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NEOPLASIA
From The Center for Blood Research, Dana-Farber Cancer
Institute, and Harvard Institutes of Medicine and Howard Hughes Medical
Institute, Harvard Medical School, Boston, MA.
Primitive hematopoietic progenitors from some patients with
Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML)
express aberrant transcripts for interleukin 3 (IL-3) and granulocyte
colony-stimulating factor (G-CSF), and exhibit autonomous proliferation
in serum-free cultures that is inhibited by anti-IL-3 and anti-IL-3
receptor antibodies. Expression of the product of the Ph chromosome,
the BCR/ABL oncogene, in mice by retroviral bone marrow
transduction and transplantation induces CML-like leukemia, and some
leukemic mice have increased circulating IL-3, and perhaps
granulocyte-macrophage colony-stimulating factor (GM-CSF). These
observations raise the possibility of autocrine or paracrine cytokine
production in the pathogenesis of human CML. Mice with homozygous
inactivation of the Il-3 gene, the Gm-csf gene,
or both, were used to test the requirement for these cytokines for induction of CML-like disease by BCR/ABL. Neither IL-3 nor
GM-CSF was required in donor, recipient, or both for induction of
CML-like leukemia by p210 BCR/ABL. Use of novel mice
deficient in both IL-3 and GM-CSF demonstrated that the lack of effect
on leukemogenesis was not due to redundancy between these hematopoietic
growth factors. Analysis of cytokine levels in leukemic mice where
either donor or recipient was Il-3 Human chronic myeloid leukemia (CML) is a
myeloproliferative disease characterized by excessive clonal production
of maturing myeloid cells.1,2 The principal genetic
abnormality in CML is the Philadelphia (Ph) chromosome, the product of
a balanced t(9;22) translocation that is found in multiple myeloid,
B-lymphoid, and sometimes T-lymphoid lineages, suggesting that the
translocation event occurred in an early multipotential or stemlike
cell. The Ph translocation fuses the c-ABL gene on
chromosome 9q34 to BCR on 22q11, generating a chimeric
BCR/ABL gene. The product of this gene, the Bcr/Abl fusion
protein, is a dysregulated nonreceptor tyrosine kinase that transforms
fibroblasts,3 cytokine-dependent hematopoietic cell
lines,4 and primary bone marrow B-lymphoid cells5 in vitro. Retroviral transduction of the
BCR/ABL gene into mouse bone marrow cells, followed by
transplantation into syngeneic recipient mice, leads to development of
CML-like leukemia in all recipients,6-8 demonstrating that
BCR/ABL is the principal cause of CML.
BCR/ABL-induced murine CML-like disease is characterized by
massive expansion of maturing myeloid cells, with infiltration of
spleen, liver, and lungs by malignant myeloid cells that carry the
BCR/ABL provirus and express Bcr/Abl protein. The cell that initiates the CML-like disease is an early multipotential hematopoietic progenitor cell.6 Murine CML-like leukemia is therefore an accurate and faithful model of human CML that has proven useful for
analyzing the molecular pathogenesis of this
disease.6,9,10
The mechanism by which Bcr/Abl induces CML is not known.
