 50
mg/m2/d given as a 72-hour continuous intravenous infusion every
14 days. There were no clinical responses, and only 3 patients
maintained stable disease through treatment. This variability may
be related to the pharmacokinetics of flavopiridol and its increased
binding to human plasma proteins. This incited a phase 1 trial in
which patients with refractory chronic lymphocytic leukemia
received flavopiridol at total dose of either 60 mg/m2 or 80 mg/m2
weekly for 4 weeks on a 6-week schedule. Half the dose was
administered as a 30-minute intravenous bolus followed by the
other half given by 4-hour continuous infusion. The responses were
promising, but life-threatening tumor lysis occurred that led to the
interruption of trial.70-72
When tested against a number of cell lines, cytotoxic synergy
between flavopiridol and various chemotherapeutic agents could be
demonstrated based on a specific sequence of drug administration.
Thus, flavopiridol has possible utility for lymphoid malignancies, likely in combination with antineoplastic drugs.8,70 The
preclinical and clinical studies with most of the other CDK
inhibitors similarly demonstrated nonselectivity of these agents,
with activity against multiple kinase proteins and CDK members and modest antitumor activity as monotherapy.69,73 Additionally, although the 2 novel agents CYC202 and BMS-387032 are
more selective CDK inhibitors, none is a potent inhibitor of CDK4,
thereby limiting their use.69,74
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1914
HACHEM and GARTENHAUS
c-Myc
Role of c-Myc in lymphomagenesis
Another attractive candidate for targeted therapy is the c-Myc
protein that is highly expressed in Burkitt lymphoma (BL). The
up-regulation of the c-Myc protein is usually the result of
chromosomal translocations that place the c-myc gene located on
8q24 under the control of immunoglobulin (Ig) enhancers. The
most frequent translocation partner, detected in 80% of BL, is the
Ig heavy chain region on chromosome 14: t(8;14)(q24;q32). The
other 2 translocation partners include the kappa light chain locus on
chromosome 2, t(2;8)(p12;q24), and the lambda light chain locus
on chromosome 22, t(8;22)(q24;q11), occurring in 15% and 5% of
cases, respectively.1,75-79 The Ig enhancers are responsible for
the inappropriately high transcription rate of the translocated
c-myc allele.75,78-81
The detailed function of c-Myc is not yet completely understood, but it is believed to involve the promotion of both cell
proliferation and apoptosis.10,78,79 The activity of c-Myc depends on
its association with the Max protein. The formation of the
Myc-Max heterodimer promotes its binding to hexameric DNA
sequence termed the E-box, with subsequent interaction with Trrap
and recruitment of histone acetylases to activate transcription.
Other proteins including Mad and Mxi-1 are involved in downregulating the c-Myc protein function, both by binding to Max and
making it unavailable for c-Myc and through forming heterodimers
(Max-Mad, and Mad-Mad) that compete with Myc-Max complexes for binding to DNA target sites. The oncogenic properties of
c-Myc are probably the result of its constitutive expression leading
to increased levels of Myc-Max heterodimers.10,75,78 Multiple
cellular processes are perturbed by c-Myc up-regulation, some
involved in promoting malignant cellular transformation and others
implicated in antagonizing it. c-Myc is an attractive therapeutic
target for several reasons. It is highly expressed only in cycling
cells, and thus the toxicities expected from its inhibition should be
limited to highly dividing tissues like the gastrointestinal tract and
bone marrow. Also, c-Myc is the convergence point of several
upstream signaling pathways that can be affected by genetic
mutations in various tumors. Finally, several in vitro studies have
shown that while c-Myc overexpression can sensitize cells to the
cytotoxic effects of some chemotherapeutic agents, inhibiting
c-Myc can enhance the sensitivity of tumor cells to several other
antineoplastic agents.82-87
Targeting c-Myc at the level of RNA and DNA
Down-regulation of c-Myc by ASOs has been tested in a large array
of cancer cells including hematologic and solid tumors.20 Reduction in cell proliferation occurred when ASOs were used either
alone or in combination with antineoplastic agents.20 Using in
vivo models of solid tumors, a better antitumor activity of ASOs
was observed when they were administered after the chemotherapeutic agent.20,88,89
Newer generations of ASOs with greater resistance to endogenous nucleases, improved cellular uptake, and increased stability
with their target sequence are being developed.82 Most promising
are the phosphorodiamidate morpholino ASOs (PMOs) with documented activity against c-Myc expression.89-92
Several other related approaches have been developed to
interfere with c-Myc function. Peptide nucleic acids (PNAs) and
locked nucleic acids (LNAs) are capable of complexing with both
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
DNA and RNA and subsequently inhibiting transcription and
translation.82,93-95 Triple helix–forming oligonucleotides (TFOs)
are purine-rich or mixed purine/pyrimidine-rich oligonucleotides
that target c-Myc gene directly. TFOs stably bind to duplex DNA
within the major groove and prevent the binding of transcription
factors.82,87 Several lymphoma cell lines were treated with these
