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Blood First Edition Paper, prepublished online January 14, 2010; DOI 10.1182/blood-2009-09-220020
Targeting the translational machinery as a novel treatment strategy for hematologic
malignancies
Patrick R. Hagner1, Abraham Schneider1, 2, and Ronald B. Gartenhaus1, 3*
1
Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine,
Baltimore, MD, 21201
2
Department of Oncology and Diagnostic Sciences, University of Maryland Dental School,
Baltimore, MD 21201
3
Veterans Administration Medical Center, Baltimore, MD, 21201
*Corresponding Author: Ronald B. Gartenhaus.
Telephone: 410-328-3691. Fax: 410-328-6559
Email: [email protected] <mailto:[email protected]>
1
Copyright © 2010 American Society of Hematology
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Abstract: The dysregulation of protein synthesis evident in the transformed phenotype has
opened up a burgeoning field of research in cancer biology. Translation initiation has recently
been shown to be a common downstream target of signal transduction pathways deregulated in
cancer and initiated by mutated/overexpressed oncogenes and tumor suppressors. The
overexpression and/or activation of proteins involved in translation initiation such as eIF4E,
mTOR and eIF4G have been shown to induce a malignant phenotype. Therefore,
understanding the mechanisms that control protein synthesis is emerging as an exciting new
research area with significant potential for developing innovative therapies. This review
highlights molecules that are activated or dysregulated in hematologic malignancies, and
promotes the transformed phenotype through the deregulation of protein synthesis. Targeting
these proteins with small molecule inhibitors may constitute a novel therapeutic approach in the
treatment of cancer.
2
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Eukaryotic cells have various mechanisms and levels by which gene expression can be
regulated including: transcription, export of mRNA messages, mRNA stability and posttranslational modifications. Protein synthesis is essential for cell viability, and controlling mRNA
translation is a critical step in regulation of gene expression. Translation can be divided between
three stages: initiation, elongation, and termination. Translational control is principally exerted
by regulating the formation of the cap-dependent translation initiation complex. Translation
initiation is comprised of a mechanism in which the eIF4F ternary complex (eIF4E, eIF4A, and
eIF4G) recruits a 43S pre-initiation complex containing a Met-tRNAi and multiple initiation
factors (eIFs 1, 1A, 2, 3, and 5) to the 5’ methyl-7-GTP cap complex on mRNA (reviewed in
Sonenberg and Hinnebusch)1. Once the pre-initiation complex scans the 5’ untranslated region
of the mRNA and reaches the AUG start codon, the large (60S) ribosomal subunit joins the
small (40S) ribosomal subunit and beings to synthesize the protein.1
Deregulated protein synthesis plays an important role in human cancer and deregulated
translational control has been recognized as an integral part of the malignant state.2,3 In the last
several years it has become clear that the efficiency of expression of key proteins involved in
cell growth regulation, proliferation and apoptosis may be controlled at the translational level by
changes in the activity of components of the protein synthesis machinery.4,5 Various classes of
mRNAs differ considerably in their translational efficiency. Typically, mRNAs coding for proteins
positively involved in regulating cell growth and survival have a high degree of secondary
structure in the 5’ untranslated region (UTR). The translation of such messages is particularly
sensitive to the activity of the cap-dependent translation-initiation machinery.6 In view of the fact
that translation factors are closely regulated by conditions that affect cell growth, it is not
surprising that, experimentally, aberrant expression of some of these factors has been shown to
induce malignant transformation of cells.
Translation initiation has recently been shown to be a common downstream target of
signal transduction pathways deregulated in cancer and initiated by mutated/overexpressed
3
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oncogenes and tumor suppressors.7 A number of previous publications indicate that aberrant
control of protein synthesis contributes to lymphomagenesis3,8 opening up possibilities for
innovative therapeutics i.e., targeting the translational machinery. Below, we present an
overview of potentially targetable translational machinery components and regulatory signaling
pathways that represent a novel approach for the treatment of hematologic malignancies.
