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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4279-4286
Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating
Factor Is Toxic to Blasts From Patients With Juvenile Myelomonocytic
Leukemia and Chronic Myelomonocytic Leukemia
By
Arthur E. Frankel,
Michael Lilly,
Robert Kreitman,
Donna Hogge,
Miloslav Beran,
Melvin H. Freedman,
Peter D. Emanuel,
Chris McLain,
Philip Hall,
Edward Tagge,
Marc Berger, and
Connie Eaves
From the Wake Forest Comprehensive Cancer Center/Bowman Gray School
of Medicine, Winston-Salem, NC; the Division of Medical Oncology, the
Department of Medicine, University of Washington, Seattle; Laboratory
of Molecular Biology, National Cancer Institute, National Institutes of
Health (NIH), Bethesda, MD; Terry Fox Laboratory, British Columbia
Cancer Agency, Vancouver, BC, Canada; the Leukemia Department,
University of Texas M.D. Anderson Cancer Center, Houston; the Division
of Haematology/Oncology, The Hospital for Sick Children, Toronto,
Ontario, Canada; Comprehensive Cancer Center, University of Alabama at
Birmingham; and the Departments of Surgery and Pharmaceutical Sciences,
Medical University of South Carolina, Charleston.
 |
ABSTRACT |
We have previously demonstrated that human granulocyte-macrophage
colony-stimulating factor fused to a truncated diphtheria toxin
(DT388-GM-CSF) is toxic to patient acute myeloid leukemia progenitors
bearing the GM-CSF receptor, but not normal marrow progenitors. We now
report that exposure of mononuclear cells from five of seven (71%)
juvenile myelomonocytic leukemia (JMML) patients and from 12 of 20 (60%) adult chronic myelomonocytic leukemia (CMML) patients to
10-9 mol/L DT388-GM-CSF for 48 hours in culture reduces the
number of cells capable of forming colonies in semisolid medium
(colony-forming units-leukemia) 10-fold to 300-fold (1 to
2.5 log decrease). In contrast, normal myeloid progenitors
(colony-forming unit-granulocyte-macrophage) from six different donors
treated and assayed under identical conditions were consistently
insensitive to the same fusion toxin even when treated as highly
purified CD34+ cells. The leukemic progenitors from the
two other JMML patients showed intermediate sensitivity to DT388-GM-CSF
and the leukemic progenitors from eight of the 20 (40%) CMML patients
were not different from normal progenitors. Parallel measurements of
the number and affinity of GM-CSF receptors on cells from the same samples showed no consistent differences between JMML, CMML, and normal
light density or CD34+ bone marrow cells. The increased
sensitivity of leukemic progenitors from all JMML progenitors and some
CMML patients to the fusion toxin is therefore not likely to be
explained by an increased density of GM-CSF receptors on these cells.
We also examined the DT388-GM-CSF sensitivity of two murine cell lines
transfected with cDNAs encoding varying portions of the human GM-CSF
receptor  and/or  chains. These studies showed that
high-affinity ligand binding was sufficient for DT388-GM-CSF-induced
toxicity, as this could occur even in the absence of functional signal
transduction and that the background of the host cell had a major
influence on the degree to which this decreased the toxicity of
DT388-GM-CSF. The selective sensitivity to DT388-GM-CSF of leukemic
progenitors from a majority of JMML and CMML patients suggests that
this agent could have therapeutic potential for some patients with
these diseases.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
CHRONIC MYELOMONOCYTIC leukemia (CMML) is
a distinct clonal hematopoietic disorder seen primarily in older men.
