|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2469-2476
In Vitro and In Vivo Effects of a Farnesyltransferase Inhibitor on
Nf1-Deficient Hematopoietic Cells
By
Nidal Mahgoub,
Brigit R. Taylor,
Mary Gratiot,
Nancy E. Kohl,
Jackson B. Gibbs,
Tyler Jacks, and
Kevin M. Shannon
From the Department of Pediatrics, University of California, San
Francisco, CA; Department of Cancer Research; Merck and Co, West Point,
PA; and Department of Biology, Howard Hughes Medical Institute,
Massachusetts Institute of Technology, Cambridge, MA.
 |
ABSTRACT |
Oncogenic RAS alleles encode proteins that accumulate in the
guanosine triphosphate (GTP)-bound state. Because post-translational processing of Ras by farnesyltransferase is essential for biologic function, inhibitors of this enzyme have been developed as rational cancer therapeutics. We have investigated farnesyltransferase inhibitor
(FTI) L-744,832 in an in vivo murine model of myeloid leukemia that is
associated with inactivation of the Nf1 tumor suppressor gene.
Nf1 encodes a GTPase activating protein for Ras, and
Nf1-deficient (Nf1 /) hematopoietic cells show
hyperactive Ras signaling through the mitogen-activated protein (MAP)
kinase pathway. L-744,832 inhibited H-Ras prenylation in cell lines and in primary hematopoietic cells and abrogated the in vitro growth of
myeloid progenitor colonies in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). This FTI also partially blocked GM-CSF-induced MAP kinase activation, but did not reduce
constitutively elevated levels of MAP kinase activity in primary
Nf1/ cells. Injection of a single dose of 40 or 80 mg/kg
of L-744,832 increased the amount of unprocessed H-Ras in bone marrow
cells, but had no detectable effect on N-Ras. Adoptive transfer of
Nf1/ hematopoietic cells into irradiated mice induces a
myeloproliferative disorder that did not respond to L-744,832
treatment. We speculate that the lack of efficacy in this model is due
to the resistance of N-Ras and K-Ras processing to inhibition by this FTI.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
RAS PROTO-ONCOGENES encode 21-kD proteins
that regulate cellular growth and differentiation by transducing
signals from the plasma membrane to the nucleus through a number of
downstream effectors.1-3 The biochemical output of Ras
proteins is tightly regulated by their ability to cycle between an
active guanosine triphosphate (GTP)-bound state (Ras · GTP) and an
inactive guanosine diphosphate (GDP)-bound state
(Ras · GDP). RAS mutations are the most common
oncogenic alterations in human cancer cells and are frequently detected
in carcinomas of the colon and pancreas, as well as in myeloid
leukemias.4,5 Mutant RAS alleles encode proteins
that accumulate in the GTP-bound conformation because of defective GTP
hydrolysis.1-3
Ras proteins undergo post-translational processing at a common
C-terminal CAAX sequence in which C is cysteine, A is an aliphatic amino acid, and X is any amino acid.6-9 Ras processing is
initiated by farnesyltransferase, which transfers the C15
isoprenoid group from farnesyl diphosphate to the cysteine residue of
the CAAX box. The 3 terminal amino acids of farnesylated Ras are then
cleaved by an endopeptidase; this reaction is followed by methylation of the now-terminal cysteine by a specific methyltransferase. Because
farnesyltransferase is essential for biologic activity of mutant and
oncogenic Ras proteins, competitive inhibitors of this enzyme have been
developed as a new class of rational anticancer therapeutics.6-9
Studies evaluating different farnesyltransferase inhibitors (FTIs) in a
number of in vitro and in vivo systems have provided preclinical data
supporting selective antitumor effects of these compounds.7,9 FTIs have been shown to block Ras-induced
transformation in tissue culture cells, to inhibit the growth of many
cancer cell lines, and to halt proliferation of Ras-activated
xenografts in nude mice.7,9 FTI L-744,832 also showed
efficacy in 2 transgenic mouse models of breast cancer in which
RAS expression is driven from a mammary tumor virus (MMTV)
promoter.10,11 Barrington et al12 recently
reported that L744-832 induced breast tumor regression was associated
with apoptotic cell death that was partially independent of P53
function in MMTV-HRAS mice.
Although the data supporting antitumor effects of FTIs are impressive,
questions remain with respect to their biochemical mechanism(s) of
action, the reason(s) that these compounds selectively inhibit the
growth of malignant cells in many models, and the spectrum of antitumor
activity. In particular, although data from C elegans and
Drosophilia indicate that FTIs can modify phenotypes attributed
to hyperactive Ras,13,14 it is unclear if inhibition of
mammalian tumor cell growth is mediated through a direct effect on Ras
or by targeting other farnesylated proteins such as
RhoB.15-17 For example, in a study showing
that a FTI inhibited the growth of about 70% of human tumor cell
lines, the presence or absence of a RAS mutation did not
correlate with responsiveness.18 Understanding the
mechanisms of FTI action is complicated because oncogenic Ras proteins
perturb signaling in a number of different downstream effector
pathways.2,3
GTPase activating proteins (GAPs) negatively regulate Ras output by
accelerating the slow intrinsic Ras GTPase activity.19 The
neurofibromatosis type 1 tumor suppressor gene (NF1) encodes neurofibromin, which functions as a GAP for Ras.19
Individuals with NF1 are predisposed to specific cancers including
malignant peripheral nerve sheath tumors (MPNST), pheochromocytomas,
and juvenile myelomonocytic leukemia (JMML).20 MPNST cell
lines derived from patients with NF1 show elevated levels of
Ras · GTP and reduced GAP activity.21,22 Treatment with
an FTI blocked growth and reverted the malignant phenotype of one of
these cell lines; this result suggested that FTIs might prove useful in
treating tumors that are associated with inactivation of
NF1.23
JMML is a myeloproliferative disorder (MPD) of young children that is
characterized by overproduction of myeloid cells, a subacute but
relentless clinical course, and by the formation of excessive numbers
of myeloid progenitor colonies in cultures stimulated with the
hematopoietic growth factor granulocyte-macrophage colony-stimulating
factor (GM-CSF).24,25 Genetic and biochemical analyses of
JMML bone marrow cells from children with NF1 showed inactivation of
NF1, an increase in the percentage of Ras · GTP, a decrease
in neurofibromin-associated GAP activity, and evidence of in vivo
mitogen-activated protein (MAP) kinase activation.24-28 Furthermore, oncogenic RAS mutations are detected in the bone marrows of 20% to 30% of children with JMML and other myeloid leukemias who do not have NF1, but are conspicuously absent in children
with NF1.29 Taken together, these data provide compelling evidence that the tumor suppressor function of NF1 in myeloid cells is mediated through the ability of neurofibromin to negatively regulate Ras signaling.