eIF4F: A major regulatory step in control of protein synthesis is translation initiation. Translation
initiation is modulated by the association of a ternary complex of proteins, eukaryotic translation
initiation factor F (eIF4F), composed of eIF4G, eIF4A and eIF4E.9 The cellular levels of eIF4E
molecules are 10-30 fold lower than other known initiation factors10,11 and its association with the
eIF4F complex is therefore the rate-limiting step in translation initiation (reviewed by Clemens,12
De Benedetti and Graff13), however the stoichiometry of eIF4F components is still debated by
some investigators.14 It was previously hypothesized that an increase in the rate of translation
would have an impact on the spectrum of mRNAs synthesized.15 Subsequently, it was shown
that overexpression of eIF4E could increase the translation of mRNAs with long highly
structured 5’ untranslated region (UTR)9 such as chloramphenical acetyl transferase and
ornithine decarboxylase.16 Early studies established that eIF4E overexpression resulted in the
transformation of immortalized cell lines as exemplified by increased proliferation, anchorage
independent growth and invasiveness.17,18 Recently, the overexpression of eIF4E has been
observed in primary human malignancies such as colon,19 breast20,21, non-Hodgkin lymphoma
(NHL)22, acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML).23
These aggregate data prompted a number of laboratories to investigate the role of eIF4E in
neoplastic transformation as well as suggesting the feasibility of targeting this molecule as a
novel therapeutic approach. Several investigators have examined the impact of overexpressing
eIF4E in transgenic mouse models, which can increase the incidence of multiple malignancies
including lymphomas.3,24
4
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The disruption of translation initiation through the modulation of either eIF4E protein
levels or formation of the eIF4F ternary complex has been examined employing RNA
interference.25,26,27,28 Graff et al. demonstrated that intravenous administration of anti-sense
oligonucleotides in a mouse xenograft model could successfully silence the expression of eIF4E
in vivo. This decrease in eIF4E levels lead to an inhibition of tumor growth and reduced cell
viability. While the decrease in eIF4E levels was not restricted to only the tumor cells (80%
reduction in normal tissues), the clinical impact on normal tissues was minimal as there were no
significant changes in body weight, liver weight or hepatic enzymes. These data demonstrated
that modulating eIF4E protein levels was a useful approach to disrupt tumor growth in vivo
without significant off target effects in normal tissues. The function of eIF4E may also be
interrupted by small molecule inhibitors that mimic the 5’ methyl-7-GTP moiety found within the
capped structure of mRNA.29,30 The small molecule Ribavirin has been shown to suppress
eIF4E induced transformation through a mechanism in which the inhibitor competes with the
endogenous 5’ methyl-7-GTP cap structure for eIF4E.
Through this competitive inhibition,
Kentsis et al. were able to demonstrate a decrease in the translation of oncogenic messages
such as ornithine decarboxylase (ODC) as shown by the absence of its mRNA in the polysomal
fraction of ribosomes.29 eIF4E has also been shown to regulate translation of mRNA by
promoting the transport of oncogenic messages.16 The use of Ribavirin to interfere with eIF4E
association to the 5’ methyl-7-GTP cap structure results in the specific disruption of eIF4E
directed transport of messages to the cytoplasm.29,30 The Ribavirin mediated inhibition of eIF4E
has also been successfully demonstrated in a xenograft mouse model with a six-fold reduction
in tumor volume when compared to controls further highlighting the critical role of eIF4E in
maintaining the malignant phenotype.
Additionally, small molecule inhibitors have been investigated targeting the proteinprotein domain that governs binding between eIF4E and eIF4G. These inhibitors work through
molecular mimicry of the domain on either eIF4G or 4E-BP1 that promotes binding of eIF4E.
5
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These 4E-BP1 functional mimetic inhibitors have been shown to be effective in promoting
apoptosis and impairing cell cycle progression in both lymphoma and leukemia.31,32 Utilizing a
high-throughput approach, Moerke et al were the first to describe an eIF4F specific inhibitor
(4EGI-1), which can disrupt the eIF4F complex and inhibit cap-dependent translation.
Importantly, treatment of T-cell acute lymphoblastic leukemia (Jurkat T-ALL) and lung cancer
(A549) cell lines with 4EGI-1 was able to induce apoptosis as well as inhibit proliferation.31 The
authors also demonstrate that 4EGI-1 preferentially inhibits the growth of cells transformed with
the Philadelphia chromosome compared to the non-transformed isogenic Ba/F3 cell line. The
use of this inhibitor has also been examined in acute myeloid leukemia (AML).32 While the
macrolide rapamycin has been an effective tool to inhibit the activity of eIF4E and capdependent translation in certain cell types,33,34 Tamburini et al. demonstrated that AML cells are
resistant to inhibition of the mammalian target of rapamycin (mTOR) by treatment with the
rapamycin derivative RAD001 or Raptor small inhibitory RNA (siRNA). Nevertheless, treatment
of cells with 4EGI-1 significantly reduced the clonogenic growth and induced apoptosis of
primary AML cells. Furthermore, 4EGI-1 also proved to be effective in disrupting eIF4F complex
formation in primary AML samples lending additional credence to targeting of the eIF4F
complex.32 Alternatively, an innovative peptidomimetic approach targeting the association
between eIF4G and eIF4E was employed in an ovarian cancer mouse model. The authors
demonstrated that the peptide could be specifically targeted to the ovary by fusing the peptide to
gonadotropin releasing hormone (GnRH).