It is characterized by peripheral monocytosis, dysmyelopoiesis, and minimal numbers of blasts in the marrow. Patients with CMML have a
median survival of only 1 to 2 years.1 Death is usually due to infection, bleeding, or transformation of the disease to an acute
leukemic phase. Intensive chemotherapy, low dose cytosine arabinoside,
hydroxyurea, and ATRA have all proven ineffective in
generating long-term remissions.2
Juvenile myelomonocytic leukemia (JMML), formerly termed juvenile
chronic myelogenous leukemia, is another rare clonal myeloproliferative disease with similar features, but occurs in early childhood. Like
CMML, it is characterized by an excessive in vitro proliferation of
myeloid progenitors that show hypersensitivity to
granulocyte-macrophage colony-stimulating factor (GM-CSF).3
JMML is clinically manifested by failure to thrive, marked
hepatosplenomegaly, adenopathy, skin rash, anemia, thrombocytopenia,
leukocytosis with monocytosis, and less than 20% marrow blasts.
Abnormalities in the neurofibromatosis and RAS genes have been found in
50% to 60% of patients' hematopoietic progenitor cells, but
chromosomal abnormalities are rare. While the use of allogeneic stem
cell transplantation in JMML has resulted in some durable remissions,
recurrent leukemia remains a significant problem, and the 5-year
disease-free survival rate is in the range of 25%.4 Thus,
novel therapeutics with unique mechanisms of action are needed for both
of these myeloid disorders.
One class of therapeutics of potential interest are targeted toxins
consisting of protein toxins covalently linked to peptide ligands. The
ligand directs the molecule to the surface of specific cell types, and
the toxin moiety then enters the cell and catalytically inactivates
protein synthesis. We previously synthesized a fusion toxin composed of
the first 388 amino acid residues of diphtheria toxin fused to human
GM-CSF (DT388-GM-CSF) and tested its activity on acute myeloid leukemia
(AML) blasts.5,6 These studies demonstrated a potent and
selective cytotoxicity of DT388-GM-CSF on leukemic progenitors from AML
patients with GM-CSF receptor-expressing blasts, whereas normal
clonogenic progenitors were relatively insensitive to DT388-GM-CSF. In
this report, we now show that the progenitors from a majority of CMML
and JMML patients also show an increased sensitivity to DT388-GM-CSF
and that this is not explained by an increased expression of GM-CSF
receptors on their blasts by comparison to highly purified
CD34+ marrow cells from normal marrow. In addition,
investigation of two murine cell lines expressing different mutant
human GM-CSF receptors showed that sensitivity to DT388-GM-CSF was
variably affected when the ligand binding activity of the transduced
receptor was retained, but the signaling function eliminated in the two different host cell types.
| |
MATERIALS AND METHODS |
JMML, CMML, and normal marrow cells.
With the approval of the respective Institutional Review Boards and
after obtaining informed patient or parental consent, heparinized blood
samples were obtained from seven patients with a diagnosis of JMML and
22 patients with CMML. Heparinized marrow aspirates were also obtained
from three allogeneic bone marrow donors and from vertebral harvests of
three normal cadaveric donors (Northwest Tissue Center, Seattle, WA).
Blood and marrow cells were diluted 1:1 with RPMI 1640 medium and
layered over 0.3 vol of Ficoll-Hypaque (1.070 g/mL;
Pharmacia, Piscataway, NJ). After density gradient centrifugation at
2,000 rpm for 30 minutes, light density cells (<1.077 g/mL) were
diluted threefold with RPMI 1640 and centrifuged again at 1,300 rpm for
10 minutes. Cells were then usually cryopreserved in 50% fetal calf
serum (FCS) with 10% dimethyl sulfoxide (DMSO). Upon thawing, cells
were suspended in RPMI 1640 medium with 15% FCS and 2 mmol/L
L-glutamine (GIBCO-BRL, Grand Island, NY), 50 U/mL penicillin G
(GIBCO), and 50 µg/mL streptomycin sulfate (GIBCO) with
1 mg/mL DNAse I (Sigma Chemicals, St Louis, MO). In some cases, normal
CD34+ cells ( 99.9% pure) were isolated by immunoaffinity
separation and fluorescence-activated cell sorting (FACS) as previously
described.7,8 These methods enriched significantly for
colony-forming unit-granulocyte-macrophage (CFU-GM). Cells were then
counted and used for GM-CSF receptor measurements and studies of
progenitor sensitivity to DT388-GM-CSF as described below.