The murine homolog of NF1 has been disrupted by targeted
homologous recombination to generate lines of Nf1 knockout
mice.30,31 Approximately 10% of heterozygous mice
(Nf1+/) develop a JMML-like MPD during the second year of
life that is associated with loss of the wild-type Nf1 allele
in bone marrow cells.31 Homozygous Nf1-deficient
embryos (Nf1/) die in utero between 12 and 14 days of
gestation from cardiac defects.30,31 Fetal
Nf1/ hematopoietic cells show a similar pattern of
aberrant in vitro myeloid progenitor colony growth as human JMML cells
in response to GM-CSF.27,32 Furthermore, adoptive transfer
of these Nf1-deficient fetal liver cells into irradiated
recipient mice consistently induces a JMML-like MPD that is
characterized by activated Ras-MAP kinase signaling in hematopoietic
cells.32,33 The subacute nature of this MPD, the central
role of hyperactive Ras in this and other myeloid malignancies, the
availability of primary hematopoietic cells for biochemical studies,
and the fact the disease phenotype does not depend on overexpressing a
RAS transgene from a heterologous promoter makes this in vivo
model appealing for preclinical studies of FTIs and other promising
anti-Ras therapeutics. Here, we report biochemical and hematologic data
from studies examining FTI L-744,832 in this experimental system.
| |
MATERIALS AND METHODS |
Compounds.
The L-744,832 used in these studies was provided by the Department of
Medicinal Chemistry, Merck Research Laboratories. Compactin was
purchased from Sigma (St Louis, MO) and was dissolved in dimethyl sulfoxide as previously described.34
Nf1 mice.
Nf1 knockout mice were produced and characterized as described
previously.31 Inbred 129/Sv mice were used as transplant recipients in experiment 1 and both 129/Sv and 129/Sv × C57BL/6 mixed
genetic background were used as recipients in the second experiment.
The fetal liver cells used as grafts were from a mixed 129/Sv × C57BL/6 genetic background. The experimental procedures were reviewed
and approved by the University of California San Francisco Committee
for Animal Research.
Transplant procedure.
Nf1+/ mice were mated to produce Nf1/ embryos.
Pregnant Nf1+/ females were killed by CO2
inhalation on day E13.5 and the embryos were removed through an
abdominal incision. Fetal livers were removed from embryonic tissues
and transferred to 1 mL of Iscove's Modified Dulbecco's Medium (IMDM)
supplemented with 20% fetal calf serum. Single-cell suspensions were
prepared by drawing the livers through progressively smaller needles
(22-gauge needle × 2; followed by a 25-gauge needle ×2). A total
of 4 to 14 × 106 mononuclear cells were injected into the
dorsal tail veins of recipients that have been conditioned with 1,000 cGy of total body irradiation. This protocol induces destruction of
recipient hematopoiesis and is associated with more than 80% survival
of animals engrafted with donor fetal liver cells (data not shown).
Treatment and observations.
The recipients of wild-type (Nf1+/+), mutant
(Nf1/) or of a mixture of Nf1+/+, and
Nf1/ cells were observed weekly and had complete blood
counts (CBCs) performed monthly for at least 2.5 months before being
assigned to the study. The genotype of circulating blood cells was
confirmed by Southern blot analysis by using a genomic Nf1
probe that distinguishes between the wild-type and targeted
alleles.31 Recipients transplanted with cells of each
genotype were assigned to either receive treatment with L-744,832 or to
an untreated control group. L-744,832 was diluted in a citrate-buffered sodium chloride solution and a volume of 0.1 to 0.5 mL was injected subcutaneously once per day. Study mice were weighed weekly and the
volume of L-744,832 injected was adjusted as necessary to maintain the
dose at 40 mg/kg (in the first experiment) or at 80 mg/kg (in the
second experiment). Mice were treated 5 days per week for 8 weeks in
the first experiment and 7 days per week for 4 weeks in the second
experiment. The animals were observed daily during treatment, and CBCs
with differential white blood cell counts were performed every 2 weeks.
Mice from the second experiment were killed after their final treatment
and their spleens were weighed. Bone marrow and spleen cells were
isolated for biochemical studies.
Hematopoietic progenitor colony growth.
Mononuclear cells from fetal livers were plated on the day they were
harvested in duplicate 35-mm plates at a final concentration of 5 × 104 cells per milliliter in culture medium consisting of
0.8% methyl cellulose supplemented with 30% fetal calf serum,
L-glutamine, and fully supplemented IMDM (Stem Cell Technologies,
Vancouver, British Columbia). Cells, recombinant murine GM-CSF, and
L-744,832 were added directly to the methylcellulose culture medium and the solution was mixed thoroughly before plating. The cells were grown
at 37°C in a humidified 21% O2, 5% CO2
incubator. Colonies derived from granulocyte-macrophage colony-forming
units (CFU-GM) were counted by indirect microscopy.
MAP kinase assay.