TFOs in vitro, with secondary cell arrest followed by apoptosis.96,97
Some researchers exploited the DNA secondary structure to use
quadruplex-forming oligonucleotides to inhibit transcription.98,99
Others identified a cationic porphyrin molecule that can specifically interact with the P1 promoter and stabilize DNA in a
quadruplex structure that represses transcription.100 Targeting cMyc by ribozymes, which are RNA molecules capable of degrading specific target RNA, and short interfering RNAs (siRNAs) are
promising techniques, yet the optimal delivery system still needs to
be determined.82,87 Introducing ribozymes into tumor cell lines by
means of retroviruses down-regulated c-Myc gene.101,102 c-Myc
inhibition by siRNAs has not yet been reported.82 Another untested
strategy is the use of decoy oligonucleotides (DOs) that contain
c-Myc DNA binding sites. DOs are short, double-stranded oligonucleotides with DNA binding sites that compete with the real
genomic sites for binding to transcription factors.82 Recently,
low-molecular-weight compounds were effectively used both in
vitro and in vivo to disrupt the formation of Myc-Max heterodimers.82,87,103,104 However, high concentrations of these compounds were needed to be effective, which carries the potential for
significant toxicities.82 An alternative approach to target the
Myc-Max interaction is to shift the heterodimerization balance by
ectopically inducing the overexpression of Mad family members.82,105,106
Interference with c-Myc function may also be potentially achieved
at the level of the TRAAP–histone acetylase complex.82,87
NF-kB family
The nuclear factor–kB (NF-kB) pathway is active in several of the
non-Hodgkin lymphomas and confers cancer cells with a survival
advantage by inhibiting apoptosis. The term “NF-kB” is used to
describe several dimeric transcription factors that belong to the
c-Rel family.107,108 Some factors are retained in the cytoplasm as
mature products by a specific inhibitor called the inhibitor of kB
(IkB), while others are derived by proteolytic cleavage of larger
intracytoplasmic precursors. After activation, NF-kB translocates
from the cell cytoplasm to the nucleus, where it participates in gene
transcription.107-110 Based on murine studies, NF-kB is believed to
play a central role in the regulation of innate and adaptive
immunity, inflammatory response, lymphoid organ development,
as well as apoptotic inhibition.108,111-113 NF-kB activity is tightly
controlled during normal B-cell development, where different
NF-kB heterodimers are differentially expressed in the B-cell
lineage, emphasizing their role as stage-specific regulators of
B-cell development, survival, division, and immunoglobulin expression.114 Additionally, the NF-kB pathway is one of the key
transcription factors activated by B-cell receptor (BCR) signaling.
Engagement of BCR with secondary activation of NF-kB is critical
for the survival of quiescent B cells in the periphery, allowing them
to evade apoptosis; it is equally important for the survival and
expansion of mitogen-activated B cells by increasing cell division
and decreasing apoptosis.114-116 The NF-kB function of inhibiting
programmed cell death is derived from its ability to induce the
expression of antiapoptotic factors including cellular inhibitor of
apoptosis (c-IAP), Fas-associating protein with death domain–like
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
interleukin-1–converting enzyme (FLICE) inhibitory protein (cFLIP), TNF receptor (TNFR)–associated factor 1 (TRAF1) and
TRAF2, as well as members of the Bcl-2 family. NF-kB is a potent
IAP that is triggered by either death receptors or the mitochondrialdependent pathway, making it an attractive target for antitumor
therapy (Figure 1).108
Role of NF-kB in lymphomagenesis
The deregulation of NF-kB pathway plays a role in the development of several NHLs. Approximately half of extranodal marginal
zone B-cell lymphomas of mucosal-associated lymphoid tissue
(MALT) type harbor 1 of 3 specific chromosomal translocations
that result in a dramatic increase in NF-kB activity.117 Constitutive
activation of NF-kB is also a common molecular feature for other
B-cell malignancies such as Hodgkin lymphomas and mediastinal
B-cell lymphomas where amplifications of c-REL are frequently
seen.118,119 About 23% of diffuse large B-cell lymphomas (DLBcls)
show amplification of the c-REL proto-oncogene (2p12-16).120
However, based on recent work by Houldsworth and colleagues in
DLBcl, the amplification of the c-REL locus did not necessarily
correlate with activation of NF-kB pathway. No significant association was found between the Rel copy number, REL nuclear
accumulation, and the differential expression of NF-kB target
genes, suggesting that Rel may not be the functional target of 2p
amplification.121 Nonetheless, constitutive activation of NF-kB is
critical for one subtype of DLBcl. DLBcl encompasses 2 distinct
diseases that differ in the expression of hundreds of genes, based on
ONCOGENES AS MOLECULAR TARGETS IN LYMPHOMA
1915
gene expression profiling, and also have markedly different clinical
outcome. One subtype, germinal center B-cell–like (GCB) DLBcl
that resembles normal germinal center B cells, has a good
prognosis, whereas activated B-cell–like (ABC) DLBcl that resembles BCR-stimulated blood B cells has a poor outcome.