Recently, aberrant production of hematopoietic cytokines has been suggested to play a role in the pathogenesis of CML. CML patients do
not typically have increased plasma or serum levels of
cytokines.11,12 However, in some patients with chronic
phase CML and predominantly Ph+ primitive progenitors,
isolated CD34+CD38 Several molecular biologic studies support a role for cytokine
production, particularly of IL-3, in BCR/ABL transformation. BCR/ABL activates many signal transduction pathways that
overlap with those induced by cytokines, including the
Ras/MAPK15 and Jak/STAT pathways.16
Expression of BCR/ABL induces the secretion of IL-3 and
granulocyte-macrophage colony-stimulating factor (GM-CSF) from
cytokine-dependent hematopoietic cell lines,4,17 although the ability of BCR/ABL to transform such cells to become
independent of cytokine for survival and proliferation does not require
autocrine secretion. Monocyte/macrophage tumors induced by
BCR/ABL after retroviral bone marrow transduction and
transplantation secrete G-CSF and GM-CSF,18 and mice with
BCR/ABL-induced CML-like myeloproliferative disease have
been reported to have increased circulating IL-3 and
GM-CSF.8 Finally, direct expression of IL-3 in mouse bone marrow by retroviral gene transfer leads to a myeloproliferative illness in recipients closely resembling that induced by transduction of BCR/ABL.19
The murine retroviral bone marrow transduction/transplantation model
system offers an ideal method to test the requirement of cytokines for
leukemogenesis by BCR/ABL. If a naturally occurring or
targeted mouse germline mutation exists in a given gene, in principal
it is straightforward to test whether that gene is necessary for
induction of CML-like leukemia by BCR/ABL, by using the
mutant mice as donors and recipients in this assay. In practice, such an experiment will be most informative if the mutant mice have relatively normal baseline hematopoiesis and bone marrow donor and
recipient function. Fortunately, these conditions are met for mutations
in the mouse Il-3 and Gm-csf genes. These loci
are closely linked on murine chromosome 11, and each has been
inactivated in mice by homologous recombination. Mice lacking IL-3 have
defective delayed-type hypersensitivity20 and decreased
mast cell/basophil responses to parasitic infection,21
whereas mice lacking GM-CSF have pulmonary aveolar
proteinosis,22,23 but both mutant mice have essentially
normal hematopoiesis. Recently, mice with mutations in both
Il-3 and Gm-csf have been generated by a serial
gene targeting approach, and these animals show only modest
perturbations in hematopoiesis.24 Here, we have used these
mutant mice as donors, recipients, or both in the bone marrow
transduction/transplantation system to determine the role of these
cytokines in the pathogenesis of BCR/ABL-induced murine
CML-like leukemia.
Mouse strains
Bone marrow transduction/transplantation
Cytokine enzyme-linked immunosorbent assays (ELISAs) Peripheral blood was obtained from diseased mice by sampling of the retro-orbital venous plexus or by cardiac puncture at autopsy. Total immunoreactive IL-3 and GM-CSF (dynamic range, 0.19-200 ng/mL) were determined using ACCUCYTE murine IL-3 and GM-CSF competitive enzyme immunoassay kits (Cytimmune Sciences, College Park, MD); equivalent results were obtained using heparinized plasma or serum. Selected samples were also tested in Quantikine M kits (R&D Systems, Minneapolis, MN) for either IL-3 or GM-CSF, dynamic range 7.8 to 500 pg/mL. For controls, samples were obtained from recipients of nontransduced marrow or recipients of marrow transduced with MSCVneo virus at 4 weeks after transplantation.
Elevated circulating IL-3, but not GM-CSF, in mice with BCR/ABL-induced CML-like leukemia We tested plasma from mice with BCR/ABL-induced CML-like disease for increased IL-3 and GM-CSF using ELISAs. Plasma or serum IL-3 levels are undetectable in normal mice using most assays. We used an ELISA that detects total immunoreactive IL-3 or GM-CSF in a complex biologic fluid such as plasma or ascites and is insensitive to interfering substances such as soluble receptors, autoantibodies, and binding proteins (see "Materials and methods"). With this assay, there were low levels of circulating IL-3 in Balb/c mice transplanted with nontransduced marrow and in recipients of bone marrow transduced with retrovirus lacking BCR/ABL (Figure 1A). We found significantly increased IL-3 in plasma of mice with BCR/ABL-induced CML-like disease (Figure 1A), in agreement with others.8 Interestingly, recipients of marrow transduced with BCR/ABL in a retroviral vector that efficiently coexpresses green fluorescent protein (GFP) had significantly higher IL-3 levels than recipients of marrow transduced with a BCR/ABL-MSCVneo virus. In contrast to the previous report,8 we detected no significant increase in circulating GM-CSF in mice with BCR/ABL-induced CML-like leukemia, relative to control mice or recipients of empty vector-transduced marrow (Figure 1B). We also tested selected samples using conventional immunoassay kits for IL-3 and GM-CSF that have a sensitivity of 2.5 to 500 pg/mL (see "Materials and methods"). In these assays, IL-3 was undetectable in control mice, with low-level (< 8 pg/mL) but detectable circulating IL-3 in a minority of mice with BCR/ABL-induced CML-like leukemia (data not shown). Circulating GM-CSF was not detected with this assay in either control or leukemic mice.