To measure endogenous MAP kinase activity, the ERK2 isoform was
selectively immunopurified by using an antibody directed to the
carboxy-terminal amino acids 345-358 (Santa Cruz Biotechnology Inc,
Santa Cruz, CA; Catalog No. sc-154). Cells were lysed in 20 mmol/L
Tris, pH 8, 137 mmol/L NaCl, 10% glycerol, 1% Triton X-100, 50 mmol/L
NaF, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 1 mmol/L vanadate, 1 mmol/L Pefabloc, 20 µg/mL leupeptin, and 10 µg/mL aprotinin, and
cell debris was removed by centrifugation. Lysates were rocked with 20 µL Protein A Sepharose FF (PAS) (Pharmacia, Piscataway, NJ) beads for
30 minutes at 4°C. Sepharose resin was pelleted and
cleared lysate was added to 10 µL ERK2 antibody and 90 µL PAS beads
and incubated overnight at 4°C with constant rotation. The beads were
pelleted and washed 2 times with lysis buffer and then once with kinase
buffer (20 mmol/L 3-[N-Morpholino] propanesulfonic acid [MOPS], pH
7.2, 30 mmol/L  glycerol phosphate, 5 mmol/L EGTA, 20 mmol/L
MgCl2, 1 mmol/L dithiothreitol [DTT], 1 mmol/L vanadate,
1 mmol/L Pefabloc, 20 µg/mL leupeptin, 10 µg/mL
aprotinin). Kinase activity was then measured
by adding 30 µL kinase buffer containing 13 µmol/L of
cold adenosine diphosphate (ATP) and 2.3 µCi of
33P -ATP (2,500 Ci/mmol) and 10 µg Elk-1 fusion protein
(New England Biolabs, Beverly, MA) and incubated for 30 minutes at
30°C. Assays were stopped by addition of sodium dodecyl sulfate (SDS)
sample buffer and incubation at 95°C for 5 minutes and resolved on
15% SDS-polyacrylamide gels. Incorporation of 33P into the
Elk-1 fusion protein was assessed by autoradiography and quantitated on
a phosphoimager.
Ras processing.
Lysates prepared as described above were rocked with 20 µL PAS FF
beads (Pharmacia) for 30 minutes at 4°C. Sepharose resin was pelleted
and the cleared lysate was incubated with Y13-259 beads at 4°C for 1 hour with constant rotation. Beads were then washed 3 times with
phosphate-buffered saline (PBS) and 2 mmol/L MgCl2,
resuspended in SDS sample buffer, and heated 95°C for 5 minutes. Ras
proteins were resolved on a 15% SDS-polyacrylamide gel and
electrotransferred to a PVDF membrane (Millipore). The membrane was
stained with Coomassie brilliant blue R, blocked for 1 hour in PBS,
0.1% Tween, 1 mmol/L EDTA (PBST), 3% bovine serum albumin (BSA), and
then incubated for 1 hour with a monoclonal antibody directed to either
H-Ras residues 157-181 (LAO69, Quality Biotech., diluted 1:10,000 in
PBST, 1% BSA) or N-Ras (F155, Santa Cruz Biotechnology, Inc.; diluted
1:400). The membrane was washed 3 times with
PBST, 1% BSA and incubated for 1 hour with goat anti-mouse IgG-peroxidase conjugate (diluted 1:10,000 in PBST, 1% BSA). Proteins were visualized with an enhanced chemiluminescence detection system (ECL; Amersham, Arlington Heights, IL).
| |
RESULTS |
We first tested the ability of L-744,832 to inhibit Ras processing in
COS 7 cells and in the THP-1 human myeloid leukemia cell line, which
contains an oncogenic NRAS mutation.35 In these and
subsequent experiments, cell lysates were first immunoprecipitated with
a pan-Ras antibody (Y13-259), followed by electrophoresis, transfer,
and blotting with a second anti-Ras antibody (see Materials and
Methods). Compactin (100 µmol/L), which blocks Ras farnesylation by
depleting farnesyl diphosphate levels, was included as a positive control.34 L-744,832 concentrations of 10 µmol/L and 25 µmol/L were tested on the basis of previous data showing that the
growth of 70% of human tumor cell lines was inhibited by 2 to 20 µmol/L of this compound.18 Figure
1 shows that all of the H-Ras detected in
untreated COS 7 and THP-1 cells has been processed, and that either
L-744,832 or compactin induced the appearance of a higher molecular
weight band, which corresponds to the unprenylated protein.
View larger version (21K):
[in this window]
[in a new window]
| Fig 1.
L-744,832 inhibits H-Ras farnesylation in cultured cell
lines. COS 7 or THP-1 cells were cultured for 48 hours, lysed,
immunoprecipitated with pan-Ras antibody Y13-259, blotted, and probed
with an antibody specific for H-Ras. Cells were either untreated (U) or
were cultured with either 10 µmol/L or 25 µM of L-744,832 or with
100 µmol/L of compactin. Unprocessed H-Ras is indicated by the white
arrow and processed Ras is designated with a black arrow.
|
|
We next investigated bone marrow cells from recipient mice that had
been reconstituted with hematopoietic cells from either wild-type
(Nf1+/+) or knockout (Nf1/) embryos. Because
these primary cells did not appear as robust as the cell lines (this was particularly true of the compactin-treated cells), the time in
culture was reduced from 48 to 20 hours. As in the cultured cell lines,
exposure to L-744,832 or compactin was associated with the appearance
of a slower-migrating band that corresponds to unprenylated H-Ras (Fig
2). The relative amount of unprocessed Ras
was lower in primary cells than in either cell line and appeared to be
concentration dependent in cells treated with L-744,832 (Fig 2).
Reprobing these blots with an antibody specific for N-Ras revealed no
inhibition of Ras processing (data not shown).
View larger version (25K):
[in this window]
[in a new window]
| Fig 2.
L-744,832 inhibits H-Ras farnesylation in murine
hematopoietic cells. Mononuclear cells harvested from the bone marrows
of mice transplanted with wild-type or Nf1/ cells were
cultured for 20 hours. Cells were either untreated (U) or were cultured
with either 10 µmol/L or 25 µmol/L of L-744,832 or with 100 µmol/L of compactin. The cell lysates were prepared and H-Ras
processing was assessed as described in Fig 1. Unprocessed H-Ras is
indicated by the white arrow and processed Ras is designated with a
black arrow.
|
|
Although these data indicate that L-744,832 can partially inhibit H-Ras
prenylation in cultured cell lines and in primary hematopoietic cells,
they provide no insights regarding the functional consequences of
treatment. To approach this question, we assayed the effects of
L-744,832 on the growth of the CFU-GM myeloid progenitor colonies from
fetal hematopoietic cells in response to GM-CSF. We were specifically
interested in GM-CSF because this growth factor is known to signal
through Ras and is implicated in the abnormal growth of human JMML
cells and of murine Nf1/ fetal hematopoietic
cells.24,25,27,32,36 In the CFU-GM assay, 10 µmol/L of
L744,732 abrogated colony growth from Nf1/ or control Nf1+/+ fetal hematopoietic cells in response to saturating
concentrations of GM-CSF (Fig 3). In
addition to a marked reduction in the number of colonies that formed in
methylcellulose cultures containing 1 µmol/L of L-744,832,
the individual CFU-GM were smaller and had many fewer cells than those
that developed in control dishes (data not shown).