CD34+ selection was not performed on leukemic samples
because of the known heterogeneity in leukemic progenitor
phenotype.9
Cell lines.
The hematopoietic growth factor-dependent M1 mouse leukemia cell
line10 and 32D mouse myeloid cell line (a gift of Dr J. Greenberger, University of Pittsburgh, Pittsburgh, PA) were maintained in RPMI 1640 with 15% FCS supplemented with 10% WEHI-3B conditioned medium.11 Both cell lines were transfected with cDNAs
encoding the human wild-type GM-CSF receptor chain, the term1
mutation of the human GM-CSF receptor chain (truncated immediately
after the transmembrane domain),12 and/or the -2
variant of the human GM-CSF receptor chain13 using
previously reported methods.14 The -2 variant is a
normally occuring splice variant of the chain and is fully
biologically active for proliferation.13 The human GM-CSF
receptor chain cDNA contained in the mammalian expression plasmid
pEF-BOS15 was a gift of Dr N. Nicola (Walter and Eliza Hall
Institute, Melbourne, Australia). The full-length human GM-CSF receptor
-2 chain cDNA was ligated into the plasmid pLXSN,16 as
was the term1 mutant chain cDNA. pLXSN plasmids containing the chain cDNAs were transfected along with a plasmid encoding the pol and
env sequences into human 293 cells. Twenty-four hours later,
supernatants containing recombinant retroviruses were used to infect
cells of the amphotropic packaging line PA317. G418-resistant clones
were then selected and analyzed for the production of chain-transducing retroviruses. These were then used to transduce M1
and 32D cells with human GM-CSF receptors. In some cases, the chain
cDNA was introduced first, via electroporation, along with a puromycin
resistance plasmid for selection. Expression of the chain was
confirmed by flow cytometry of puromycin-resistant clones, using a
murine monoclonal antibody to an external domain of the chain
(AMRAD, Melbourne, Australia). Positive clones or M1 cells were then
retrovirally infected (by cocultivation for 24 hours on the appropriate
packaging cell line) to introduce either the -2 chain or the
truncated chain (term1). Again, positive clones were selected by
FACS analysis of G418-resistant clones, using a murine monoclonal
antibody to the GM-CSF receptor chain (Santa Cruz Biochemical,
Santa Cruz, CA).
Fusion toxin.
DT388-GM-CSF was prepared and purified as previously
described17 and stored as 830 µg/mL in phosphate-buffered
saline (PBS) plus 1% human serum albumin (HSA) at  20°C. The
material used in this study was found to kill human HL60 cells at an
inhibitory concentration (IC)50 of 2 × 10 12 mol/L using a 48-hour thymidine incorporation
assay to assess HL60 cell kill. The preparation at
109 mol/L reduced the clonogenic activity of HL60
cells in a semisolid medium by a factor of 3,000-fold.6
GM-CSF receptor density measurements.
Aliquots of 1 to 6 × 106 cells in RPMI 1640 plus
2.5% bovine serum albumin (BSA), 20 mmol/L Hepes, and 0.2% sodium
azide were mixed with varying amounts of 125I Bolton-Hunter
labeled human GM-CSF (80 to 120 µCi/µg, NEX249; DuPont, Boston, MA)
with or without excess (1,500 ng) cold GM-CSF (Immunex, Seattle, WA) in
a total volume of 150 µL in 1.5-mL Eppendorf tubes. Cells were
incubated at 37°C for 30 minutes and then layered over a 200-µL
oil phthalate mixture (1 part dioctylphthalate and 1.5 parts
dibutylphthalate, Aldrich, Milwaukee, WI). After centrifugation at
12,000 rpm for 1 minute in a microfuge at room temperature, both
pellets and supernatants were saved and counted in an LKB-Wallac 1260 Multi-gamma counter (Turku, Finland) gated for
125I with 50% counting efficiency. Background cpm were
calculated by linear extrapolation from incubations with excess cold
GM-CSF. Scatchard plots of specific bound/free versus specific bound
cpm were made. Experiments were performed in duplicate. Receptor
number/cell was calculated by dividing the x intercept by (specific
activity in µCi/µg times the cell number times 4.2 × 108); dissociation constant (kd) was
calculated by multiplying the x-intercept by 2.7 × 1013 divided by (the y-intercept times the specific
activity). A statistical software package (Statsoft, Tulsa, OK) was
used to perform linear regressions. Receptor densities of 0 were
recorded when there was no specific 125I binding or when
the kd values measured were negative.