The Il-3 gene is not required for induction of CML-like leukemia by BCR/ABL We used mice with homozygous inactivation of the Il-3 gene as donors or recipients in the BCR/ABL bone marrow transduction/transplantation model. After transduction with BCR/ABL, bone marrow from donor Il-3 / mice induced CML-like leukemia in
wild-type Balb/c recipient mice with 100% efficiency within 4 weeks
after transplantation (Figure 2).
Similarly, CML-like disease developed in all
Il-3/ recipients of
BCR/ABL-transduced wild-type marrow and when both donor and
recipient lacked IL-3. There was no significant difference in survival
when either donor or recipient was Il-3/,
and actually a slight but significant acceleration in the disease process when both donor and recipient were of
Il-3/ genotype (Figure 2). When both donor
and recipient lacked IL-3, the CML-like disease was efficiently
transferred to irradiated Il-3/ or wild-type
secondary recipients by transplantation of marrow and/or spleen (data
not shown), as previously reported in a wild-type background.6 The histopathology of the disease process was similar in all groups (Figure 3 and Table
1). There was massive splenomegaly of
similar magnitude in all recipients, due to extensive infiltration by
maturing myeloid cells with total disruption of the splenic
architecture, accompanied by increased megakaryocytes and
erythropoiesis (Figure 3A,D). The liver sinusoids were heavily infiltrated with maturing myeloid and erythroid cells (Figure 3B,E).
The cause of morbidity or death in all cases appeared to be extensive
myeloid infiltration of the lung parenchyma with hemorrhages (data not
shown). Southern blot analysis of gDNA from blood- or spleen-derived
myeloid cells from leukemic mice demonstrated that multiple (average of
9) independent proviral clones contributed to the myeloproliferative
disease in these animals (Figure 4A and
data not shown), similar to observations in a wild-type
background.6 Despite the overall similarity of the organ
histopathology, there were some notable differences in the
myeloproliferative process in the blood of some recipient cohorts. When
donor marrow was Il-3/, the average blood
leukocyte count was lower, and there was an increase in the percentage
of circulating immature erythoid cells (Table 1). When both donor and
recipient were Il-3/, recipients also had
lower peripheral blood leukocyte counts, but exhibited an increased
percentage of monocytes and macrophage-like cells in the peripheral
blood (average 48% monocyte/macrophages for
Il-3/ donor/recipient versus 11% for
wild-type, Figure 3F and Table 1). These results demonstrate that IL-3
is not required for induction of CML-like myeloproliferative disease in
mice by BCR/ABL, but monocytosis is observed in the absence
of this cytokine.
The Gm-csf gene is not required for induction of CML-like leukemia by BCR/ABL We performed a similar set of transduction/transplantation experiments using mice with homozygous inactivation of the Gm-csf gene. As with IL-3, we found that all recipients developed CML-like leukemia on transplantation with BCR/ABL-transduced marrow, regardless of whether the donor, recipient, or both were of Gm-csf/ genotype (Figure
5). There was a slight but statistically
significant prolongation in survival when both donor and recipient were
Gm-csf/. There was no difference in
peripheral blood leukocyte count or differential, spleen weight, or
histopathology of the myeloproliferative disease in the absence of
GM-CSF (Figure 3G-I and data not shown). The disease was polyclonal by
Southern blot analysis in all cases (data not shown), except when both
donor and recipient were Gm-csf/,
when there appeared to be fewer proviral clones contributing to the
leukemia (Figure 4A). These results demonstrate that GM-CSF is not
required for induction of CML-like disease by BCR/ABL.
Lack of a compensatory increase of the alternative cytokine in
Il-3 / donors had increased plasma IL-3
levels that were similar to those found in wild-type recipients of
transduced wild-type marrow, whereas Il-3/
recipients of BCR/ABL-transduced wild-type marrow had low to normal levels of IL-3 (Figure 6A). This suggests that the source of the
increased circulating IL-3 in mice with BCR/ABL-induced CML-like leukemia is the recipient, rather than the
donor-derived, BCR/ABL-expressing leukemic cells. There was
no significant increase in circulating GM-CSF levels in diseased mice
from transplants with an Il-3/ donor or
recipient (Figure 6B), demonstrating that a compensatory increase in
GM-CSF did not occur in the absence of IL-3. Conversely, when the
transduced donor cells were of Gm-csf/
genotype, the levels of both IL-3 and GM-CSF in wild-type recipients were low (Figure 6A,B). This suggests that the majority of
circulating GM-CSF in mice shortly after transplantation is donor
derived, and GM-CSF production from the BCR/ABL-transduced
marrow is necessary to elicit an IL-3 response from
recipients.
Lack of redundancy of IL-3 and GM-CSF in BCR/ABL-induced CML-like leukemia The above experiments demonstrate that IL-3 and GM-CSF are not required individually for induction of murine CML-like leukemia by BCR/ABL, but do not exclude the possibility that these 2 hematopoietic growth factors may have redundant or overlapping functions in the pathogenesis of the myeloproliferative disease. To address this, we used novel mice with homozygous inactivation of both the Il-3 and Gm-csf genes ("double-knockout" mice), which have only modest perturbations in hematopoiesis.24 When Il-3/Gm-csf double-knockout mice, in a Balb/c background, were used as both donors and recipients in the BCR/ABL transduction/transplantation model system, recipient mice developed typical CML-like disease, with significantly shorter latency than wild-type transplants (Figure 7). The clinical features and histopathology of the myeloproliferative disease in the double-knockout background were identical to those of wild-type (Figure 3), except that some mice exhibited peripheral blood monocytosis, similar to that observed in the Il-3/ donor/recipient
transplant (Figure 3L and Table 1). Although the Il-3/Gm-csf
double-knockout mice exhibit moderate eosinophilia under normal
conditions,24 we did not observe excess eosinophils in the
myelproliferative disease induced by BCR/ABL in this
background. A similar number of proviral clones contributed to the
leukemia in the double-knockout transplant as in a wild-type background (Figure 4A). This demonstrates that there is no redundancy in the
function of these cytokines in the pathogenesis of
BCR/ABL-induced myeloproliferative disease. Interestingly,
about half the double-knockout recipients of
BCR/ABL-transduced double-knockout marrow transplanted with
5 × 105 cells died before 14 days after transplantation
because of failure to engraft, whereas more than 90% of mice that
received 1 × 106 cells engrafted and developed leukemia
(data not shown). The failure to engraft was dependent on
BCR/ABL transduction, because vector-transduced
double-knockout marrow efficiently engrafted all double-knockout
recipients at a dose of 1 × 105 cells (data not
shown).
BCR/ABL induces CML-like disease inefficiently in C57Bl/6 mice, and IL-3 and GM-CSF are not required for leukemogenesis in this strain To extend these observations, we used a different inbred strain of mice, C57Bl/6, for which congenic Il-3/
Gm-csf/ double-knockout mice are also
available. In an earlier, less efficient model of BCR/ABL
leukemogenesis, it was reported that C57Bl/6 mice did not develop
CML-like disease on transplantation with syngeneic
BCR/ABL-transduced bone marrow, but instead developed leukemias of B-lymphoid, monocyte/macrophage, mast cell, and erythroid lineages.18 Using our current optimized model
system,6 we observed that BCR/ABL did induce
CML-like myeloproliferative disease in C57Bl/6 mice, but inefficiently
(Figure 8). Relative to Balb/c mice,
C57Bl/6 (B6) recipients of BCR/ABL-transduced marrow
exhibited prolonged survival, with 50% of B6 mice developing fatal
leukemia by 39 days after transplantation, versus 22 days for Balb/c
(Figure 8A,B). In addition, the B6 recipients no longer developed
exclusively CML-like leukemia, with some animals developing B-lymphoid
leukemia or macrophage tumors in addition to, or instead of,
myeloproliferative disease (Figure 8A). The myeloproliferative illness
induced by BCR/ABL in B6 mice was characterized by lower
peripheral blood leukocyte counts and spleen weights than that observed
in Balb/c mice (Figure 8B), and fewer BCR/ABL-transduced
proviral clones contributed to the disease (Figure 4B), but was
otherwise clinicopathologically similar. As in Balb/c
mice,6 the same spectrum of proviral clones was observed
in gDNA from neutrophils, macrophages, erythroid precursors, and
B-lymphoid cells purified from diseased B6 mice, indicative of a
multipotential target cell (Figure 4C). However, B6 mice with CML-like
leukemia did not have significant elevations in circulating IL-3 (or
GM-CSF) relative to B6 recipients of nontransduced marrow (data not
shown). When congenic C57Bl/6 Il-3/
Gm-csf / mice were used as bone marrow
donors and recipients, CML-like myeloproliferative leukemia developed
in 6 of 7 recipients, with a similar prolonged latency (Figure 8A).
These results demonstrate that IL-3 and GM-CSF are not required for the
CML-like myeloproliferative disease induced by BCR/ABL in 2 different inbred mouse strains.
Mice with transplanted with BCR/ABL-transduced bone marrow develop a myeloproliferative disease that is a very close pathophysiologic match to human CML. Diseased mice were reported to have elevated circulating IL-3 and GM-CSF,8 which are not characteristic of most patients with chronic phase CML, but the mechanism of production of these cytokines and their contribution to leukemogenesis were unclear. Here, we confirmed that mice with BCR/ABL-induced CML-like disease have variable but significant elevations in plasma IL-3, but we did not detect increased GM-CSF in these mice. Increased circulating IL-3 and sometimes GM-CSF are also observed in mice with myeloproliferative disease induced by oncogenic fusions of several other tyrosine kinases with the Tel transcription factor, found in human myelodysplastic syndromes, atypical CML, and acute leukemia.28 Our findings suggest that most of the increased IL-3 in mice with BCR/ABL-induced myeloproliferative disease originates from the recipient, not the donor. The actual source of the cytokine is not known, but may be produced by radioresistant recipient T lymphocytes. Interestingly, a wild-type Gm-csf gene was required in donor marrow for the recipient IL-3 response. We also noted that IL-3 levels were significantly higher in mice with CML-like disease induced with a retroviral vector that efficiently coexpresses Bcr/Abl and GFP, compared with a BCR/ABL-MSCVneo vector, which coexpresses neomycin phosphotransferase at low and variable levels from an internal pgk promoter.25 Collectively, these results suggest that the increased IL-3 represents a reaction of the recipient to transplantation of bone marrow expressing foreign proteins. A several-fold elevation in circulating IL-3 is unlikely to account for
the massive expansion of myelopoiesis in mice with BCR/ABL-induced CML-like disease, but this does not preclude
a pathophysiologic role for cytokines, because IL-3 and other growth factors can stimulate autocrine proliferation without requiring secretion.29 To definitively test the role of IL-3 and
GM-CSF in the pathogenesis of BCR/ABL-induced CML-like
disease, we used Balb/c mice with homozygous inactivation of the
Il-3 gene, the Gm-csf gene, and both genes, in
the retroviral transduction/transplantation model system. Our results
clearly establish that neither IL-3 nor GM-CSF is required individually
for induction of CML-like disease by BCR/ABL. In the absence
of IL-3, the myeloproliferative disease induced by BCR/ABL
was characterized by lower peripheral blood leukocyte counts with a
higher percentage of monocyte/macrophage cells, somewhat reminiscent of
chronic myelomonocytic leukemia. The mechanism of the shift in
myelopoiesis in these mice is not known, but the monocytic cells were
derived from the same spectrum of BCR/ABL-transduced clones
as the neutrophils (data not shown), and the histopathology of the
spleen and liver was identical to wild-type transplants (Figure 3). The
use of Il-3 Interestingly, we observed a decreased efficiency of engraftment of the transplanted marrow when both donor and recipient were of double-knockout genotype. The decreased engraftment appeared to be dependent on BCR/ABL transduction, because double-knockout marrow transduced with insertless retrovirus efficiently engrafted double-knockout recipients transplanted with 5-fold fewer marrow cells. It is not clear whether the defect in engraftment requires the absence of IL-3 or GM-CSF or both in the recipient. Because the impaired engraftment was only observed on transduction of BCR/ABL, it is possible that this reflects decreased efficiency of establishment of BCR/ABL-transduced stem cells after transplantation in the absence of IL-3 and GM-CSF. However, this is unlikely for 2 reasons. First, it is doubtful that the transduction efficiency of stem cells in our experiments was 100%, so that untransduced normal stem cells are present in the donor marrow population and would be expected to efficiently engraft