View larger version (26K):
[in this window]
[in a new window]
| Fig 3.
L-744,832 inhibits CFU-GM colony growth from fetal liver
cells. Fetal hematopoietic cells from Nf1+/+ ( ),
Nf1+/ ( ), or Nf1/ ( ) embryos were
cultured in methylcellulose medium supplemented with 1 unit/mL of
recombinant murine GM-CSF with either 0, 1, 10, or 25 µmol/L of
L-744,832. CFU-GM colonies were counted after 7 days in
culture.
|
|
We also measured MAP kinase activities in Nf1+/+ and
Nf1/ hematopoietic cells isolated from recipient mice
after adoptive transfer. As recently reported for purified c-kit
positive cells,33 basal MAP kinase activities were
generally higher in the unfractionated Nf1/ hematopoietic
cells than in Nf1+/+ cells that we studied, with some variation
noted between individual experiments. Treatment with 25 µmol/L
L-744,832 had no effect on the baseline MAP kinase activity of either
Nf1/ or Nf1+/+ cells; however, the FTI blunted activation in response to either GM-CSF or interleukin-3 (IL-3) (Fig
4). In contrast, treatment with compactin
both reduced baseline MAP kinase activities and eliminated the response
to growth factor stimulation (Fig 4).
View larger version (25K):
[in this window]
[in a new window]
| Fig 4.
L-744,832 blunts MAP kinase activation in response to
hematopoietic growth factors. Mononuclear cells collected from
recipients engrafted with either Nf1+/+ (left 6 lanes) or
Nf1/ (right 9 lanes) cells were incubated at 37°C
overnight with medium alone (), medium plus 25 µmol/L L-744,832
(L) or medium and 100 µmol/L compactin (C). The cultures were then
split and either not stimulated with any growth factors () or
stimulated with 10 ng/mL of either GM-CSF (G) or IL-3 (I). The cells
were lysed and MAP kinase activity was measured. The top graph shows a
phosphoimager plot (counts per minute [CPM]-background) for each
condition over the raw data from the autoradiograph. Loss of
Nf1 is associated with constitutive activation of MAP kinase
(compare lane 1 with 7) that is unaffected by L-744,832 (compare lane 7 with 10) but is inhibited by compactin (compare lane 7 with 13). In
this experiment, L-744,832 blunted MAP kinase activation induced by
GM-CSF by 31% in Nf1+/+ cells (compare lane 2 with 4) and
by 30% in Nf1/ cells (compare lane 8 with 11).
Similarly, FTI treatment decreased the kinase activity measured in
Nf1/ cells stimulated with IL-3 by 37% (compare lane 9 with 12). In contrast, compactin completely abrogated this induction of
MAP kinase activity (compare lane 2 with 6, lane 8 with 15, and lane 9 with 16).
|
|
To assess the in vivo effects of FTI treatment on Ras processing in
hematopoietic cells, a single L-744,832 dose of either 40 mg/kg or 80 mg/kg was administered to a group of untransplanted mice, and the
animals were then sacrificed at defined time intervals. L-744,832-treated animals showed a progressive increase in the proportion of unprenylated H-Ras that was dose and time dependent (Fig
5A). However, when this blot was stripped
and reprobed with an antibody specific for N-Ras, a single band
corresponding to processed Ras was detected (Fig 5B).
View larger version (25K):
[in this window]
[in a new window]
| Fig 5.
L-744,832 inhibits H-Ras, but not N-Ras, farnesylation in
vivo. Wild-type mice were injected with a single dose of either 40 mg/kg of 80 mg/kg of L-744,832, then killed 2, 4, 16, 24, or 48 hours
after treatment. Mononuclear cells were isolated from hematopoietic
tissues, lysed, immunoprecipitated, subject to gel electrophoresis, and
blotted as described in Materials and Methods. Bands corresponding to
unprocessed and processed Ras are labeled with open and closed arrows,
respectively. Cells from untreated mice are labeled "U" and a
lysate prepared from a compactin-treated cell line is labeled
"C." (A) Western blot probed with an H-Ras antibody. (B) The same
Western blot was stripped and reprobed with an N-Ras antibody.
|
|
In a breast cancer model in which expression of an HRAS
oncogene is driven by an MMTV promoter, treatment with L-744,832 at a
daily dose of 40 mg/kg was associated with objective tumor responses that were superior to doxorubicin with no evidence of
toxicity.10 Therefore, we assigned mice that had been
engrafted with either Nf1/ or with Nf1+/+ fetal
liver cells to either receive treatment with L-744,832 (at a dose of 40 mg/kg/d 5 days each week for 8 weeks) or vehicle for 8 weeks. Two mice
of each transplant genotype were assigned to each arm (total: n = 8).
All of the Nf1/ recipients were entered at least 3 months
after adoptive transfer and had markedly elevated white blood cell
counts (Table 1). Although recipient mice
tolerated FTI administration with no apparent adverse effects, there
was no improvement in white blood cell counts (Table 1).
Based on the dose-response effect of L-744,832 on H-Ras prenylation
(Fig 5) and on the absence of toxicity in the initial cohort, a second
experiment was performed to evaluate an intensive treatment protocol in
which the FTI was administered daily at a dose of 80 mg/kg/d for 4 weeks. In addition to 10 mice that had been transplanted with
Nf1/ or Nf1+/+ cells alone, we studied 4 animals
that had been transplanted with a mixture of Nf1/ and
Nf1+/+ cells to determine if treatment with L-744,832 might selectively inhibit the growth of Nf1/ cells in vivo.