Cellular sensitivity to DT388-GM-CSF.
Sensitivity to DT388-GM-CSF of progenitors in normal and leukemic
samples was tested by exposing the cells in suspension culture for 48 hours and then assessing their residual ability to form colonies in
semisolid cultures.6,18 For this, aliquots of 5 × 105 CMML, JMML, normal marrow light density, or purified
CD34+ cells were incubated with different concentrations of
DT388-GM-CSF (0 to 4 × 108 mol/L) in 150 µL
of RPMI 1640 medium plus 15% FCS supplemented with 50 ng/mL G-CSF
(Amgen, Thousand Oaks, CA) in 96-well flat-bottomed Costar plates at
37°C/5% CO2 in air. After 16 to 48 hours, 100-µL samples from each well were mixed with 3 mL of RPMI 1640 plus 15% FCS
plus 50 ng/mL human G-CSF, 50 ng/mL human GM-CSF, 10% human 5637 bladder carcinoma cell line conditioned medium (5637 CM) and 0.3%
agarose (SeaPlaque; FMC Bioproducts, Rockland, ME) and the mixture then
poured into 35-mm gridded Petri dishes (Nunc, Naperville, IL) and
allowed to solidify before incubation at 37°C/5% CO2
in air and the number of colonies containing greater than 20 cells
assessed 10 to 20 days later. Both of the concentrations of toxin
reducing colony formation by 50% (IC50) and the maximal (log) cell kill values compared with control cells not exposed to toxin
were calculated as previously described.6 All experiments were performed in duplicate, and control cells were treated identically to DT388-GM-CSF-treated cells, but with the absence of any toxin.
Receptor properties and DT388-GM-CSF sensitivity of mutant GM-CSF
receptor cell lines.
The six different murine cell lines expressing different chains of the
human GM-CSF receptor used in the present studies are listed (see Table
4). Receptor affinity and density were measured as described above for
patients' cells. Their sensitivities to DT388-GM-CSF were determined
using inhibition of 3H-leucine incorporation to assess
effects on protein synthesis and 3H-thymidine incorporation
to assess effects on proliferation after 48 hours incubation of the
cells with varying concentrations of fusion proteins as previously
described.5,19 These assays correlate well with
measurements of clonogenicity for most myeloid cell
lines.5,19
| |
RESULTS |
Clinical history of JMML and CMML patients studied.
Peripheral blood cells from 7 JMML and 22 previously untreated CMML
patients were studied. The age, peripheral blood cell counts,
percentage of blasts in the marrow at the time of collection of the
samples, and the subsequent response of each patient to therapy are
summarized in Table 1.
Progenitor cells from some JMML and CMML patients show increased
sensitivity to DT388-GM-CSF in vitro.
As shown in Table 2, all of the leukemic
and normal samples showed colony growth in semisolid medium in the
absence of fusion toxin. Exposure of JMML and CMML cells to
DT388-GM-CSF for 48 hours resulted in a significant (P < .05, Student's t-test) loss of progenitor activity in five of the
seven JMML cases (71%) and in 12 of the 20 CMML cases studied (57%,
Fig 1 and Table 2). The cells
in the other two JMML cases showed intermediate sensitivities to
DT388-GM-CSF with less than 1 log cell kill and an IC50 of 1010 mol/L to 1011 mol/L. In
experiments with the other eight CMML samples, there was no significant
loss of progenitor activity even in the presence of 4 × 108 mol/L DT388-GM-CSF. The DT388-GM-CSF sensitivity
of the progenitors from two other CMML samples was not studied. A lack
of sensitivity was exhibited by the clonogenic progenitors in six
normal marrow samples treated and assessed under identical conditions.