Interference of eIF4F complex formation with a
targeted peptide strongly inhibited proliferation and induced apoptosis in tumor cells when
compared to both saline and GnRH treated controls, with no toxicity observed in normal
tissues.35 Interfering with eIF4F activity and translation initiation has also been shown to restore
the chemo-sensitivity of tumors in pre-clinical lymphoma models.36,37
The small molecule inhibitor Ribavirin, is currently used in the therapeutic regimen for
viral infections such as Lassa fever virus and hepatitis C virus (HCV).38 Treatment of patients
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with HCV related non-Hodgkin lymphoma39,40 using Ribavirin resulted in a dramatic clinical
response in some patients, these responses often correlated with a reduction in HCV viral load.
The first clinical trial evaluating the targeting of eIF4E as a monotherapy utilizing Ribavirin was
recently conducted in 11 patients with acute myeloid leukemia. The results were striking with
complete remission (CR), partial remission (PR) and other hematological improvements
obtained in patients.30 These exciting recent clinical data, along with earlier animal models,
together suggest that Ribavirin shows great potential to be an effective new therapeutic agent in
the treatment of hematological malignancies.
mTOR: The evolutionarily conserved Target of Rapamycin (TOR) is a large serine/threonine
protein kinase that belongs to a family of kinases known as the phosphatidylinositol kinaserelated kinase family.41 TOR was originally identified in the yeast species, Saccharomyces
cerevisiae, because of its sensitivity to the compound rapamycin (sirolimus), a macrocyclic
lactone with immunosuppressive properties initially isolated from the bacterial strain
Streptomyces hygroscopicus.42 The mammalian homologue, mTOR, is a key component of two
functionally and structurally distinct multiprotein complexes, mTOR complex 1 (mTORC1) and
mTORC2. While mTORC1 is responsive to the inhibitory effects of rapamycin, mTORC2
appears to be insensitive in a tissue-specific manner.43 Interestingly, prolonged treatment with
rapamycin derivatives reduce mTORC2 signaling in AML cells resulting in a decrease in AKT
signaling.44 Compelling evidence demonstrates that mTORC1 is a prime signaling node in many
pathways that regulate cell growth and metabolism by transducing signals associated with
environmental, nutritional, growth factor, and other bioenergetic cues.
mTORC1 is a multimeric protein complex that is comprised of mTOR together with the
associated proteins raptor (regulatory associated protein of mTOR), mLST845 and PRAS40.46
Upon activation, the catalytic subunit of mTOR directly controls protein synthesis by
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phosphorylating key regulators of the translational machinery such as the eukaryotic initiation
factor 4E-binding protein-1 (4EBP1), an inhibitor of cap-dependent translation, and the
ribosomal p70 S6 kinase (S6K), a critical factor that may participate in ribosomal biogenesis
through activation of ribosomal protein S6 (RPS6).47 In this regard, S6K is phosphorylated by
mTOR at threonine 389 which in concert with another phosphorylating event carried out by the
serine/threonine kinase phosphoinositide-dependent kinase-1 (PDK1) promotes full activation of
S6K through phosphorylation of threonine 229. Once activated, S6K phosphorylates the RPS6
to enhance ribosomal biogenesis and overall protein synthesis (see below). While mTOR
phosphorylation of p70 S6 kinase results in the activation of the kinase, mTOR controls capdependent translation by phosphorylating 4EBP1 which is a suppressor of eIF4E.43 Due to the
fact that the strong binding of a hypophosphorylated 4EBP1 prevents the assembly of the
translation initiation complex to eIF4E, mTOR-mediated phosphorylation of 4EBP1 facilitates the
release of eIF4E from its inactive state to form, together with eIF4G and eIF4A, the eIF4F
complex. Therefore, by inhibiting mTOR activity, multiple components of protein translation are
suppressed, resulting in cell growth retardation and eventually cell death.48
Cellular processes commonly deregulated in human cancer, such as cell proliferation,
cell cycle progression, survival and motility are closely related to mTORC1 function.49 In
addition, several upstream (PTEN, AKT, TSC1/2) and downstream (eIF4E) mediators within the
mTORC1 signaling axis are often mutated or activated in a number of human malignancies
including cancers of the hematopoietic system.50,51 These findings have triggered an intense
search for novel therapeutic strategies to target the mTOR signaling pathway in conditions such
as leukemia, lymphomas and multiple myeloma. Indeed, this is evident by the increasing
number of preclinical studies and the approximately 20 clinical trials that are either active or
about to be initiated on the use of sirolimus, rapamycin analogs “rapalogs”, or second
generation derivatives, such as temsirolimus, everolimus, and deferolimus alone or in
8
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combination
with
other
agents
on
hematological
malignancies,
as
described
on
www.ClinicalTrials.gov at the time this review was being written.