recipients. Second, efficient engraftment and induction of CML-like disease in the double-knockout recipients was achieved with just a 2-fold higher dose of donor marrow, and mice that developed CML-like disease in the double-knockout background at either dose of donor marrow exhibited about the same number of proviral clones contributing to the disease as in a wild-type background (Figure 4A), rather than the monoclonal or oligoclonal disease expected if limiting numbers of BCR/ABL-transduced stem cells were engrafting. It seems more plausible that the early mortality in this transplant reflects a negative or toxic effect of BCR/ABL expression on the donor marrow population as a whole, and on the ability of the marrow to radioprotect the recipient mice. Further experiments are necessary to understand the precise mechanism of this phenomenon, but the central conclusion that IL-3 and GM-CSF are not required for BCR/ABL leukemogenesis is not affected. The data presented here argue that autocrine production of IL-3 does
not play an essential role in the pathogenesis of established human
chronic phase CML. However, it cannot be concluded that IL-3 does not
contribute at all to this disease process. Perhaps because bone marrow
cells must be actively cycling to be transduced by ecotropic
retroviruses, the murine CML-like disease does not exhibit the long
latent period (18-60 months) characteristic of human
CML,32,33 which may reflect the ability of primitive Ph+ progenitors to be quiescent.34 It is
therefore possible that IL-3 could be involved in the slow expansion of
the Ph+ clone that is required for the eventual development
of peripheral blood leukocytosis and clinical symptoms in human CML.
Modifications to the retroviral model system may allow this latent
period to be modeled in mice, and permit the role of cytokines in this
process to be tested. Our results also do not exclude a role in the
pathogenesis of murine CML-like disease for other cytokines instead of,
or in addition to, the 2 analyzed here. Notably, human CML progenitors also express aberrant G-CSF transcripts,13 whereas
oncostatin M has been implicated in the pathogenesis of
myeloproliferative disease induced in mice by the TEL/JAK2 tyrosine
kinase fusion.35 Mice lacking G-CSF are available,
allowing the role of G-CSF in BCR/ABL leukemogenesis to be
tested. However, unlike the Il-3 and Gm-csf
mutant mice, Gcsf In conclusion, we have demonstrated here that neither IL-3 nor GM-CSF is required, alone or together, for induction of CML-like myeloproliferative disease in mice by BCR/ABL. Our results demonstrate the value of accurate and faithful mouse models of human cancer. In this instance, the retroviral bone marrow transduction/transplantation model of human CML has definitively answered a question about the pathogenesis of CML that would be difficult if not impossible to settle by studying primary human CML cells. Further application of this model system should continue to provide important new knowledge about the molecular pathophysiology of the human Ph+ leukemias.
G.D. is a Clinical Scholar of the Leukemia and Lymphoma Society. D.G.G. is an Associate Investigator of the Howard Hughes Medical Institute. R.A.V. is a Scholar of the Leukemia and Lymphoma Society and the Carl and Margaret Walter Scholar in Blood Research at Harvard Medical School.
Submitted July 21, 2000; accepted December 7, 2000.
Supported in part by National Institutes of Health grants CA81197 (M.H.T.), CA74886 and CA39542 (G.D.), DK50654 and CA66996 (D.G.G.), and CA57593 (R.A.V.), and grants from the Leukemia and Lymphoma Society (G.D. and R.A.V.), Swiss National Science Foundation (S.G.), Swiss Cancer League (S.G.), Cancer Research Institute/Partridge Foundation and the MarJo Foundation (G.D.), and the Howard Hughes Medical Institute (D.G.G.).
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: Richard A. Van Etten, The Center for Blood Research, 200 Longwood Ave, Boston, MA 02115; e-mail: vanetten{at}cbr.med.harvard.edu.
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Top
Abstract
Introduction
to Gm
),
peripheral blood neutrophils (p. blood), total, B220+, and
B220
R,