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NEOPLASIA
From the Institute for Nutrition Research, University
of Oslo, and Department of Immunology, Molecular Medicine Group, the
Norwegian Radium Hospital, Oslo, Norway; and the Division of
Hematology/Oncology, University of Alabama at Birmingham.
Juvenile myelomonocytic leukemia (JMML) is an
aggressive childhood disorder with few therapeutic options.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor
necrosis factor- Juvenile myelomonocytic leukemia (JMML) is a
rare childhood malignancy with a poor prognosis.1
Substantial improvement has not been observed in patients offered
either conventional chemotherapy or treatment with
retinoids.1,2 Only allogeneic bone marrow transplantation
can induce durable remissions, but the relapse frequency is high and
serious side effects are relatively common.3
A major feature of this disease is that the cytokines
granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor- Cytokines signal via surface receptors and then through multiple
intracellular cytoplasmic signaling systems to the nuclear effector
mechanisms. The Ras family of proteins plays an important role in
relaying these signals through the protein kinase Raf-1 and the
mitogen-activated protein kinase (MAPK) cascades.12,13 The
neurofibromin protein, encoded by the neurofibromatosis type 1 tumor
suppressor gene (NF1), inhibits Ras by hydrolyzing active guanosine triphosphate (GTP)-Ras to the inactive guanosine
diphosphate-bound form. Several independent findings link a
dysregulated Ras signaling pathway to the pathogenesis of JMML. There
are increased frequencies of both NF1 and RAS
gene mutations among JMML patients,14,15 and these groups
appear to be mutually exclusive. Allogeneic transplantation of
hematopoietic fetal liver cells from mice with a targeted disruption of
the Nf1 gene led to a myeloproliferative disorder resembling JMML.16 Moreover, the loss of the Nf1 gene in
these cells rendered them hypersensitive to GM-CSF, similar to JMML
cells,16,17 and hematopoietic fetal liver cells lacking
both the Nf1 and the GM-CSF genes were dependent
on exogenous GM-CSF for efficient homing and proliferation in recipient
murine bone marrows.18 Furthermore, inhibition of the
prenylation of Ras, mandatory for Ras cell transforming activity,
markedly inhibits JMML colony growth.19
These results suggest that GM-CSF is an important regulator of
JMML cell growth and indicate that GM-CSF-induced proliferation is
mediated via the Ras/Raf-1 signaling system. Specific Raf-1-related inhibition might therefore be a therapeutic option in this fatal childhood leukemia. The advent of catalytic nucleotides, such as
ribozymes and DNA enzymes, offers a unique opportunity to selectively inhibit synthesis of individual proteins by degradation of their mRNAs.20 In the present study, we used a DNA enzyme
designed to specifically cleave mRNA for Raf-1. The enzyme was tested
on JMML cells cultured both in vitro and in a xenograft model of JMML.
Synthesis of DNA enzymes and in vitro cleavage activity
The DNA enzyme (100 nM) and the in vitro-transcribed RNA (900 nM) were
mixed in a reaction buffer containing 50 mM Tris, pH 7.5, and 10 mM
MgCl2. The reaction was performed at 37°C for various time periods. Following cleavage, a stop solution was added, and then
samples were analyzed by 10% polyacrylamide gels containing 7 M urea.
Sampling of donor cells
Transfection of cells, colony formation, and cytokine and mRNA measurements We used cationic liposomes (25 µg/mL, DOTAP; Boehringer Mannheim, Mannheim, Germany) alone or complexed with the Raf-1 DNA enzyme or its inactive form. Cells were transfected with the liposomes for 10 to 15 hours in liquid culture before they were plated in semisolid methylcellulose to grow for 2 weeks. Colonies (>40 cells per clone) were then stained supravitally and scored.10Aliquots were sampled from the liquid medium after a 24-hour
culture, and the concentrations of GM-CSF, TNF- To determine mRNAs, we used RNAse protection assays after extraction of total RNA, as described.9 We used mRNA-reduced glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal standard. Immunoprecipitation, Western blotting, and kinase assays Immunoprecipitation of intracellular molecules and Western blotting of phosphorylated molecules were performed according to methods described previously.22 Briefly, 10 million cells per milliliter sample were incubated in liquid medium with or without cytokines for selected time periods before they were harvested and lysed. Total Raf-1, JNK-1, and ERK-1 proteins as well as their respective phosphorylated forms were immunoprecipitated with MoAbs (Pharmingen, San Diego, CA) and resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5%). Phosphorylation of the immunoprecipitated molecules was examined using an antiphosphotyrosine MoAb (Boehringer Mannheim) and an enhanced chemiluminescence detection kit (ECL; Amersham, Little Chalfont, United Kingdom).The activities of JNK-1 and ERK-1 were measured as
described.22 In short, the JMML samples (107
cells/mL) were cultured with GM-CSF (10 ng/mL) or TNF- Transplantation of JMML cells into NOD/SCID mice and phenotyping of engrafted cells Immunodeficient mice underwent transplantation with primary JMML bone marrow cells, as described.11 Four weeks after transplantation, an osmotic mini-pump (0.25 µL/h; Alzet, Palo Alto, CA) was inserted into the peritoneal cavity. The pump contained 1 of 2 DNA enzymes (active or inactive; 10 µg/d) or saline, and the mice were randomly assigned to 1 of these 3 experimental groups. After death, the femoral bone marrows from the recipient mice were removed, stained with a human-specific anti-CD45 MoAb, and sorted with flow cytometry (FACScan; Becton Dickinson, Mountain View, CA). The extent of engraftment was assessed as the fraction of CD45+ cells among the total number of nucleated bone marrow cells. We have previously shown that this measure correlates well with values obtained from either Southern blotting with a human-specific DNA probe or fluorescence in situ hybridization using specific human chromosomal markers.11Statistics Each measurement was made in triplicate, and the resulting median value was used to calculate the mean and SEM for the 8 JMML patients and the 6 healthy controls. Differences were evaluated with 2-tailed Wilcoxon rank sum tests and assumed significant for P < .05.
In vitro functional analysis of the DNA enzyme The catalytic core of the DNA enzyme was derived from the in vitro selected 10-23 DNA enzyme.20 This catalytic motif can recognize and cleave mRNA sequences at a phosphodiester bond located between unpaired purine and paired pyrimidines. For the RNA ribozymes,20 the specificity of DNA enzymes was also determined by their antisense arms, which bind to target mRNA through Watson-Crick base pairing. In vitro screening experiments using AU- and GU-cleaving DNA enzymes with random antisense arms identified potential RNA sites that were accessible to DNA enzyme binding and cleavage (data not shown). In this study, we targeted a site located at the translation start AUG. To increase the DNA enzyme stability in biologic fluids, we replaced the hydroxy groups of the phosphate backbone within the DNA antisense arms with sulfur atoms to make it a phosphorothioate-modified DNA enzyme. Incubation of the in vitro-transcribed mRNA-Raf-1 with the DNA enzyme, which was designed to cleave its 5'-end, resulted in significant cleavage activity (Figure 1). This result underscores the in vitro accessibility of the targeted site.
GM-CSF, but not TNF-  .
Similarly, the MAPKs ERK-1 and JNK-1 were also present within the JMML
cells, and GM-CSF, but not TNF-, increased the
phosphorylation of both molecules (Figure 2B,C).
The lack of effect on Raf-1/MAPK activation by TNF-
The DNA enzyme reduced Raf-1 gene expression and colony formation of JMML cells, but not of normal bone marrow progenitor cells Figure 4 shows that the active DNA enzyme markedly impaired the expression of the Raf-1 gene in the JMML cells, whereas the inactive form was without any apparent effect.
We next tested the DNA enzyme on the proliferative capacity of
JMML cells in colony assays. As expected, the JMML cells spontaneously formed a large number of colonies, and this was further enhanced upon
addition of GM-CSF and TNF-
Given the known hypersensitivity of JMML cells to
GM-CSF,5 we examined this in JMML cells cocultured with
cytokines and the active and inactive DNA enzymes. Figure
5A indicates that this GM-CSF
hypersensitivity was virtually lost upon treatment with the active DNA
enzyme, being only partially restored with high concentrations of
exogenous GM-CSF. JMML cells did not show any apparent hypersensitivity
to TNF-
The lack of effect of the DNA enzymes on the formation of colony
subsets by normal bone marrow progenitor cells is shown in Table
2. This suggests the specificity and
possible safety of this DNA enzyme-based treatment of JMML.
The DNA enzyme inhibited GM-CSF and TNF- has
been suggested as a pathogenic mechanism in JMML.10 The
activation of Raf-1 by GM-CSF, but not TNF- (Figures 2 and 3),
prompted us to investigate the levels of GM-CSF and TNF- in JMML
cells following treatment with the Raf-1 DNA enzyme. The active DNA enzyme exerted a marked repression of the mRNAs for both GM-CSF and
TNF-, but not for mRNA-G-CSF (Figure
6A,B). JMML cell production of both
GM-CSF and TNF- protein, but not of G-CSF protein, was accordingly
diminished (Figure 6C). There were no significant differences between
GM-CSF and TNF- in the relative reductions of either the transcripts
or proteins.
Antileukemic effect of the DNA enzyme given to immunodeficient mice engrafted with JMML cells We employed the previously described xenograft model of JMML to test the efficacy of the active DNA enzyme in inhibiting leukemogenesis.11 Four weeks after transplantation, the mice developed overt leukemia (data not shown). At that time, a 2-week continuous treatment with the active DNA enzyme commenced, which led to a reduction in the JMML cell mass, as evidenced by a reduction from about 90% to 70% of the total number of nucleated femoral bone marrow cells (Figure 7). Importantly, mice receiving treatment with active DNA enzyme for a total of 4 weeks had a further substantial reduction in the leukemic cell mass to about 20%. All mice treated for 4 weeks with the active DNA enzyme survived; however, untreated mice or those treated with the inactive DNA enzyme all died of leukemia between 2 to 3 weeks after implantation of the peritoneal pump. Thus, none of the mice in these 2 latter groups were alive at the 4-week analysis (Figure 7).
JMML is a rare hematologic malignancy with few therapeutic options and a high mortality rate.1 There is considerable evidence linking GM-CSF stimulation of a dysregulated Ras pathway to the pathogenesis of JMML.4-8,14-19 Recent studies have revealed a crucial role of the MAPKs, downstream of Ras, in the proliferation and survival of many cell types.23 Hence, there is a rationale for a mechanism-based therapy targeting the Ras/Raf-1/MAPK pathway in this disease.24 For many chemical drugs, such as those directed against Ras, the mechanism of action is not well defined. By contrast, the specificity of Watson-Crick hybridization is the basis for rational drug design of nucleic acid enzymes, including ribozymes.25,26 Because of Watson-Crick base pairing, it is possible to specifically inhibit the expression of related proteins such as isoenzymes.27 Specific targeting of Raf-1 gene expression by a DNA enzyme inhibited JMML cell growth both in vitro and in vivo. Thus, the Raf-1 signaling pathway seems to be important in JMML. GM-CSF exerts its biologic effects by first binding to its cognate
receptor, resulting in activation of intracellular signaling pathways.
Although it has been shown that mutated GM-CSF receptor subunits can
induce autonomous proliferation of various cell lines, pathogenic
mutations in the genes coding for the GM-CSF ligand-receptor binding stimulates the Ras pathway at least partly via the Shc adaptor protein, so that the active Ras-GTP-bound form can associate with the inner surface of the cell membrane.30,31 This reaction is necessary for activation of molecules downstream of Ras, such as Raf-1 and the MAPKs, and it requires addition of a farnesyl group to Ras through a prenyl reaction. We recently showed that addition of specific farnesyl transferase inhibitors reduces JMML cell proliferation in vitro.19 Whether farnesyl transferase inhibitors are specific for RAS-mutated cells and whether they can be effective therapies for JMML patients are the subjects of an active JMML clinical protocol (no. AAML0122) in the Children's Oncology Group in North America. It has been shown that GM-CSF can phosphorylate Raf-1 in hematopoietic
cells and thereby activate the MAPK signaling cascade, leading to
increased proliferation and cell survival.12,13 In
contrast, TNF- To further explore the role of GM-CSF in stimulating Raf-1 in JMML, we
treated the JMML cells with a DNA enzyme designed to selectively cleave
mRNA-Raf-1 and thereby block protein synthesis. This active DNA enzyme
efficiently reduced the Raf-1 gene expression in JMML cells,
leading to a reduced proliferation in these cells. In contrast, growth
of normal bone marrow progenitor cells was not affected, and addition
of an inactive DNA enzyme had no effect on either JMML cells or normal
bone marrow progenitor cells in any assay. These data indicate that
this DNA enzyme may eventually prove to be a specific, and potentially
safe, therapeutic agent. Using growth-kinetic studies and colony assays
of JMML cells treated with the active DNA enzyme, we demonstrated an
apparent loss of GM-CSF hypersensitivity, the most consistent hallmark
of this disease.5 In contrast to these findings, JMML
cells showed no hypersensitivity to TNF- Collectively, these findings strongly suggest that GM-CSF is a
chief regulator in JMML cell proliferation. The data also argue that
the stimulatory action of GM-CSF is at least partly conveyed via the
Ras/Raf-1/MAPK signaling cascade. In the present study, we included
JMML patients who had either RAS or NF1 gene
mutations, as well as some who had neither mutation. The inhibitory
effect of the DNA enzyme was similar regardless of specific mutational status. This indicates that targeted therapeutic inhibition of Raf-1
can potentially be accomplished regardless of the mutation that
activates the Ras pathway upstream of Raf-1. Whether there is a
possible additional dysregulated activation of the JAK/STAT pathway,34 or aberrant cross-talk between the Ras/Raf-1
and JAK/STAT pathways upon GM-CSF stimulation in JMML cells, is not known. Although the action of TNF- In conclusion, the active DNA enzyme effectively led to the degradation of mRNA-Raf-1 with disruption of the downstream MAPK signaling cascade and the subsequent inhibition of JMML cell growth. Whether this mechanism-based approach holds promise as a clinical therapeutic in this fatal childhood leukemia warrants further examination.
Submitted November 14, 2001; accepted January 30, 2002.
Supported by the Norwegian Cancer Society, the Norwegian Research Council, and the Throne Holst Foundation, and by National Institutes of Health grant CA80916.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Per Ole Iversen, Institute for Nutrition Research, University of Oslo, PO Box 1046 Blindern, 0316 Oslo, Norway; e-mail: poiversen{at}hotmail.com.
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  S. Schubert, D. C. Gul, H.-P. Grunert, H. Zeichhardt, V. A. Erdmann, and J. Kurreck RNA cleaving '10-23' DNAzymes with enhanced stability and activity Nucleic Acids Res., October 15, 2003; 31(20): 5982 - 5992. [Abstract] [Full Text] [PDF]
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 R. J. Arceci, B. J. Longley, and P. D. Emanuel Atypical Cellular Disorders Hematology, January 1, 2002; 2002(1): 297 - 314. [Abstract] [Full Text]
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Top
Abstract
Introduction
-32P]-ATP (adenosine
triphosphate; Amersham) and 20 µg of either the fusion protein c-Jun
[1-169]-glutathione-S-transferase (GST) (Upstate
Biotechnology, Lake Placid, NY) or myelin basic protein (Sigma, St
Louis, MO). A 30-minute kinase reaction (30°C) was allowed before
termination with an equal volume of Laemmli sample buffer.
Phosphorylation of products was examined after SDS-PAGE and quantified
with a PhosphoImager (Molecular Dynamics, Sunnyvale, CA).
common subunits
of the GM-CSF receptor in primary JMML cells have not yet been
identified.