Nf1/ cells have a proliferative advantage over
Nf1+/+ cells, and mice that receive these transplants develop a
myeloid disorder that is similar to what is seen in mice that are given
Nf1/ cells only (data not shown). As in the first
experiment, there were no differences in white blood cell counts
between FTI-treated and control mice (Table 2). At sacrifice, the
spleens of both FTI-treated and control Nf1/ recipients
were enlarged and were infiltrated with myeloid cells (Table 2).
Immunoprecipitation and Western blotting experiments showed partial
inhibition of H-Ras processing in bone marrow cells from mice that
received L-744,832 with no effect on N-Ras (data not shown). MAP kinase activities varied widely in unstimulated bone marrow cells from treated
and untreated mice; however, bone marrow cells from mice that had been
given the FTI showed a blunted MAP kinase response to GM-CSF (1.7-fold
above baseline kinase activity v 3.3-fold for cells from
untreated mice). This higher dose of L-744,832 was associated with
significant clinical toxicity that included decreased activity, weight
loss, abscess formation, and crusted skin lesions at the sites of
injections and blood draws.
| |
DISCUSSION |
A number of observations have suggested that Ras is not the primary
biochemical target of FTIs in mammalian cells.6-9 In a
comprehensive survey in which L-744,832 inhibited the growth of over
70% of 42 human tumor cell lines, in vitro efficacy did not correlate
with the presence of a RAS mutation.18
Farnesylation of lamin B was inhibited to a similar degree by FTI
treatment in 1 resistant cell line and 1 sensitive line; however, MAP
kinase activation in response to epidermal growth factor was only
blocked in sensitive cells.18 Studies of K-Ras processing
have also cast doubt on the notion that FTIs act as Ras inhibitors in
mammalian cells. In contrast to H-Ras, the carboxy termini of K-Ras and N-Ras proteins are good substrates for processing by geranylgeranyl transferase type 1 (GGTase-1),7,9,37 and
geranylgeranylation of K-Ras has recently been demonstrated in
vivo.38 Consistent with this, treatment with both an FTI
and a GGTase inhibitor were required to block K-Ras prenylation in 5 human tumor cell lines.39 Furthermore, in both tissue
culture and in nude mouse xenograft assays, FTI treatment inhibited the
growth of 2 cancer cell lines with oncogenic KRAS mutations
(A-549 and Calu-1), but had no effect on K-Ras
processing.40
If FTIs do not function as Ras inhibitors, what other farnesylated
proteins might account for the dramatic antiproliferative effects of
these compounds in many assays? Studies performed by Prendergast et
al15 and Lebowitz et al16,17 implicate RhoB, which is essential for Ras-induced transformation, as a potential in
vivo target of FTIs. Their results are consistent with a
recent study showing that RhoB function is required for oncogenic Ras to stimulate DNA synthesis in fibroblasts.41 In the absence of RhoB function, activated Ras induces p21Waf1 and, in
turn, blocks proliferation.41 These data further suggest that cells expressing oncogenic Ras might be highly sensitive to
FTI-induced growth arrest because they efficiently induce
p21Waf1 in response to inhibition of RhoB.
These questions regarding the mechanisms of FTI action underscore the
need for studies in relevant immunocompetent animal models. A major
advantage of the JMML-like MPD that follows adoptive transfer of
Nf1-deficient fetal cells for testing therapeutics is that
clinical responses can be correlated with biochemical effects by
measuring Ras prenylation and MAP kinase activity in primary
hematopoietic cells. Furthermore, compelling genetic and biochemical
data implicate deregulated Ras signaling as playing a central role in
these and other myeloid leukemias.5,42,43 We found that
L-744,832 partially inhibited H-Ras processing in hematopoietic cells
but had no effect on N-Ras. Although we did not have a reliable
antibody that is specific for K-Ras, Western blotting with pan-Ras
antibodies indicated that most of the Ras was fully processed in vivo
and, therefore, suggested that K-Ras was also resistant to L-744,832
(data not shown). This is consistent with other data showing that K-Ras
is relatively insensitive to inhibition by FTIs.37,39,40 At
the doses tested in this study, L-744,832 had no effect on MAP kinase
activity in unstimulated hematopoietic cells, but blunted the normal
induction of this kinase in response to GM-CSF. Given these biochemical
data and the observation that almost all of the RAS mutations
in myeloid leukemia involve KRAS or NRAS, it is not
surprising that FTI treatment was clinically ineffective in a model in
which hyperactive Ras underlies abnormal myeloid cell growth.
In 2 previous studies, FTIs inhibited the in vitro growth of THP-1
cells and of all 6 other human leukemia cell lines
tested.18,44 Similarly, we found that 10 µmol/L 744,832 abrogated CFU-GM colony formation in methylcellulose cultures of
wild-type and Nf1-deficient fetal hematopoietic cells. In this
system, colony formation from individual myeloid progenitor cells
requires extensive proliferation in response to high concentrations of
cytokine growth factors. It is likely that the profound inhibition of
colony formation that we observed was because of the ability of
L-744,832 to partially block MAP kinase activation in response to
GM-CSF. This interpretation is supported by our finding of relatively
small colonies containing few cells in cultures containing 1 µmol/L
L-744,832. Our results also differ from a previous report in which FTI
treatment induced growth inhibition and morphologic reversion in an
NF1-deficient MPNST cell line.23 Potential
explanations include: (1) FTIs might differentially affect the growth
of neural crest and hematopoietic cells, (2) genetic variations between
immortal tumor-derived cell lines and primary cells, and (3) higher
concentrations of drug may be achievable in tissue culture than in
whole animals. Whatever the reason, the discordant results seen in
vitro and in this relevant animal model emphasize the importance of
testing experimental therapeutics in an in vivo preclinical setting.