These included three light density normal marrow cell preparations, as
well as three highly purified (>99%) CD34+ cell
preparations isolated from another three normal donors.
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| Fig 1.
Effect of exposing cells to DT388-GM-CSF in liquid
culture for 48 hours followed by measurement of the remaining
progenitor activity in semisolid assays. (A through C) Cells from
representative CMML patients; (D) cells from normal marrow samples and
HL60 cells (plated at 2 × 104 cells/dish); (E) cells from
a representative JMML patient. (A) ( ), KP; ( ), RM; ( ), SB;
( ), EB. (B) ( ), ED; ( ), LP; ( ), WB; ( ), FB. (C) (),
PP; (), GW; (), GN; (), DJ. (D) (), U11811; (), U11454;
(), U11802; (), HL60; (E) (), AJ.
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GM-CSF receptor numbers and affinities on JMML and CMML cells are
normal.
To determine whether the increased DT388-GM-CSF sensitivity of the
progenitors from all JMML and some CMML patients might reflect an
increased expression of GM-CSF receptors, receptor densities (and
affinities for GM-CSF) were determined by Scatchard analysis for most
of the samples studied biologically. Representative Scatchard plots are
shown in Fig 2. The results for five of the seven JMML patients' samples and all 21 CMML patients' samples and
for cells from five normal marrow donors are shown in
Table 3. The light density JMML and CMML
cells all showed 120 high-affinity GM-CSF receptors/cell with a mean
GM-CSF receptor density of 260 ± 90 (mean ± standard error of
mean [SEM]) for the JMML cells and of 590 ± 100 for the CMML
cells. The number of GM-CSF receptors per normal light density marrow
cell was 340 ± 130 (n = 5). The average values for GM-CSF receptor
numbers and affinities for light density JMML and CMML peripheral blood
cells and normal marrow light density and CD34+ cells are
not significantly different (P > .05, Student's
t-test). There was no correlation between DT388-GM-CSF
sensitivity (IC50) and the level of GM-CSF receptor
expression among the JMML or CMML samples studied (r = .2).
Thus, the lack of a toxic action of DT388-GM-CSF on leukemic cells from
some patients with JMML or CMML cannot be attributed to a decreased
level of GM-CSF receptor expression as appears to be the case in
AML.6
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| Fig 2.
Scatchard plot of JMML cells (patient CF) obtained using
1.3 × 106 cells per aliquot and
125I-GM-CSF at a specific activity of 54 µCi/µg.
r2 = .92 using 0.1 to 2 pmol
125I-GM-CSF, kd = 6 × 1012 mol/L and
receptor density = 164 GM-CSF receptors/cell.
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Investigations of the role of altered GM-CSF receptors on
DT388-GM-CSF sensitivity.
To evaluate other types of alterations in GM-CSF receptor structure or
signaling that might enhance cellular sensitivity to DT388-GM-CSF,
several murine factor-dependent hematopoietic cell lines expressing
different parts of the and/or chains of the human
GM-CSF receptor were created as described in Materials and Methods. As
expected, cell lines expressing only the or chain of the human
GM-CSF receptor failed to bind human GM-CSF
(Table 4). In contrast, all four cell lines
expressing both the extracellular part of the chain and the chain of the human GM-CSF receptor did bind ligand and with a
high-affinity. The numbers of ligand-binding receptors per transduced
cell ranged from 2,700 to 9,400; ie, approximately 10-fold higher than
seen with primary normal or JMML/CMML human hematopoietic cells (Table
3). Interestingly, all four mouse cell lines capable of binding human
GM-CSF were sensitive to DT388-GM-CSF at concentrations similar to
those that kill primary human leukemia cells (Table 2 and Hogge et
al6). However, the presence of an intracytoplasmically
truncated chain, which blocks GM-CSF-induced signaling did cause a
fivefold reduction in sensitivity to DT388-GM-CSF in M1 cells and a
100-fold reduction in sensitivity in 32D cells exposed to the same
agent. Subunit alone expressed at high levels (10,000 to
20,000/cell) showed reduced but present sensitivity to DT388-GM-CSF.