Constitutive activation of the NF-kB pathway is required for
survival of ABC DLBcl as illustrated in the work by Davis and
colleagues. The ABC subtype expressed known NF-kB target
genes and showed high levels of NF-kB DNA binding activity, with
constitutive IkB kinase (IKK) activity and IkB degradation,
validating the constitutive activation of NF-kB pathway. On the
other hand, this molecular profile was not observed in the GCB
subtype. Inhibition of the NF-kB through the introduction of a
super-repressor IkB that cannot be phosphorylated by IKK was
selectively toxic to ABC but not GCB DLBcl, causing apoptosis
and G1-phase cell cycle arrest.122
The translocation t(11;18)(q21;q21) is the most common translocation, occurring in 18% to 35% of MALT lymphomas cases. It
results in the juxtaposition of the apoptosis inhibitor-2 (API2) gene
on chromosome 11 and MLT (MALT lymphoma–associated translocation) gene on chromosome 18. API2 encodes for an IAP
protein, and MLT encodes for the MALT1 protein. IAP family
proteins inhibit activated caspases, resulting in arrest of programmed cell death, while MALT1 contains a caspaselike domain
with a death domain that recruits the protein to the cell death
signaling pathway.117,123,124 The AIP2-MALT1 fusion protein is
also capable of directly activating IKK, a function that requires the
Figure 1. Inhibition of molecular targets for the treatment of non-Hogkin lymphomas. Activation of oncogenes is a critical event in the development of non-Hodgkin
lymphomas. Oncogenic proteins are key effectors in signaling pathways that regulate cell proliferation, cell cycle progression, and apoptosis. Their inhibition via specific small
molecules and pharmacologic agents offers a novel approach in the targeted treatment of non-Hodgkin lymphomas. While several have already entered clinical trials with
promising preliminary results, other agents are still awaiting preclinical validation. LMW indicates low molecular weight; CAK, CDK-activating kinase.
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1916
HACHEM and GARTENHAUS
presence of both proteins of the fusion product, with resulting
increased translocation of NF-kB into the nucleus.117,124 The other
translocations reported in MALT lymphomas are t(1;14)(p22;q32),
which brings the entire coding region of the Bcl-10 gene under the
control of enhancer elements of the IGH locus on chromosome 14,
and t(14;18)(q32;q21), with resultant IGH-MLT rearrangement.
Bcl-10 normally activates the NF-kB pathway through binding to
MALT1 and subsequent activation of IKK complex. The upregulation of Bcl-10 in t(1;14) or MALT1 in t(14;18) will increase
the amount of the protein required to activate IKK with secondary
increase in translocation of NF-kB to the nucleus.117,124,125
Proteasome inhibitors
Several molecular therapeutic approaches have been designed to
inhibit NF-kB. Among these, the proteasome inhibitors have shown
promising results. The proteasome is a large protease complex that
possesses a central role in cellular metabolism through the elimination of intracellular proteins that have been tagged for degradation.
The process requires the initial modification of the proteins by
conjugation to ubiquitin prior to proteolytic degradation within the
core of the proteasome. Among the targets for ubiquitination and
proteolysis are proteins involved in cell cycle regulation, transcription factor activation, apoptosis, and control of chemotaxis,
angiogenesis, and cell adhesion.8,126 Inhibition of NF-kB is one of
several mechanisms through which proteasome inhibitors derive
their antitumor effect. These mechanisms have been best studied in
the context of multiple myeloma cells lines treated with PS-341 or
bortezomib, a boronic acid peptide that is a specific inhibitor of the
26S proteasome. Specific inhibition of NF-kB resulted in apoptosis
via mitochondrial cytochrome c release and caspase-9 activation,
decreased expression of angiogenic cytokines and adhesion molecules, and growth factors such as interleukin-6 (IL-6). Several
other signaling molecules were also affected by PS-341 independent of NF-kB inhibition, including the induction of apoptosis via
activation of c-jun NH2-terminal kinase (JNK) and a Fas/caspase8–dependent apoptotic pathway, increasing p53 activity by p53
protein phosphorylation and degrading Mdm2, cleaving the DNA
protein kinase catalytic subunit and ataxia telangiestasia mutated
protein (ATM) involved in DNA repair, activation of BAX
proteins, and accumulation of CDK inhibitors p21 and p27.
Additionally, PS-341 down-regulates several effectors of the
protective cellular response to genotoxic stress, like topoisomerase
II, restoring the sensitivity of myeloma cells to DNA-damaging
chemotherapeutic agents.127-129
As a single agent, bortezomib induced apoptosis in many types
of human cancer cell lines, including those derived from hematologic malignancies as well as in a murine model of BL.8,130
Moreover, the drug demonstrated marked synergy with chemotherapeutic agents like irinotecan, gemcitabine, and doxorubicin, and
ionizing radiation.8,126 Lactacystin, a bacterial metabolite with a
natural ability to inhibit proteasome, has been tested in DLBcl cells
with proven ability to prevent IkB degradation.131 Constitutive
activation of NF-kB in MCL has been observed by Lan Pham et
al.132 Treatment of the MCL cells with PS-341 or the specific IkBa
inhibitor BAY 11-7082 resulted in decreased NF-kB DNA binding
activity and stabilization of IkB. Cell growth inhibition occurred
through both cell cycle arrest and induction of apoptosis.132
Proteasome inhibition in MCL may carry therapeutic potentials
through other pathways unrelated to NF-kB inhibition. There are
data to suggest that the low levels of the CDK inhibitor p27 may be
a consequence of increased proteasome-mediated degradation.133
This is supported by experiments in cell models where proteasome
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
inhibition resulted in up-regulation of p27.126,132 In human phase 1
and 2 trials, bortezomib was well tolerated, with toxic effects being
limited to diarrhea, thrombocytopenia, neutropenia and peripheral
neuropathy.134 Currently, bortezomib has a definite role in the
treatment of myeloma. In a phase 2 trial in patients with relapsed
and refractory myeloma, bortezomib was administered at a dose of
1.3 mg/m2 intravenously twice weekly for 2 weeks every 21 days,
with addition of dexamethasone of 20 mg on the day of and day
after each dose of bortezomib for patients with progressive disease
after 2 cycles. The rate of response was 35% and included a CR of
4%. The most common adverse events were thrombocytopenia,
fatigue, peripheral neuropathy, and gastrointestinal side effects.135
According to preliminary data from phase 1 trials, several patients
with NHLs are experiencing responses to bortezomib.126,131,136
Similar encouraging results were seen in a preliminary report from
a phase 2 trial by O’Connor et al37 in patients with indolent
lymphomas, particularly FL and MCL, and in a phase 2 study in
relapsed or refractory B-cell NHL showing overall response rate of
41% among patients with MCL.126,137,138 A trial combining bortezomib with EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin) in patients with NHLs is also being
studied.134
Other inhibitors of the NF-kB pathway
Inhibiting the NF-kB pathway has been also achieved through
other routes. One group identified curcumin (diferuloylmethane) as
an effective agent that blocks constitutive NF-kB in myeloma
cells.139 Curcumin, which has been shown to possess chemopreventive activity against various tumors in animal models, is a safe
agent in humans as demonstrated in phase 1 clinical trials.140
Curcumin inhibited IKK activity with secondary down-regulation
of NF-kB and activation of caspases leading to apoptosis of
myeloma cells.139 G06976, protein kinase C inhibitor, has been
shown to inhibit IKK activity in estrogen receptor–negative (ER-)
human and mouse mammary adenocarcinoma cell line with
demonstrable antitumor activity in vivo (Table 1).141
PI3K/Akt/mTOR pathway
Protein kinases are generally classified as either tyrosine or
serine/threonine specific, although some are capable of phosphorylating both tyrosine and serine/threonine residues. The proteintyrosine kinases (PTKs) include several families of transmembrane-spanning receptors (receptor protein tyrosine kinases
[RPTKs]) as well as cytoplasmic, nonreceptor PTKs. The
catalytic activity of PTKs is tightly controlled by multiple
autoinhibitory mechanisms, and the oncogenic transformation
of PTKs can be viewed as a consequence of the relief of these
normal autoinhibitory constraints. The loss of these regulatory
restraints can result from several mechanisms, including chromosomal translocations, gain of function (GOF) mutations or small
deletions, and gene amplification.142 However, malignant transformation of cells can also arise from chromosomal perturbations that involve the downstream effectors of PTKs, including
the Ras–Raf–extracellular signal-regulated kinase (Ras-RafERK) and the lipid kinase phosphoinositide 3-OH kinase (PI3K)
signaling pathways. PI3K is a family of proteins that phosphorylate the 3⬘-OH group of the inositol ring in inositol phospholipids, and hence they are lipid kinases. This protein family can be
subdivided in 3 classes, but class I is the most studied of PI3Ks
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
ONCOGENES AS MOLECULAR TARGETS IN LYMPHOMA
1917
Table 1. Targets for oncogenic signaling pathway inhibition
Oncogene/pathway
Bcl-2
NHL subtype
Targeting agents
Follicular lymphoma, diffuse large B-cell lymphoma
Oligonucleotides
G3139 (Bcl-2-specific ASO)
Bispecific ASOs (Bcl-2 and Bcl-XL)
Low molecular weight compounds
HA14-1
Hydrocarbon-stapled BH3 helix BID-like
Antibodies
Anti-Bcl-2-scFv-TAT
Bcl-XL
?
Oligonucleotides
ASOs
Bispecific ASOs (Bcl-2 and Bcl-XL)
ASOs that shift splicing of pre-mRNA
Low molecular weight compounds
Cyclin D1
Mantle cell lymphoma
CDK inhibitors
c-Myc
Burkitt lymphoma
Oligonucleotides
Flavopiridol, R-roscovitine (CYC202), BMS-387032, UCN-01
ASOs, PMOs, PNAs, LNAs, TFOs
Quadruplex-forming oligonucleotides
Ribozymes, siRNAs, DOs
Low molecular weight compounds
Cationic porphyrin that binds P1 promotor
NF-kB
MALT lymphoma, diffuse large B-cell lymphoma, multiple myeloma,
mediastinal B-cell lymphoma, Hodgkin lymphoma
Proteasome inhibitors
Bortezomib (PS-341), lactacystin
IkB␣ inhibitors
BAY 11–7082
IKK inhibitors
Curcumin, G06976 (PKC inhibitor)
PI3K/Akt/mTOR
?
PI3K inhibitors
Wortmanin, LY294002
Akt inhibitors
Phosphatidylinositol analogs
OSU-03012 (celecoxib analog)
OSU-03013 (celecoxib analog)
mTOR inhibitors
rapamycin, RAD001, AP23573, CCI-779
Raf
?
Oligonucleotides
ISIS 5132 (ASO)
Raf kinase inhibitors
BAY 43–9006
Bcl-6
Diffuse large B-cell lymphoma, follicular lymphoma
Bcl-6 peptide inhibitor (BPI)
siRNA
Trichostatin A (HDAC inhibitor)
Nicotinamide (SIR-2 inhibitor)
Multiple signaling pathways are abnormally activated in non-Hodgkin lymphomas and are considered to play a major role in lymphomagenesis. The events leading to the
perturbed oncogenic activity of these pathways are diverse but include several well-defined chromosomal translocations. While some have been exclusively associated with
specific NHL subtypes, others may prove to be more global. The targeting agents used to interfere with these pathways for antilymphoma effect are wide ranging and employ
different mechanisms of action (see text for details). They can be directed against the molecule of interest specifically or other effectors that are critical in the respective
pathway. ASOs indicates antisense oligonucleotides; CDK, cyclin-dependent kinase; PMOs, phosphorodiamidate morpholino ASOs; PNAs, peptide nucleic acids; LNAs,
locked nucleic acids; TFOs, triple helix-forming oligonucleotides; siRNAs, short interfering RNAs; DOs, decoy oligonucleotides; IkB, inhibitor of kB; and IKK, IkB kinase.
and the most important in hematopoietic cells. Class I PI3Ks are
heterodimers consisting of a catalytic subunit (p100) and a
regulatory subunit, and their preferred substrate is phosphoinositide (4,5) diphosphate (PtdIns(4,5)P2). The activation of class I
PI3Ks starts with their recruitment to the plasma membrane,
where they associate with an activated RPTK or its substrates.
These interactions lead to allosteric activation of the catalytic
subunit with secondary production of PtIns(3,4,5)P3 and
PtIns(3,4)P2. The phospholipid products of PI3K activate
downstream targets, including 3⬘-phosphoinositide-dependent
kinase (PDK-1), Akt/protein kinase B (PKB), and PKC. The
activation of Akt also requires its interaction with PDK with
subsequent phosphorylation of threonine 308 and serine 473 in
Akt. Activated Akt is then translocated to the nucleus where
several of its substrates reside, including regulators of glucose
transport/metabolism, apoptosis, and cell cycle transit. In this
manner, the PI3K/PDK-1/Akt pathway is viewed as the controlling mechanism allowing the cell to undergo cell cycle transit
provided that there are sufficient nutrients. Several phosphatases
negatively regulate the PI3K/PDK-1/Akt pathway, including the
tumor-suppressor gene PTEN. High levels of Akt activation
have been described in multiple cancers including NHLs.142-147
The transforming effects of PI3K/Akt are dependent on the
regulation of another downstream target, the mammalian target
of rapamycin (mTOR), also called FK506-binding protein
(FKBP)–rapamycin-associated protein (FRAP). Activation of
mTOR results in phosphorylation of p70S6 kinase (p70S6K),
required for ribosome biogenesis, and 4E-BP1 translational
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HACHEM and GARTENHAUS
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
repressor with secondary release of eIF-4E that is critical for
assembly of a translational complex. The end result is capdependent translation of proteins required for cell cycle transit,
specifically cyclin D1 and c-Myc.142,147,148 However, there is
evidence that cells are capable of expressing these proteins
through cap-independent mechanisms, providing a salvage
pathway that allows the cells to survive during starvation
conditions.147 This is important because it implies that inhibiting
mTOR may not lead to sufficient decreased levels of cell cycle
proteins leading to G1 arrest. Gera et al147 demonstrated in cell
cultures and mouse xenografts with high Akt activity that mTOR
inhibitors, rapamycin and CCI-779, resulted in cell cycle arrest
through down-regulation of cyclin D1 and c-Myc. These data
suggested simultaneous inhibition of both the cap-dependent
and salvage pathways. When tested in cells with a low Akt
activity, mTOR inhibitors resulted in up-regulation of cyclin D1
and c-Myc expression through activation of the salvage or
cap-independent translation pathway.147
against a variety of solid tumors.162,163 Preclinical studies in human
multiple myeloma cells using a xenograft model confirmed the
activity of CCI-997 in myeloma cells through inhibition of
proliferation, angiogenesis, and induction of apoptosis. The sensitivity of the myeloma cells to the growth inhibitory effects of
CCI-997 was also shown to correlate with high Akt activity.164
Wendel and colleagues used a mouse model of B-cell lymphoma to
demonstrate that activated Akt promoted lymphomagenesis by
disabling apoptosis. The phenotype was resistant to conventional
chemotherapy agents when used alone. However, the combination
with rapamycin restored the cells sensitivity to these drugs. Similar
activity of rapamycin was not demonstrated in chemoresistant
lymphoma cells that depended on Bcl-2 for survival, again
underlying the selective activity of mTOR inhibitors.165
Role of PI3K/PDK-1/Akt/mTOR pathway in lymphomagenesis
The Ras/Raf/Mek/Erk or MAPK pathway is another potential
target for the development of new therapeutic agents in NHLs. The
MAPK pathway possesses dual roles in promoting cellular proliferation. It regulates the production of various cytokines and growth
factors, and then it relays the message generated by these cytokines
and growth factors by mediating primarily mitogenic and antiapoptotic signals.166 The Ras proteins are located at the inner surface of
the plasma membrane and are inactive in their guanosine diphosphate (GDP)–bound state but are active in the guanosine triphosphate (GTP)–bound state. By means of cell surface receptor
tyrosine kinases (RTKs) and other membrane receptors, diverse
extracellular ligand–mediated stimuli transiently promote the formation of the active GTP-bound state as regulated by guanine
nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). The interaction between activated Ras and Raf leads
to localization of Raf to the plasma membrane. Activated Raf
subsequently phosphorylates and activates dual-specificity kinase
MEK (MAPK kinase) that phosphorylates and activates the
ERK1/2 family of MAPK. The activated ERKs then translocate to
the nucleus where they phosphorylate transcriptional regulatory
proteins.167 Constitutive activation of the MAPK pathway with
subsequent deregulation of the cell proliferation program can occur
via 3 main routes. These include activating mutations in Ras,
activating mutations in Raf, as well as up-regulation of RTK. The
MAPK pathway is involved in the regulation of normal hematopoietic cell proliferation and differentiation.166 Its role in the pathogenesis of lymphomas is suggested by studies of lymphoma-derived
cell lines.168 One study suggested that rituximab sensitizes NHL
B-cell lines to chemotherapeutic agents by down-regulaing the
Erk1/2 signaling pathway.169 Another MAPK implicated in lymphomagenesis, specifically transformed FL and EBV-related lymphomas, is p38 MAPK.170-172 Targeting the MAPK pathway has
already been accomplished at 2 important levels, Ras and Raf. The
arsenal against Raf currently includes ASO of Raf-1, ISIS 5132,
that has been tested in phase 1 trials and the Raf-1 kinase inhibitor
BAY 43-9006 (Bayer, West Haven, CT).173,174 BAY 43-9006 was
shown to have in vitro activity against a multitude of kinases
including wild-type and mutant (V599E) B-Raf and Raf-1 kinase
isoforms as well as vascular endothelial growth factor receptor-2
(VEGFR-2). The antitumor activity of BAY 43-9006 in NHLs is
awaiting the results of phase 2 trials.174,175
The PI3K/PDK-1/Akt/mTOR pathway plays an important role in
lymphomagenesis. Three Akt members are associated with different types of malignancies, but Akt1 specifically has been mapped to
chromosome 14q32, a region frequently involved in translocations
in leukemia and lymphoma.143,149 The role of PI3K in cell survival
and proliferation of immortalized B lymphocytes was examined in
Burkitt lymphoma (BL) cells and Epstein-Barr virus (EBV)–
immortalized B cells, lymphoblastoid cell lines (LCLs), which are
a model for the cells in posttransplantation lymphoproliferative
disease (PTLD). Treatment of these cells with the PI3K inhibitor,
LY294002, induced apoptosis in BL cells and cell cycle arrest at G1
phase in LCL cells.150 Some investigators suggested that induction
of apoptosis in B-cell lymphoma cell lines by anti-IgM and
anti-IgD is mediated through inhibitory effects on the PI3K
pathway.151 Others investigated apoptosis in DLBcl as mediated by
inhibition of the PI3K/Akt pathway.152 There are also data showing
interplay between eIF-4E and c-Myc in promoting lymphomagenesis153 as well as data to suggest that the T-cell leukemia 1 (TCL1)
proto-oncogene, which is highly expressed in a variety of NHLs,
induces B-cell malignancies by potentiating the activity of Akt.154
Recently, there was a report that inhibition of apoptosis by
activation of the AKT signaling pathway is an important mechanism involved in a subset of NHLs associated with MCT-1
overexpression.155 Several agents have been investigated to block
this important pathway in search of new dugs for cancer therapy.
Although some showed disappointing results, other agents proved
to be promising.
Inhibitors of mTOR
Inhibition of PI3K catalytic subunit with wortmannin and LY294002
carries several limitations. These include the very short half-life of
wortmannin, nonspecificity of LY294002, and the potential for
disrupting glucose transport and metabolism in normal cells.156,157
Several Akt inhibitors have been developed and shown to inhibit
the growth of various cancer cell lines.158-161 There are 3 mTOR
inhibitors in clinical development, RAD001, AP23573, and CCI779. All 3 are rapamycin analogs that act through binding to the
intracytoplasmic receptor FK506-binding protein-12 (FKBP12)
with subsequent inhibition of the kinase activity of mTOR. In phase
1 and 2 trials, the mTOR inhibitors demonstrated clinical activity
The MAPK pathway
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BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
Bcl-6
Role of Bcl-6 in lymphomagenesis
The Bcl-6 protein is a potent sequence-specific transcriptional
repressor that is involved in the regulation of lymphoid development. Bcl-6 is normally expressed in B cells within the germinal
centers (GCs) but is absent in pre-GC and differentiated progenies.
This finding highlights the function of Bcl-6; it is needed to license
the B cells for the GC reaction (class switch recombination and
somatic hypermutation) but is subsequently down-regulated to
permit B-cell exit from GCs and terminal differentiation.1,10,176
Consistent with its role in modulating B-cell responses in GCs,
Bcl-6 has been shown to repress signal transducer and activator of
transcription-2 (STAT-6)–dependent IL-4–induced induction of Ig
epsilon transcripts and expression of the p27kip1, cyclin D2, and
Blimp-1 target genes, with the later being involved in B-cell
differentiation. On the other hand, Bcl-6 protein, in response to
activation of BCR, is down-regulated by MAPK-induced phosphorylation, which marks Bcl-6 for degradation by the ubiquitinproteasome pathway. Also, Bcl-6 RNA is down-regulated by CD40
receptor signaling.1,176 The continued expression of Bcl-6, by
means of chromosomal translocations of 3q27, will result in
repression of genes involved in B-cell activation, differentiation,
cell cycle arrest, and apoptosis, contributing to the development of
NHL. The deregulation of Bcl-6 expression is the result of
promoter substitution, wherein the 5⬘ noncoding sequence in the
Bcl-6 gene is truncated and promoters from immunoglobulin (Ig)
genes or a non-Ig gene are placed adjacent to the intact coding
exons of Bcl-6, preventing normal down-regulation of Bcl-6
transcription that occurs during B-cell terminal differentiation.
Such translocations are reported in 20% to 40% of DLBcl and 6%
to 14% of FL (and have also been observed in cases of MCL,
MALT lymphoma, Hodgkin lymphoma, and chronic lymphocytic
leukemia/small lymphocytic lymphoma).1,10,176 Yet, somatic mutations in the 5⬘ noncoding region of the gene may also cause
deregulated expression of Bcl-6. Generally, mutations in this
region can be found in one third of normal GC and memory B cells
and up to 75% of DLBcl cases, but specific nucleotide substitutions
capable of disrupting a negative autoregulatory circuit have been
associated with DLBcl.176
Targeting Bcl-6
Interfering with Bcl-6 function for potential therapeutic benefits
in NHL has been explored at several levels. The regulation of
gene expression by Bcl-6 involves the recruitment of corepressor complexes by means of its broad complex/tramtrack/bric-abrac (BTB) domain. A lateral groove motif specific to Bcl-6 BTB
domain binds to a conserved sequence called the BTB binding
domain (BBD) found in silencing mediator of retinoid and thyroid
receptors (SMRTs), nuclear receptor corepressor (N-CoR), and
Bcl-6 corepressor (BCoR). Bcl-6 also binds through its middle
region to metastases-associated genes (MTA3) protein, a subunit of
nucleosome remodeling and deacetylase (NuRD) complex that
contains a histone deacetylase (HDAC). These repressor programs
may have different sets of target genes with different functions,
where the former permits the B cells to undergo rapid proliferation
and survive the DNA damage occurring as part of the GC reaction
and the latter blocks differentiation.177 Using crystallographic data,
peptides were designed to disrupt the interaction between BTB
lateral groove and BBD motif. The Bcl-6 peptide inhibitors (BPIs)
ONCOGENES AS MOLECULAR TARGETS IN LYMPHOMA
1919
efficiently penetrated cells, localized to the nucleus, and competed
with corepressors for binding to Bcl-6 lateral groove, with no
binding to other BTB proteins. They were also shown to disrupt
Bcl-6–mediated transcriptional expression with secondary reactivation of target genes and induce apoptosis and cell cycle arrest in
Bcl-6–positive DLBcl cells. Similar results were also obtained by
using siRNA. Targeting the NuRD repression mechanism may also
possess therapeutic potentials.177 Recent observations have found
that the acetylation of Bcl-6 inhibits its function and that 2
deacetylase pathways including HDAC-2 and silent information
regulator SIR-2 control the level of Bcl-6 acetylation. Using
HDAC inhibitor trichostatin A and SIR-2 inhibitor nicotinamide
can induce Bcl-6 acetylation and therefore inhibit its function.176
Conclusions and future directions
The recent interest in developing new antineoplastic treatments,
based on the molecular features of particular tumors, has been
driven by several recent accomplishments in understanding the
pathogenesis of cancer. Among those was the discovery of the role
played by activation of oncogenes and inactivation or loss of tumor
suppressor genes in tumorgenesis. The proteins encoded by these
genes are mostly involved in regulation of cell proliferation, cell
cycle control, and apoptosis, which make them ideal targets for the
development of cancer therapy. Targeted therapeutic agents are
therefore more likely to be specific and associated with fewer
deleterious side effects. They are also expected to be highly active.
Because tumor cells accumulate additional genetic changes, multiple factors are likely to contribute toward the abnormal proliferation of cancer cells. This implies that disruption of multiple cellular
pathways may be needed to achieve therapeutic efficacy. While no
single pathway can solely determine whether a cell will survive or
undergo programmed death, the ultimate fate of the cell will
depend on the complex balance of multiple antiapoptotic to
proapoptotic factors As a consequence, targeted therapy aiming at
shifting the balance toward the proapoptotic pathway may require
the interruption of multiple antiapoptotic signals to be effective.
The new paradigm may involve combining molecular targeted
therapy with conventional cytotoxic therapy. Currently, there is a
large arsenal of targeted agents of potential use in the treatment of
NHLs. Some are still in preclinical testing, but others have already
entered the clinical arena.
Oligonucleotide-based therapy, including ASOs, DOs, and
other oligonucleotide-mediated therapy, has several unresolved
problems. They all share a short half-life and undergo extensive
degradation by endocytosis and nucleases. DOs additionally are
required to translocate from the cytoplasm into the cell nucleus to
exert their effects. The design of a suitable vector to ensure efficient
delivery of the oligonucleotides to target cells and enhancing their
efficacy and specificity are also critical to minimize nonspecific
effects of high doses of oligonucleotides. Several steps have
already been undertaken to circumvent these barriers, including the
chemical modification of oligonuleotides.
Proteasome inhibition holds promise as a novel effective
therapy for several subtypes of NHLs with minimal toxicities.
Intuitively, it would seem that proteasome inhibition should be
associated with significant toxicities to normal tissues, because
the ubiquitin-proteasome pathway is essential to the survival of
all cells. However; several reports have documented the greater
susceptibility of transformed cells to proteasome inhibition as
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1920
BLOOD, 15 SEPTEMBER 2005 䡠 VOLUME 106, NUMBER 6
HACHEM and GARTENHAUS
compared with nonmalignant cells.178,179 Several factors probably contribute to such selectivity, but the exact mechanisms are
not fully understood. The proteasome inhibitor is also very
effective in inducing cell cycle arrest and apoptosis in different
types of cancer cells. This is derived from its ability to affect
many pathways involved in promotion of tumorigenesis and
apoptosis, resulting in decreased NF-kB–dependent gene transcription, increased level of the tumor suppressor p53, accumulation of p21 and p27 with secondary induction of cell cycle
arrest, accumulation of the proapoptotic protein BAX, and
down-regulation of signaling through p44/42 MAPK.134
Small-molecule inhibitors or low-molecular-weight compounds
will probably prove to be a useful class of therapeutic agents in
NHLs. The nonpeptidic compounds are attractive because they
share excellent molecular properties in terms of stability, bioavailability, cell permeability, and central nervous system penetration.13
Using structure-based methods, several of these small molecules
can be designed or screened for in chemical directories and
subsequently tested for inhibitory effects on oncogenes or their
products and for in vivo antitumor activity.
Targeting oncogenes represents an important step forward in
the treatment of NHLs. The strategies employed, albeit imperfect, continue to evolve. The careful selection of patients based
on their molecular profile holds great potential for increased
efficacy and diminished toxicities in the future therapy of
lymphoid malignancies.
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2005 106: 1911-1923
doi:10.1182/blood-2004-12-4621 originally published online
March 31, 2005
Oncogenes as molecular targets in lymphoma
Ali Hachem and Ronald B. Gartenhaus
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