The efficacy of L-744,832 has been evaluated previously in 2 murine
cancer models in which tumors arise spontaneously in immunocompetent mice.10,11 In a line of transgenic mice that overexpress
oncogenic HRAS from an MMTV promoter, Kohl et al10
found that treatment with 40 mg/kg/d of L-744,832 induced tumor
regression in 100% of the animals. The FTI was much more effective
than the conventional chemotherapeutic agent doxorubicin in this model,
and a subsequent study showed an increased rate of apoptosis in tumors
from L-744,832-treated animals.12 In a recent study, the
MMTV promoter was also used to drive overexpression of a wild-type
NRAS gene.11 The response was not as dramatic as in
the previous study; however, tumor growth was reduced in mice that
received L-744,832 and this was associated with an increase in the
percentages of apoptotic cells within the tumors. Importantly, almost
all of the N-Ras was processed normally in the tumor cells of these
FTI-treated mice.11
We conclude that treatment with FTI L-744,832 partially inhibits H-Ras
processing and blunts GM-CSF-induced MAP kinase activation in a murine
model of Ras-activated myeloid leukemia. However, N-Ras and K-Ras
proteins were processed normally even at an FTI dose of 80 mg per kg,
and there was no clinical efficacy. These data provide
direct evidence that L-744,832 (and perhaps other FTIs presently in
preclinical and clinical trials) does not efficiently inhibit N-Ras or
K-Ras processing at clinically tolerable doses in mice. Our finding
that H-Ras prenylation is partially inhibited in vivo is consistent
with the impressive antitumor effects of L-744,832 seen in an
MMTV-HRAS model of breast cancer.10 However, L-744,832 reduced the growth of MMTV-NRAS-driven breast tumors without inhibiting N-Ras processing.11 It remains to be
determined if the discrepant therapeutic results are explained by
differences between how breast cancer and myeloid cells respond to
FTI-induced inhibition of RhoB or other non-Ras proteins, by the
presence or absence of specific cooperating mutations in tumor cell
clones, and/or by genetic differences in the way hyperactive Ras is
induced in these murine models. The growing evidence that the antitumor effects of FTIs are not caused by inhibition of K-Ras or N-Ras and the
markedly different results obtained in murine breast cancer and myeloid
leukemia models, emphasize both the importance of identifying the
authentic in vivo targets of this promising class of anticancer agents
as well as the need to test the clinical efficacy of these compounds
against a broad spectrum of human tumors.
| |
ACKNOWLEDGMENT |
We are grateful to Allen Oliff and George Hartman for support and
helpful discussions, to Gideon Bollag for the gift of antibody Y13-259
and for advice on performing the kinase assays, and to Connie Gebbia
and Jennifer Alkire for administrative assistance.
| |
FOOTNOTES |
Submitted April 6, 1999; accepted May 28, 1999.
Supported by the Leukemia Society of America Translational Research
Grant Award No. 6306-97 and by the National Institutes of Health (NIH)
Grant No. R01 CA72614. N.M. was supported by NIH Training Grant No. DK07636.
N.M. and B.R.T. contributed equally to this work.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kevin M. Shannon, MD, Room HSE-302, Box
0519, University of California, San Francisco, CA 94143; e-mail:
kevins{at}itsa.ucsf.edu.
| |
REFERENCES |
1.
Bourne HR, Sanders DA, McCormick F:
The GTPase superfamily: A conserved switch for diverse cell functions.
Nature
348:125, 1990[Medline]
[Order article via Infotrieve]
2.
Khosrabi-Far R, Der CJ:
The Ras signal transduction pathway.
Cancer Metastasis Rev
13:67, 1994[Medline]
[Order article via Infotrieve]
3.
Downward J:
Control of ras activation.
Cancer Surv
27:87, 1996[Medline]
[Order article via Infotrieve]
4.
Bos JL:
ras oncogenes in human cancer: A review.
Cancer Res
49:4682, 1989[Abstract/Free Full Text]
5.
Rodenhuis S:
ras and human tumors.
Semin Cancer Biol
3:241, 1992[Medline]
[Order article via Infotrieve]
6.
Gibbs JB, Oliff A, Kohl NE:
Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic.
Cell
77:175, 1994[Medline]
[Order article via Infotrieve]
7.
Gibbs JB, Oliff A:
The potential of farnesyltransferase inhibitors as cancer chemotherapeutics.
Annu Rev Pharmacol Toxicol
37:143, 1997[Medline]
[Order article via Infotrieve]
8.
Kohl NE, Conner MW, Gibbs JB, Graham SL, Hartman GD, Oliff A:
Development of inhibitors of protein farnesylation as potential chemotherapeutic agents.
J Cell Biochem
22:145, 1995
9.
Cox AD, Der CJ:
Farnesyltransferase inhibitors and cancer treatment: Targeting simply ras?
Biochim Biophys Acta
1333:F51, 1997[Medline]
[Order article via Infotrieve]
10.
Kohl NE, Omer CA, Conner MW, Anthony NJ, Davide JP, DeSolms J, Giuliani EA, Gomez RP, Graham SL, Hamilton K, Handt LK, G.D. H, Koblan KS, Kral AM, Miller PJ, Mosser SD, O'Neill TJ, Rands E, Schaber MD, Gibbs JB, Oliff A:
Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice.
Nat Med
1:792, 1995[Medline]
[Order article via Infotrieve]
11.
Mangues R, Corral T, Kohl NE, Symmans WF, Lu S, Malumbres M, Gibbs JB, Oliff A, Pellicer A:
Antitumor effect of a farnesyl protein transferase inhibitor in mammary and lymphoid tumors overexpressing N-ras in transgenic mice.
Cancer Res
58:1253, 1998[Abstract/Free Full Text]
12.
Barrington RE, Subler MA, Rands E, Omer CA, Miller PJ, Hundley JE, Koester SK, Troyer DA, Bearss DJ, Conner MW, Gibbs JB, Hamilton K, Koblan KS, Mosser SD, O'Neill TJ, Schaber MD, Senderak ET, Windle JJ, Oliff A, Kohl NE:
A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.
Mol Cell Biol
18:85, 1998[Abstract/Free Full Text]
13.
Hara M, Han M:
Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorabditis elegans.
Proc Natl Acad Sci USA
92:3333, 1995[Abstract/Free Full Text]
14.
Kauffmann RC, Qian Y, Vogt A, Sebti SM, Hamilton AD, Carthew RW:
Activated Drosophila ras 1 selectively suppressed by isoprenyl transferase inhibitors.
Proc Natl Acad Sci USA
92:10919, 1995[Abstract/Free Full Text]
15.