This suggests that the induction of cell kill in these cells is
enhanced by the activation of internal receptor signaling events.
| |
DISCUSSION |
JMML and CMML are both heterogeneous disorders with variable
cytogenetics and clinical courses generally affecting younger children
(<5 years old) and older men (>50 years old),
respectively.3,4,20,21 Nevertheless, for both, the
prognosis without allogeneic bone marrow transplantation is dismal.
Among the 22 CMML patients studied here, there was marked variability
in the peripheral white blood cell (WBC) count, the extent of
monocytosis, and the percentage of blasts in the marrow. Also, none
showed the t(5;12) cytogenetic abnormality that has been associated
with CMML. The majority were older men in agreement with the reported
prevalence of the disease in this group.1 The WBC was, on
average, higher and the platelet count, on average, lower than
previously reported1; however, a skewing of these values
may have been incurred by the biassed selection of peripheral blood
samples suitable for cryopreservation.
The presence of high-affinity GM-CSF receptors was demonstrated on the
light density cells present in both JMML and CMML blood, as well as
light density and highly purified CD34+ cells from normal
marrow samples. In all cases, the GM-CSF receptor numbers measured
represent averages for heterogeneous mixtures of different cell types,
only a fraction of which possess progenitor activity.9 The
fact that the progenitors from a majority of the JMML and CMML patients
(70% and 60%, respectively) that we studied were inactivated after
exposure of the cells to DT388-GM-CSF confirms that these cells also
express GM-CSF receptors. Similarly, mouse cells engineered to express
a human GM-CSF-binding human GM-CSF receptor acquired sensitivity to
DT388-GM-CSF, whereas those expressing only the human GM-CSF receptor
chain did not (Table 4) and, in human AML, sensitivity to
DT388-GM-CSF was seen only in patients whose blasts showed evidence of
GM-CSF receptor expression at normal (or higher) levels.6
GM-CSF receptor expression, although prerequisite for DT388-GM-CSF
sensitivity, is, however, likely not to be the only factor involved.
Normal human progenitors are known to express GM-CSF receptor mRNAs and
respond to GM-CSF activation22,23 despite their
insensitivity to DT388-GM-CSF,5,17,24,25 as confirmed here.
Moreover, our assessment of GM-CSF receptor numbers on normal CD34+ cells (enriched for CFU-GM)7,8 suggests
that similar values would be obtained for those with progenitor
activity, despite their relative resistance to DT388-GM-CSF. However,
we were not able to measure receptors quantitatively on individual
progenitors. On the other hand, it is interesting to note that normal
mouse CFU-GM have been found to be sensitive to a similar fusion toxin (DT389-mGM-CSF).26 Similarly, why some AML progenitors
become much more sensitive to DT388-GM-CSF than their normal
counterparts does not appear to be explained by differences in GM-CSF
receptor expression.6 In the present study, an increased
sensitivity to DT388-GM-CSF of JMML and CMML progenitors could also not
be attributed to an abnormally elevated expression of GM-CSF receptors on their cells. Two studies have shown that some adult CMML patients display a hypersensitivity of their progenitors to GM-CSF stimulation in vitro, similar to JMML, although others have failed to show such
GM-CSF hypersensitivity.1,27 It will thus be of interest to
determine whether this feature is associated with DT388-GM-CSF sensitivity in a subgroup of CMML patients.