Early studies using mTOR inhibitors in hematological conditions provided key positive
findings to launch further investigations in the field. Brown et al. initially reported on the
proapoptotic activity of sirolimus on B-precursor acute lymphoblastic leukemia cell lines. They
were also able to demonstrate that by using sirolimus as monotherapy, survival of Eμ-RET
transgenic mice, an animal model of leukemia/lymphoma, could be extended when compared to
littermate controls.52 Later, the same group investigated whether the use of a rapamycin analog,
temsirolimus, was effective in preventing the in vitro growth of primary adult acute lymphoblastic
leukemia cells. Compared to untreated cells, temsirolimus-treated cells showed a marked
decrease in cell proliferation and an increase in apoptosis. They further validated the in vitro
data in a Non Obese Diabetic Severe Combined Immunodeficiency (NOD/SCID) xenograft
model by establishing tumors with the same patient samples. A significant reduction in
peripheral blood blasts and splenomegaly was observed in the temsirolimus treated animals
when compared to the control group.53 The effectiveness of rapamycin in chronic lymphocytic
leukemia (CLL) has been investigated in an Eμ-TCL1 transgenic mouse model which gives rise
to a CD5+ B-cell lymphoproliferative disorder similar to human CLL. The authors demonstrate
that rapamycin could significantly extend the life of rapamycin treated mice when compared to
controls.54 Xu et al performed the definitive work that provided the rationale for combining
rapamycin with chemotherapeutic agents.
The authors demonstrated that mTOR was
responsible for cell survival after primary AML cells were treated with etoposide and that
addition of rapamycin to the treatment could dramatically increase the cytotoxic effects of
etoposide.55 Overall, these studies were critical to establishing the promising role mTOR
inhibitors may exert against hematological cancers.
In this regard, the results of a phase II clinical trial on the use of deferolimus as a single
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agent in patients with relapsed or refractory hematologic malignancies such as; ALL, CML, CLL
and mantle cell lymphoma (MCL) have recently been reported.56 Out of the 52 evaluable
patients with various types of leukemia/lymphoma who were previously heavily pretreated, 40%
responded positively to deferolimus treatment through hematologic improvement or stable
disease. The effects of deferolimus on the translational machinery controlled by mTOR activity
was demonstrated by the presence of decreased levels in the phosphorylated status of 4EBP1
in tumor cells from a subset of 9 out of 11 patients with acute myelogenous leukemia,
confirming the inhibition of mTOR.
Similar positive outcomes and biochemical end-points
relative to the mTOR pathway were obtained in another phase I/II clinical study evaluating
responses to everolimus, another second-generation mTOR inhibitor, in patients with relapsed
or recurrent hematological cancers. Overall, everolimus was well tolerated at a daily dose of 10
milligrams. As expected, the phosphorylation status of downstream targets of mTOR, 4EBP1
and S6K was inhibited in 6 of 9 patient samples. The authors concluded that everolimus in
combination with other agents targeting components of the phosphatidylinositol 3-kinase
(PI3K)/AKT/mTOR pathway are warranted.57 Rapamycin has also been shown to be effective in
imatinib resistant chronic myeloid leukemia. A major hematologic response was reported in two
out of seven patients. The investigators also demonstrated in Ba/F3 cells, which expressed
multiple forms of imatinib resistant BCR-ABL kinases, that rapamycin could inhibit the cell cycle
progression.58
As a matter of fact, more recent preclinical and clinical studies have been addressing the
use of combination therapies in hematologic malignancies by adding other targeted agents or
cytotoxic chemotherapeutics to protocols that include mTOR inhibitors.50,59,60,61 Recently,
multiple groups have developed and described mTOR kinase specific small molecule inhibitors;
Torin1, PP242 and PP30.34,62 Recently, the Gartenhaus laboratory has established a paradigm
in which chronic ethanol exposure inhibits mTOR signaling in lymphocytes with a significant
repression of cap-dependent translation and reduction in the tumorigenic capacity of NHL in a
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human lymphoma xenograft system.63 The observed ethanol-mediated repression of mTOR
signaling coupled with a decrease in lymphoma growth presented underscore the critical role of
mTOR signaling and translation in lymphoma.63
The role of rapamycin and its derivatives and their mechanism of action are still under
investigation. A recent report demonstrated that rapamycin treatment increased the capdependent translation of cervical carcinoma and embryonic kidney cells lines33 illustrating how
rapamycin can have cell type specific effects.
Rapamycin is also reportedly effective in
inhibiting mTOR signaling in either 4E-BP1 or p70 S6 Kinase 1 null cells. However, another
publication demonstrated that RPS6 phosphorylation was still present in MEF cells (p70 S6
Kinase 1 -/-) and was likely due to a compensatory signal from p70 S6 Kinase 2.47 A possible
explanation may be that related family member proteins may be differentially inhibited by
rapamycin treatments. Certain cell types have been observed to be resistant to rapamycin
mediated mTOR inhibition; the mechanism(s) of resistance are still to be elucidated (reviewed in
Kurmasheva et al.).64 The earliest resistance mechanism observed was mutations in the
FKBP12 gene that prevented the formation of FKBP12-rapamycin complex formation and
rendered cells rapamycin resistant.65 Decreased AKT activity has also been shown to induce
rapamycin resistance. This may be due to continued cap-independent protein synthesis of cMYC and cyclin D1 through internal ribosomal entry sites (IRES) that are negatively regulated
by AKT signaling and that this ongoing translation permits cell cycle progression.66 New
strategies are being developed to overcome resistance to small molecule inhibitors including the
targeting of multiple proteins within the same pathway (reviewed in Weinstein and Joe)67.
The further development and understanding of novel molecules specifically inhibiting
mTOR are likely to be even more effective at suppressing tumor growth than rapamycin and
rapamycin derivatives. It is anticipated that results obtained from ongoing pre-clinical and
clinical trials will provide valuable information on how to optimize the incorporation of mTOR
inhibitors into therapeutic regimens.
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p70 S6 Kinase (S6K): As discussed above, mTOR plays a major role governing the rate and
efficiency of translation by signaling through the S6K family members.
S6K is generally
implicated in ribosomal biogenesis and activity by phosphorylating RPS6, which is thought to be
the rate-limiting step in the synthesis of new ribosomal RNA.43 However, the role(s) of p70 S6
Kinase and RPS6 has become an area of keen investigation recently as multiple publications
have presented conflicting data as to the function of these proteins in ribosomal biogenesis (see
below). Other S6K substrates have been identified including the eukaryotic elongation factor 2
kinase (eEF2K) and the eukaryotic translation initiation factor 4B (eIF4B). The phosphorylation
of eEF2K results in inhibition of kinase activity, which leads to an increase in translation
elongation. The phosphorylation of eIF4B by S6K leads to an increase of eIF4B protein levels
recruited to eIF4A (reviewed in Ruvinsky and Meyuhas).68 This increased association promotes
the helicase function of eIF4A and potentially increases the scanning ability of the ribosome.
Due to the fact that p70 S6 Kinase can regulate components of the translational machinery, the
targeting of S6K should have a detrimental effect on leukemias and lymphomas that are
dependent upon an abnormal rate of protein synthesis. Early indications of a plausible role of
S6K in hematological malignancies, in particular T-cell lymphoma, were first described by
Dumont et al. nearly 15 years ago. They observed that rapamycin inhibited several biologic
responses in the YAC-1 T cell lymphoma, including proliferation and cell cycle progression. By
generating stable somatic mutants with altered sensitivities to rapamycin, they further
demonstrated that the growth inhibitory effects of the drug were in fact associated with the
inhibition of S6K activity.69
More recent studies have validated these earlier findings by demonstrating that inhibition
of S6K following rapamycin treatment results in both growth inhibition and apoptotic cell death of
B-precursor acute lymphoblastic leukemia cell lines in vitro and improved survival in a
leukemia/lymphoma transgenic mouse model.52 S6K hyperactivation has also been suggested
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as a critical mediator underlying the pathogenesis of the malignant Hodgkin/Reed-Sternberg
cells of Hodgkin lymphoma (HL). Dutton et al. showed that constitutive activation of the
PI3K/AKT pathway was common in HL primary tumors and cell lines. As a consequence of this
activation, the mTOR substrates 4E-BP1 and S6K were also hyperphosphorylated. This
relationship was demonstrated not only by the clear inhibitory actions of rapamycin on these
proteins but also by the use of LY294002, a commonly utilized inhibitor which targets PIKK
family members, including mTOR (reviewed in Liu et al).70,71 These results highlight the critical
role of the PI3K/AKT/mTOR pathway in sustaining the growth of leukemia/lymphoma cells. The
inhibition of p70 S6 Kinase has been shown to activate other multiple signaling pathways
through a S6K/PI3K feedback mechanism. Therefore, the targeting of p70 S6 Kinase with small
molecule inhibitors would optimally be incorporated with other therapies to disrupt signaling
pathways known to be affected by this feedback such as mitogen activated protein kinase
(MAPK), MAP kinase signal integrating kinase (MNK), and AKT.72,73,74
RPS6: Ribosomes in higher eukaryotic cells are large multimeric ribonucleoprotein complexes
consisting of four ribosomal RNA (rRNA) molecules and approximately 80 ribosomal proteins
which make up a large (60S) and small (40S) subunit.75,76 In view of the fact that a significant
proportion of the cells energy is spent in ribosomal biogenesis77 it is reasonable to expect that
the proteins and rRNA that are the constituents of ribosomes be tightly regulated. Ribosomal
biogenesis and assembly is extremely complex with ribosomal proteins themselves being
involved in the regulation of other ribosomal proteins. Experimental evidence has recently
demonstrated that the lack of a specific ribosomal protein, RPS6 can be catastrophic for the
formation of a nascent ribosomal subunit. Due to the interdependency of ribosomal protein
regulation, the loss of RPS6 can lead to an overabundance of other ribosomal proteins and the
activation of extra-ribosomal functions of these proteins secondary to loss of regulation.78 Of all
the essential ribosomal proteins, RPS6 has attracted the most attention since it was the first
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ribosomal protein shown to be phosphorylated in response to stimuli.79,80,81 The significant body
of research conducted on RPS6 over the past few decades has implicated RPS6 in the
regulation of protein synthesis (through the regulation of 5’ terminal oligopyrimidine (5’ TOP)
RNA synthesis) and cell size. These messages containing an oligopyrimidine tract are important
for ribosomal biogenesis as they encode most of the translational apparatus.82,83
A selective
increase in the translation of these messages can be induced in mitogen stimulated cells80,81,84
and can be selectively decreased through treatment with rapamycin or its derivatives,85
suggesting that this group of mRNAs may have a important role in the control of cellular growth.
However, the role of RPS6 and its upstream regulatory molecules have come into question in
recent years with publications demonstrating that 5’ TOP mRNA are still regulated in mouse
embryonic fibroblasts (MEF) in which all five possible phosphorylation sites on RPS6 had been
mutated to alanines86 or in p70 S6 Kinase 1 null mice.87 Conversely, another report
demonstrated that RPS6 phosphorylation was still present in MEF cells (p70 S6 Kinase 1 -/-)
due to a compensatory signal from p70 S6 Kinase 2.47 In order to resolve these discrepancies a
more thorough molecular understanding of the phosphorylation status of RPS6 is needed.
The haploinsufficiency of RPS6 has been linked to multiple different phenotypes
including cancer,88,89 and specifically cancers of the hematopoietic system.90 In contrast, the
Gartenhaus lab has recently found that RPS6 is highly expressed in primary diffuse large B-cell
lymphoma (DLBCL) samples when compared to normal lymph node samples (manuscript in
preparation).
As RPS6 may regulate the stoichiometry of other ribosomal proteins, it is
plausible that RPS6 levels are increased in lymphoma to confer a larger number of ribosomes
and therefore greater capacity to translate oncogenic messages. The impact on the cellular
phenotype vis-à-vis the absence or decrease of RPS6 has previously been investigated using
genetic methods such as small inhibitory RNAs (siRNA) or conditional gene knockouts
supporting RPS6 as a negative regulator of 5’ TOP message translation. A number of
publications have revealed a mechanism in which an increase in the ribosomal protein RPL11
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following suppression of RPS6 function leads to an inhibition in proliferation and an increase in
cell apoptosis through a p53 dependent pathway.78,91,92 Since the inactivation of p53 function in
non-Hodgkin lymphoma is relatively low,93,94 targeting RPS6 function in non-Hodgkin lymphoma
may represent a potential therapeutic paradigm.
MCT-1: The oncogene Multiple Copies in T-cell Lymphoma-1 (MCT-1) is found on chromosome
Xq22-24 and has been shown to be overexpressed in a number of B-cell and T-cell lymphoma
lines and a small subset of diffuse large B-cell lymphoma (DLBCL), the most common form of
non-Hodgkin lymphoma.95,96 Recent data demonstrates that MCT-1 protein levels are highly
elevated in the vast majority of primary DLBCL samples compared with indolent lymphoid
malignancies when examined by tissue microarray (TMA) analysis.97 The overexpression of
MCT-1 has been previously implicated in cellular transformation through its ability to stimulate
cell proliferation, suppress apoptosis and enhance signaling pathways involved in cell
survival.95,96,98 Knocking down MCT-1 protein levels in DLBCL significantly reduced cell viability
through an apoptotic mechanism,97 providing the first direct genetic evidence that interfering
with MCT-1 function was able to induce apoptosis in lymphoma cells with high endogenous
levels of MCT-1 protein.
MCT-1
belongs
to
a
family
of
PseudoUridine
synthase
and
Archaeosine
transglycosylase (PUA) containing proteins, which have the ability to interact with RNA.98,99
MCT-1 protein interacts with the cap complex, m7GpppN (where N is any nucleotide) found at
the 5’-terminus of all cellular eukaryotic mRNAs which recruits the Density Regulated Protein
(DENR/DRP) in an eIF4E dependent manner.100 DENR/DRP contains a SUI1 domain and is
also found in the translation initiation factor eIF1.98 The recruitment of DENR can increase the
translation initiation of a subset of mRNAs, containing a long and highly structured 5’ UTR,
typically found in cancer-related messages.100 The mechanism(s) underlying this selective
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increase in translation has been described and is attributed to the increased scanning capability
of the 40S ribosome and increased recognition of the correct initiation codon conferred by the
DENR.98,100,101 This increased scanning ability and AUG start codon recognition allows for these
selectively upregulated messages to increase their presence in heavier polysomes and facilitate
subsequent rounds of translation leading to significantly increased levels of oncogenic proteins
present in the cell.
Earlier work demonstrated that a MCT-1 deletion mutant containing only the PUA
domain still retained the ability to interact with the eIF4F/5’ methyl-7-GTP capped structure, but
not enhance translation.100 A recent report demonstrated that the PUA domain mutant protein
could act in a dominant-negative (DN) manner competing with wild-type MCT-1 for the
translation initiation complex.102 The DN-mutant protein attenuated the malignant phenotype of
lymphoma cells through modulation of the translational profile and sensitized cells to both
irradiation and anthracyclines, two common therapeutic modalities utilized for the treatment of
NHL.102 Intriguingly, MCT-1 is unique in that it is the only known PUA-domain containing protein
to also not contain a SUI1 motif.98 Employing a peptide-based or small molecule approach to
interrupt the physical association of MCT-1 and DENR proteins is a potentially useful approach
in order to prevent the recruitment of SUI1 domain-containing DENR/DRP protein to the capcomplex. Currently, there are no specific small molecule inhibitors that can directly inhibit MCT1 protein function. Nandi et al. previously demonstrated that phosphorylation of MCT-1 protein
on threonine 81 by ERK1/2 is critical for protein stability and for its ability to promote cell
proliferation.103 Taking advantage of a recently identified ERK1/2 small molecule inhibitor104
MCT-1 function was disrupted by targeting its upstream kinase with significant impact on
lymphoma cell viability and clonogenic capacity. Importantly, employing a human lymphoma
xenograft model Dai et al. were able to demonstrate significant anti-lymphoma activity in vivo
using this approach, thereby supporting the development of novel small inhibitory molecules
directed towards MCT-1 as a promising approach in DLBCL.97
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These exciting findings support both the clinical relevance of potentially targeting the
MCT-1 oncogene and provide additional evidence linking MCT-1 with lymphoma development
and/or progression. The current challenge is to identify the complete repertoire of translated
mRNAs regulated by MCT-1 and how they participate in lymphomagenesis.
MNK: In addition to the availability of eIF4E protein to form the eIF4F translation initiation
complex, eIF4E activity is also regulated by phosphorylation events. Phosphorylation of eIF4E
is know to be enhanced when cells are treated with specific stimuli including; serum, phorbol
esters and growth factors.105 The signals from these stimuli were determined to be transduced
through the mitogen activated protein kinase pathway (MAP kinase),105 specifically the MAP
kinase signal integrating kinase (MNK).106,107
The exact mechanism as to how the phosphorylation of eIF4E regulates protein
translation is still unclear. Prior data suggested that the phosphorylation of eIF4E enhanced the
5’ methyl-7-GTP cap binding potential of the protein.108 However, more recent experimental
evidence has since shown that the phosphorylation of eIF4E actually decreases its affinity for a
capped substrate.109,110 In contrast, the physiological role of MNK phosphorylation of eIF4E may
be better defined.
Wendel et al, have demonstrated that the insertion of a constitutively
activated MNK1 into an Eµ-Myc mouse model produced lymphomas of a mature B-cell
phenotype.111 Ueda et al created a MNK1/2 double knockout mice and demonstrated that there
was no phosphorylated eIF4E levels in all tissues examined. These results demonstrated that
eIF4E phosphorylation is not necessary for general protein synthesis and cap-dependent
translation.112
With the advent of a novel small molecule inhibitor specific to the MNK kinases,
CGP57380, these data may become more easily reconciled. Treatment of prostate cancer cell
lines with CGP57380 reduced the levels of phospho-eIF4E and reduced the amount of mRNA in
17
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the polysomal fraction of translating ribosomes and inhibited proliferation.113 CGP57380 has
also been shown to be effective in inhibiting polysome associations with mRNA in chronic
myelogenous leukemia cells and that treatment with this inhibitor enhances the activity of
imatinib and induces apoptosis in imatinib resistant CML cells.114 The inhibition of MNK has also
been shown to affect the ability of eIF4E to transport oncogenic messages from the nucleus to
the cytoplasm.115,116 Topisirovic et al, convincingly demonstrated that phosphorylation of eIF4E
contributes to the oncogenic potential of eIF4E by mutating the residues which can be
phosphorylated.
These mutants were less able to form anchorage dependent foci when
compared to the wild type eIF4E.115 A theory proposed by Morley and Naegele could explain the
molecular mechanism and the phenotypic evidence presented here, they propose that
phosphorylation of eIF4E and its subsequent release from the 5’ methyl-7-GTP cap complex
could allow for a second molecule of eIF4E to interact with the cap and prime another round of
translation initiation seen in polysomes.117
The overexpression of ERK1/2 and p38 kinases, which are responsible for the activation
of MNK, has been demonstrated in follicular lymphoma118 and DLBCL.97 In addition, the
absence of microRNA’s (miRNA) that regulate the MAP Kinase pathway has also been
observed in primary DLBCL samples while still present in matched normal B-cell lymphocytes
from the same patients.119 Dai et al. also found increased levels of ERK protein in a large
percentage of human DLBCL samples.97 These observations in conjunction with the
experimental evidence of using small molecule inhibitors in CML cells demonstrate that the
MNK kinases are viable targets for future treatment strategies in hematologic malignancies.
Future Directions: Although significant data has been accumulated to date, the potential of
targeting components of the translational machinery in hematologic malignancies has yet to be
fully realized.
The seminal work accomplished in targeting the eIF4F complex28,29,30,31
demonstrates that inhibition of translation initiation should be given high priority for future
18
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development of new and more effective small molecule inhibitors. While the work on rapamycin
and its analogs is broad and has finally reached the clinical settings in a number of cases, we
are excited to see work from laboratories that are working to develop molecules that target the
mTOR kinase domain directly.34,62
We have shown that specific targeting of MCT-1 has
significant preclinical activity and experiments are in progress designed to disrupt the MCT1/DENR association observed at the cap complex during translation initiation. Finally, the
improved understanding and discovery of novel molecules involved in translation will provide
innovative clinical approaches towards the treatment of lymphoma and leukemia.
Acknowledgements: This work was supported in part by a Merit Review Award from the
Department of Veterans Affairs (R.B.G.) and R01AA017972 from the NIH (R.B.G.). We
apologize to those authors whose original articles could not be cited due to space constraints.
Author Contributions: RBG designed the review; and all authors contributed to writing and
proofreading of the paper. The authors have no conflict of interest to declare.
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31
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Figure and Table Legends:
Table 1: Small molecule inhibitors involved in the suppression of translation.
Figure 1: Targeting the translational machinery. Multiple drugs have been developed to target
molecules involved in the regulation of protein translation. Rapamycin and “rapalogs”
(temsirolimus, everolimus, and deforolimus) restrict the recruitment of the FKBP12
protein to the mTOR complex inhibiting mTORC1 signaling.
Other small molecule
inhibitors have been developed to target the mTOR kinase domain (Torin1, PP242, and
PP30), which may inhibit both mTORC1 and mTORC2 signaling pathways. CGP57380
has been developed as an ATP competitive inhibitor of the MNK kinases, which may
prevent the release of eIF4E from the 5’ methyl-7-GTP cap structure preventing a
subsequent round of translation on the same mRNA. Other drugs have been found to
block the recruitment of eIF4E to the eIF4F ternary complex including 4EGI-1 and
Ribavirin to inhibit both translation initiation and eIF4E-mediated transport of mRNA.
The PUA dominant negative has also been shown to block the interaction of MCT-1 with
the Translation Initiation Complex (TIC).
32
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Table 1
Drug
Binding Site
Mechanism of action
Reference(s)
Ribavirin
5’ methyl-7-GTP
cap
Competitive inhibitor of
eIF4E binding to capped
mRNA
29,30
4EGI-1
eIF4G
Competitive inhibitor of
eIF4E binding to eIF4G
31,32
GnRH4EBP fusion
peptide
eIF4E
Peptidomimetic inhibitor
of eIF4E binding to
eIF4G
35
Rapamycin,
analogs and
derivatives
FKBPrapamycinbinding (FRB)
domain of mTOR
Inhibits recruitment of
FKBP12 protein to
mTOR
Torin1
mTOR kinase
domain
Inhibition of ATP binding
to kinase domain
34
PP242
mTOR kinase
domain
Inhibition of ATP binding
to kinase domain
62
PP30
mTOR kinase
domain
Inhibition of ATP binding
to kinase domain
62
CGP57380
MNK kinase
domain
Inhibition of ATP binding
to kinase domain
113-116
Reviewed in
45
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Prepublished online January 14, 2010;
doi:10.1182/blood-2009-09-220020
Targeting the translational machinery as a novel treatment strategy for
hematologic malignancies
Patrick R. Hagner, Abraham Schneider and Ronald B. Gartenhaus
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