Prendergast GC, Davide JP, deSolms J, Giuliani EA, Graham SL, Gibbs JB, Oliff A, Kohl NE:
Farnesyltransferase inhibition causes morphological reveresion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.
Mol Cell Biol
14:4193, 1994[Abstract/Free Full Text]
16.
Lebowitz PF, Davide JP, Prendegast GC:
Evidence that farnesyltransferase inhibitors suppress Ras transformation by interfering with Rho activity.
Mol Cell Biol
15:6613, 1995[Abstract]
17.
Lebowitz PF, Casey PJ, Prendegast GC, Thissen JA:
Farnesyltransferase inhibitors alter the prenylation and growth-stimulation function of RhoB.
J Biol Chem
272:15591, 1997[Abstract/Free Full Text]
18.
Sepp-Lorenzino L, Ma Z, Rands E, Kohl NE, Gibbs JB, Oliff A, Rosen N:
A peptidomimetic inhibitor of fernesyl protein transferase blocks the anchorage dependent and independent growth of human tumor cell lines.
Cancer Res
55:5302, 1995[Abstract/Free Full Text]
19.
Boguski M, McCormick F:
Proteins regulating Ras and its relatives.
Nature
366:643, 1993[Medline]
[Order article via Infotrieve]
20.
Side LE, Shannon KM:
The NF1 gene as a tumor suppressor, in
Upashyaya M,
Cooper DN
(eds):
Neurofibromatosis type 1, Human Molecular Genetics series. Oxford, UK, Bios Scientific Publishers, 1998, p 133.
21.
DeClue JE, Papageorge AG, Fletcher JA, Diehl SR, Ratner N, Vass WC, Lowy DR:
Abnormal regulation of mammalian p21ras contributes to malignant tumor growth in von Recklinghausen (type 1) neurofibromatosis.
Cell
69:265, 1992[Medline]
[Order article via Infotrieve]
22.
Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J:
Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients.
Nature
356:713, 1992[Medline]
[Order article via Infotrieve]
23.
Yan N, Ricca C, Fletcher J, Glover T, Seizinger BR, Manne V:
Fernesyltransferase inhibitors bloch the neurofibromatosis type I (NF1) malignant phenotype.
Cancer Res
55:3569, 1996[Abstract/Free Full Text]
24.
Emanuel PD, Shannon KM, Castleberry RP:
Juvenile myelomonocytic leukemia: Molecular understanding and prospects for therapy.
Mol Med Today
2:468475, 1996
25.
Arico M, Biondi A, Pui C-H:
Juvenile myelomonocytic leukemia.
Blood
90:479, 1997[Free Full Text]
26.
Shannon KM, O'Connell P, Martin GA, Paderanga D, Olson K, Dinndorf P, McCormick F:
Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders.
N Engl J Med
330:597, 1994[Abstract/Free Full Text]
27.
Bollag G, Clapp DW, Shih S, Adler F, Zhang Y, Thompson P, Lange BJ, Freedman MH, McCormick F, Jacks T, Shannon K:
Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in murine and human hematopoietic cells.
Nat Genet
12:144, 1996[Medline]
[Order article via Infotrieve]
28.
Side L, Taylor B, Cayouette M, Connor E, Thompson P, Luce M, Shannon K:
Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders.
N Engl J Med
336:1713, 1997[Abstract/Free Full Text]
29.
Kalra R, Paderanga D, Olson K, Shannon KM:
Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras.
Blood
84:3435, 1994[Abstract/Free Full Text]
30.
Brannan CI, Perkins AS, Vogel KS, Ratner N, Nordlund ML, Reid SW, Buchberg AM, Jenkins N, Parada L, Copeland N:
Targeted disruption of the neurofibromatosis type 1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues.
Genes Dev
8:1019, 1994[Abstract/Free Full Text]
31.
Jacks T, Shih S, Schmitt EM, Bronson RT, Bernards A, Weinberg RA:
Tumorigenic and developmental consequences of a targeted Nf1 mutation in the mouse.
Nat Genet
7:353, 1994[Medline]
[Order article via Infotrieve]
32.
Largaespada DA, Brannan CI, Jenkins NA, Copeland NG:
Nf1 deficiency causes Ras-mediated granulocyte-macrophage colony stimulating factor hypersensitivity and chronic myeloid leukemia.
Nat Genet
12:137, 1996[Medline]
[Order article via Infotrieve]
33.
Zhang Y, Vik TA, Ryder JW, Srour EF, Jacks T, Shannon K, Clapp DW:
Nf1 regulates hematopoietic progenitor cell growth and Ras signaling in response to multiple cytokines.
J Exp Med
187:1893, 1998[Abstract/Free Full Text]
34.
James GL, Brown MS, Cobb MH, Goldstein JL:
Benzodiazepine peptidomimetic BZA-5B interrupts the MAP kinase pathway in H-Ras-transformed Rat-1 cells, but not in untransformed cells.
J Biol Chem
269:27705, 1994[Abstract/Free Full Text]
35.
Murray MJ, Cunningham JM, Parada LF, Lebowitz P, Weinberg R:
The HL-60 transforming sequence: A ras oncogene coexisting with altered myc genes in hematopoietic tumors.
Cell
33:749, 1983[Medline]
[Order article via Infotrieve]
36.
Satoh T, Nakafuku M, Miyajima A, Kaziro Y:
Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.
Proc Natl Acad Sci USA
88:3314, 1991[Abstract/Free Full Text]
37.
James G, Goldstein JL, Brown MS:
Resistance of K-RasBv12 proteins to farnesyltransferase inhibitors in Rat1 cells.
Proc Natl Acad Sci USA
93:4454, 1996[Abstract/Free Full Text]
38.
Rowell CA, Kowalczyk JJ, Lewis MD, Garcia AM:
Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo.
J Biol Chem
272:14093, 1997[Abstract/Free Full Text]
39.
Lerner EC, Zhang T-T, Knowles DB, Qian Y, Hamilton AD, Sebti SM:
Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX prptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines.
Oncogene
15:1283, 1997[Medline]
[Order article via Infotrieve]
40.