Progenitors that are not sensitive to DT388-GM-CSF may simply resemble
their normal counterparts where a postbinding step involving, for
example, receptor internalization or expression of antiapoptotic
proteins5,6,28 may contribute to the observed resistance.
Higher intracellular concentrations of antiapoptotic proteins in normal
as compared with AML progenitors has been reported29,30 and
could explain the failure of normal cells to undergo apoptosis after
exposure to concentrations of DT388-GM-CSF that are sufficient to
engage their GM-CSF receptors and which can effectively kill other
cells with similar GM-CSF receptor densities. The fact that two cell
lines expressing human GM-CSF receptors capable of binding ligand, but
not signaling, were still susceptible to DT388-GM-CSF-induced killing,
albeit with differently reduced sensitivities, further underscores the
likelihood that other intracellular factors are important determinants.
Additional studies with other mutant or transduced cell lines should
help to identify what these are and their mechanisms of action.
JMML is a disorder with deregulated signal transduction through the Ras
signaling pathway resulting in hypersensitivity to GM-CSF, but normal
sensitivity to interleukin-3 and G-CSF.31 Previous studies have demonstrated a normal GM-CSF receptor on JMML
cells by fluorescence-labeled GM-CSF binding and by subunit sequence
analysis.32,33 Hypersensitivity to GM-CSF
appears to be a unifying characteristic in JMML patients. The
present finding of an intermediate to high sensitivity toDT388-GM-CSF of the progenitors in all JMML patient samples tested suggests these
two biological features may be mechanistically related.
Whatever the mechanisms that underlie the elevated DT388-GM-CSF
sensitivity of some leukemic progenitors, its wide therapeutic index
makes it an attractive therapeutic agent for consideration. The
sensitivity of these rare myeloid leukemias resembled that of AML based
on IC50 and log cell kill.6 13-cis
retinoic acid demonstrates an overall 40% to 50% response rate in
JMML, but does not appear to be sufficient to induce durable
remissions.31,34 Hydroxyurea can prolong the survival of
some CMML patients better than etoposide, but the median survival is
still less than 20 months, and patients with this disease continue to
die of bleeding, infection, and transformation to AML.35
The use of topotecan in CMML has produced some complete remissions, but
has also been found to cause severe mucositis, diarrhea, infections,
and a poor overall median survival (<1 year).36 All trans
retinoic acid (ATRA) significantly reduces CMML colony formation in
vitro, but in vivo, the ATRA syndrome was seen and survival was not
improved.37 Thus, in the absence of effective strategies
for the treatment of either JMML or CMML, the experiments in this study
underscore the need for further preclinical development of DT388-GMCSF
as a new therapeutic agent that may have some promise for patients with
a broad spectrum of poor prognosis leukemias.
| |
ACKNOWLEDGMENT |
We thank members of the Stem Cell Assay Service and the Division of
Hematology of the B.C. Cancer Agency and Vancouver Hospital for
provision of patient materials, normal marrow CD34+ cell
purification, and clinical information. We thank Drs C. Chomienne and
B. Cassinat (Laboratoire de Biologie Cellulaire Hematopoietique,
University of Paris, Paris, France) for access to JMML sample AJ and
Drs J. Prchal and R. Mayor (Birmingham, AL) for clinical information.
We also thank J. Nicholson for photography and graphic analysis.
| |
FOOTNOTES |
Submitted June 5, 1998;
accepted July 27, 1998.
Supported by Leukemia Society of America Grant No. 6114-98 (to A.F.), NIH Grants No. R01CA76178 (to A.F.), CA45672 (to M.L.), U01CA60407 (to P.E.), and R01CA54116 (to E.T.) and grants from the
National Cancer Institute of Canada (NCIC) (to C.E. and D.H.) with
funds from the Terry Fox Run. C.E. is a Terry Fox Cancer Research
Scientist of the NCIC.
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 Arthur E. Frankel, MD, Hanes 4046, Bowman
Gray School of Medicine, Med Center Drive, Winston-Salem, NC 27157.
| |
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Abstract
Introduction