Sun Y, Qian Y, Hamilton AD, Sebti SM:
Both farnesyltransferase geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts.
Oncogene
16:1467, 1998[Medline]
[Order article via Infotrieve]
41.
Olson MF, Paterson HF, Marshall CJ:
Signals from Ras and Rho GTPases interact to regulate expression of p21 Waf1/Cip1.
Nature
394:295, 1998[Medline]
[Order article via Infotrieve]
42.
Sawyers CL, Denny CT:
Chronic myelomonocytic leukemia: Tel-a-kinase what its all about.
Cell
77:171, 1994[Medline]
[Order article via Infotrieve]
43.
Shannon KM:
The Ras signaling pathway and the molecular basis of myeloid leukemogenesis.
Curr Opin Hematol
3:305, 1995
44.
Nagasu T, Yoshimatsu K, Rowell C, Lewis MD, Garcia AM:
Inhibition of human tumor xenograft growth by treatment with the farnesyl transferase inhibitor B956.
Cancer Res
55:5310, 1995[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. H. Gutmann
Using Neurofibromatosis-1 to Better Understand and Treat Pediatric Low-Grade Glioma
J Child Neurol,
October 1, 2008;
23(10):
1186 - 1194.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. M. Wahlstrom, B. A. Cutts, M. Liu, A. Lindskog, C. Karlsson, A.-K. M. Sjogren, K. M. E. Andersson, S. G. Young, and M. O. Bergo
Inactivating Icmt ameliorates K-RAS-induced myeloproliferative disease
Blood,
August 15, 2008;
112(4):
1357 - 1365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. W. Wojtkowiak, F. Fouad, D. T. LaLonde, M. D. Kleinman, R. A. Gibbs, J. J. Reiners Jr., R. F. Borch, and R. R. Mattingly
Induction of Apoptosis in Neurofibromatosis Type 1 Malignant Peripheral Nerve Sheath Tumor Cell Lines by a Combination of Novel Farnesyl Transferase Inhibitors and Lovastatin
J. Pharmacol. Exp. Ther.,
July 1, 2008;
326(1):
1 - 11.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. C. Daginakatte and D. H. Gutmann
Neurofibromatosis-1 (Nf1) heterozygous brain microglia elaborate paracrine factors that promote Nf1-deficient astrocyte and glioma growth
Hum. Mol. Genet.,
May 1, 2007;
16(9):
1098 - 1112.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. F. Khalaf, F.-C. Yang, S. Chen, H. White, W. Bessler, D. A. Ingram, and D. W. Clapp
K-ras Is Critical for Modulating Multiple c-kit-Mediated Cellular Functions in Wild-Type and Nf1+/- Mast Cells
J. Immunol.,
February 15, 2007;
178(4):
2527 - 2534.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. P. Olive and D. A. Tuveson
The use of targeted mouse models for preclinical testing of novel cancer therapeutics.
Clin. Cancer Res.,
September 15, 2006;
12(18):
5277 - 5287.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Barkan, S. Starinsky, E. Friedman, R. Stein, and Y. Kloog
The Ras Inhibitor Farnesylthiosalicylic Acid as a Potential Therapy for Neurofibromatosis Type 1.
Clin. Cancer Res.,
September 15, 2006;
12(18):
5533 - 5542.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. R. Mattingly, J. M. Kraniak, J. T. Dilworth, P. Mathieu, B. Bealmear, J. E. Nowak, J. A. Benjamins, M. A. Tainsky, and J. J. Reiners Jr.
The Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Inhibitor PD184352 (CI-1040) Selectively Induces Apoptosis in Malignant Schwannoma Cell Lines
J. Pharmacol. Exp. Ther.,
January 1, 2006;
316(1):
456 - 465.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Meuwissen and A. Berns
Mouse models for human lung cancer
Genes & Dev.,
March 15, 2005;
19(6):
643 - 664.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Dasgupta, W. Li, A. Perry, and D. H. Gutmann
Glioma Formation in Neurofibromatosis 1 Reflects Preferential Activation of K-RAS in Astrocytes
Cancer Res.,
January 1, 2005;
65(1):
236 - 245.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. T. Le, N. Kong, Y. Zhu, J. O. Lauchle, A. Aiyigari, B. S. Braun, E. Wang, S. C. Kogan, M. M. Le Beau, L. Parada, et al.
Somatic inactivation of Nf1 in hematopoietic cells results in a progressive myeloproliferative disorder
Blood,
June 1, 2004;
103(11):
4243 - 4250.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Lancet and J. E. Karp
Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy
Blood,
December 1, 2003;
102(12):
3880 - 3889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Ingram, M. J. Wenning, K. Shannon, and D. W. Clapp
Leukemic potential of doubly mutant Nf1 and Wv hematopoietic cells
Blood,
March 1, 2003;
101(5):
1984 - 1986.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. J. Houghton, P. C. Adamson, S. Blaney, H. A. Fine, R. Gorlick, M. Haber, L. Helman, S. Hirschfeld, M. G. Hollingshead, M. A. Israel, et al.
Testing of New Agents in Childhood Cancer Preclinical Models: Meeting Summary
Clin. Cancer Res.,
December 1, 2002;
8(12):
3646 - 3657.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Demiroglu, E. J. Steer, C. Heath, K. Taylor, M. Bentley, S. L. Allen, P. Koduru, J. P. Brody, G. Hawson, R. Rodwell, et al.
The t(8;22) in chronic myeloid leukemia fuses BCR to FGFR1: transforming activity and specific inhibition of FGFR1 fusion proteins
Blood,
December 15, 2001;
98(13):
3778 - 3783.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Morgan, O. Dolp, and C. W. M. Reuter
Cell-cycle-dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling
Blood,
March 15, 2001;
97(6):
1823 - 1834.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. W. M. Reuter, M. A. Morgan, and L. Bergmann
Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?
Blood,
September 1, 2000;
96(5):
1655 - 1669.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. D. Emanuel, R. C. Snyder, T. Wiley, B. Gopurala, and R. P. Castleberry
Inhibition of juvenile myelomonocytic leukemia cell growth in vitro by farnesyltransferase inhibitors
Blood,
January 15, 2000;
95(2):
639 - 645.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION