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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0244.
NEOPLASIA
From The Center for Blood Research and Department of
Genetics and Department of Pathology, Brigham and Women's Hospital;
Harvard Institutes of Medicine; and Howard Hughes Medical Institute,
Harvard Medical School, Boston, MA.
Several patients with clinical features of chronic myeloid leukemia
(CML) have fusion of the TEL (ETV6) gene on
12p13 with ABL on 9q34 and express a chimeric Tel-Abl
protein that contains the same portion of the Abl tyrosine kinase fused
to Tel, an Ets family transcription factor, rather than Bcr. In a
murine retroviral bone marrow transduction-transplantation model, a Tel
(exon 1-5)-Abl fusion protein induced 2 distinct illnesses: a CML-like
myeloproliferative disease very similar to that induced by Bcr-Abl but
with increased latency and a novel syndrome characterized by
small-bowel myeloid cell infiltration and necrosis, increased
circulating endotoxin and tumor necrosis factor Chronic myeloid leukemia (CML) is a
myeloproliferative disease characterized by excessive clonal production
of maturing myeloid cells.1 The direct cause of CML is the
product of the t(9;22) Philadelphia chromosome translocation, the
BCR-ABL oncogene, and its corresponding protein, the Bcr-Abl
tyrosine kinase. Retroviral transduction of the BCR-ABL gene
into mouse bone marrow cells, followed by transplantation into
irradiated syngeneic mice, induces CML-like myeloproliferative leukemia
in all recipients within 4 weeks after transplantation.2-4
Mice with BCR-ABL-induced CML-like disease have massive
polyclonal expansion of maturing myeloid cells, principally
neutrophils, which express Bcr-Abl protein and infiltrate the spleen,
liver, and lungs. Although neutrophils are the predominant
hematopoietic lineage overproduced in murine CML-like disease,
macrophages, erythroid progenitors, B lymphocytes, and sometimes T
lymphocytes from diseased mice carry the same spectrum of
BCR-ABL proviral clones as the granulocytes, thereby demonstrating that the cells initiating the CML-like leukemia are early
multipotential progenitors.2
The CML-like disease is efficiently transferred by transplantation of
bone marrow or spleen cells from a primary mouse to irradiated
secondary recipients.2-4 Interestingly, of the many different BCR-ABL-transduced clones that contribute to the
leukemia in the primary mouse, only a small subset are capable of
generating day-12 spleen colonies and of engrafting and inducing
CML-like disease in secondary recipients,2,4 suggesting
that the cells initiating CML-like disease are heterogeneous for
self-renewal. Murine CML-like leukemia is an accurate and faithful
model of human CML that has been proved to be useful for analysis of
the molecular pathophysiologic characteristics of the human
disease.2,5,6
The TEL gene (also known as ETS-variant gene 6 [ETV6]), located on chromosome 12p13, was originally
identified at the breakpoint of a (5;12) translocation in a patient
with chronic myelomonocytic leukemia, where it was fused to the
platelet-derived growth factor The TEL-ABL fusion oncogenes produce 2 different Tel-Abl
proteins; in the patients with B-ALL and atypical CML, the first 4 exons of TEL were found to be fused to ABL exon
2, whereas the other 4 patients had TEL exons 1 to 5 fused
to ABL exon 2. The resulting chimeric proteins contain Tel
amino acids 1 to 154 and 1 to 336, respectively, fused to the same 1104 carboxy (COOH)-terminal amino acids of c-Abl found in the Bcr-Abl
fusion proteins. The Tel-Abl fusion proteins show increased tyrosine
kinase activity in vitro10 and are constitutively
phosphorylated on tyrosine in vivo.11,17 Although Tel is a
predominantly nuclear protein,7 Tel-Abl is localized to
the cytoplasm and F-actin cytoskeleton,11,17 similar to
Bcr-Abl. An amino (NH2)-terminal region of Tel between amino acids 60 and 120 shares homology with the Ets proteins ETS-1, FLI-1, PNTP2, and Yan and has been referred to as the PNT domain. In
Tel, the PNT domain mediates oligomerization11,18 and is required for activation of the Tel-PDGF We are interested in Tel-Abl not because of the clinical importance of
Tel-Abl-associated human leukemia but because careful comparative
analysis of signaling and leukemogenesis by Tel-Abl may help illuminate
the pathophysiologic mechanism of Bcr-Abl-induced CML. Studies of
Tel-Abl transformation in vitro failed to identify any significant
differences between Tel-Abl and Bcr-Abl.22,23 However,
studies of Bcr-Abl signaling mutants demonstrated that transformation
studies in cultured cells do not correlate well with leukemogenesis in
vivo.5,6 In the study described here, we directly compared
leukemogenesis by Tel-Abl and by Bcr-Abl in mice and demonstrated that
Tel-Abl induces distinct hematologic diseases that differ both in
phenotype and ability to be transplanted.
DNA constructs
In vitro kinase assay
Bone marrow transduction and transplantation Generation and titering of retroviral stocks was done as described previously;2 all stocks had titers of 3 to 5 × 106 neomycin-resistant colony-forming units (CFUs)/mL, and yielded equivalent proviral copy numbers, as assessed by Southern blotting on transduction of National Institutes of Health (NIH) 3T3 cells or primary bone marrow. Balb/c mice (Taconic Farms, Germantown, MD) were used for all experiments. Bone marrow from male donors treated with 5-fluorouracil (200 mg/kg) was transduced and 5 × 105 cells were transplanted into lethally irradiated (900 cGy) female recipients as described previously;2 in some experiments, recipients received only 450 cGy of irradiation. For secondary transplantation, a 1:1 mixture of 3 to 6 × 106 marrow and spleen cells was injected intravenously into pairs of lethally irradiated (900 cGy) female Balb/c secondary recipient mice. The viability of all cells used for secondary transplantation was confirmed by trypan blue staining to be greater than 90%. For isolation of day-12 spleen colonies, pairs of lethally irradiated recipient mice were injected with 1 × 105, 3 × 105, and 1 × 106 bone marrow cells, spleens were isolated 12 days after injection, and macroscopic colonies were dissected out. Colonies that were not visibly red were assessed by cytospin and Wright-Giemsa staining to confirm that they were composed of mature myeloid cells.Southern blotting and lineage analysis Purification of neutrophils, spleen erythroid progenitors, and spleen or lymph node B lymphocytes employed lineage-specific monoclonal antibodies and immunomagnetic beads (Miltenyi Biotec, Germany) as described previously,2 except in some cases in which microbeads directly conjugated with TER119 antibodies were used for isolation of erythroid cells. Peritoneal or bone marrow macrophages were isolated by adherence and by culture in medium containing macrophage colony-stimulating factor. The purity of the selected populations was assessed by Wright-Giemsa staining of cytospin preparations and, in some cases, by fluorescence-activated cell-sorter scanning, and only samples with at least 80% purity were used for Southern blot analysis. Genomic DNA was prepared, digested with BglII, and hybridized with radioactive probes from the neomycin-resistance gene to detect distinct proviral integration events. It was then stripped and reprobed with a human ABL probe that detects a common 2.2-kb fragment from all integrants and allows determination of total proviral content of each sample, as described previously.2Western blotting For assessment of in vivo kinase activity, NIH 3T3 cells were transduced with retrovirus expressing p210 BCR-ABL, TEL-ABL, TEL-ABL PNT, TEL-ABL K581R, or empty
MSCVneo vector. All stocks had equivalent titer in
fibroblasts, based on G418 resistance. Transduced cells were selected
in 1.0 mg/mL G418 for 48 hours, starved for 4 hours in Dulbecco
modified Eagle medium with 0.5% bovine serum albumin, and then lysed
in radioimmunoprecipitation assay buffer. Extracts from peripheral
blood (PB) leukocytes or spleen cells (predominantly maturing myeloid
cells) were prepared by direct boiling as described
previously2 and clarified by centrifugation at
100 000g for 15 minutes. Equivalent amounts of protein
(verified by Ponceau-S staining) were fractionated by 5% to 20%
gradient SDS-PAGE, transferred to nitrocellulose membranes, and blotted
with anti-Abl (3F12, a gift of Dr R. Salgia, Dana-Farber Cancer
Institute, Boston, MA) and antiphosphotyrosine (4G10; Upstate
Biotechnology, Charlottesville, VA) monoclonal antibodies.
Tel-Abl has increased in vivo and in vitro tyrosine kinase activity relative to p210 Bcr-Abl Previous studies demonstrated that Tel-Abl had tyrosine kinase activity in vitro that was similar to that of Bcr-Abl10 and was constitutively tyrosine phosphorylated in fibroblasts11 and hematopoietic cells,17,22 whereas mutants of Tel-Abl lacking the NH2-terminal PNT homology oligomerization domain lacked phosphotyrosine in vivo.11 To assess quantitatively the kinase activity of Tel-Abl relative to Bcr-Abl, we compared Tel-Abl (containing Tel amino acids 1-336 fused to Abl), a deletion mutant of Tel-Abl (PNT) lacking most of the PNT domain, and a catalytically inactive mutant Tel-Abl protein (K581R) with p210 Bcr-Abl (Figure
1) in an immune complex kinase assay.
Tel-Abl reproducibly had more kinase activity for a GST-Crk substrate
than did p210 Bcr-Abl (Figure 2A),
whereas the Tel-Abl PNT mutant had significantly lower activity but
still more than c-Abl. Immunoprecipitates of the Tel-Abl K581R
protein had very low kinase activity that likely originated from
endogenous c-Abl. When these fusion kinases were stably expressed in
NIH 3T3 fibroblasts by retroviral transduction, cells expressing
Tel-Abl had slightly higher levels of tyrosine phosphoproteins than
cells expressing p210 Bcr-Abl, whereas cells expressing Tel-Abl PNT had lower but significantly higher levels than cells expressing Tel-Abl
K581R or nontransduced cells (Figure 2B). These results show that
Tel-Abl has as much or higher tyrosine kinase activity than p210
Bcr-Abl in vivo and in vitro that depends partly on the PNT
oligomerization domain.
TEL-ABL induces CML-like myeloproliferative disease and a novel disease of the small bowel in mice We tested the ability of the TEL-ABL oncogenes to induce leukemia in mice in the retroviral bone marrow transduction-transplantation model system.2 Bone marrow from donors pretreated with 5-flurouracil was transduced with retrovirus expressing either p210 BCR-ABL, TEL-ABL (containing the first 5 exons of TEL fused to ABL exon 2), the kinase-inactive TEL-ABL mutant (K581R), or the TEL-ABLPNT
mutant. The PNT deletion mutation was previously shown to abolish
transformation of fibroblasts and primary bone marrow B-lymphoid cells
by TEL-ABL.11 All recipients of
TEL-ABL-transduced bone marrow died within 42 days after
transplantation, 1 to 2 weeks later than recipients of p210
BCR-ABL-transduced marrow (Figure
3). This difference in survival
was highly significant (P < .001 on Mantel-Cox test). No
recipients of marrow transduced with either TEL-ABL K581R or
TEL-ABL PNT had onset of disease during this period;
these mice had normal peripheral blood (PB) counts at 42 and 86 days
after transplantation (data not shown) and remained healthy at 6 months
after transplantation. These results show that both the Abl tyrosine
kinase activity and the Tel PNT oligomerization domain are required for
induction of fatal hematologic disease by TEL-ABL.
Two different fatal outcomes were observed in recipients of
TEL-ABL-transduced marrow. In some recipients (7 of 22), a
progressive myeloproliferative disease developed that was very similar
to the disease induced by p210 BCR-ABL (Figure 3, black
squares). This disease mimicked the distinctive features of human CML:
leukocytosis with maturing neutrophils, splenomegaly, hepatomegaly, and
myeloid cell infiltration into peripheral tissues, including the
spleen, liver, and lungs (Table 1).
In most mice (13 of 22) given transplants of
TEL-ABL-transduced marrow, a distinct fatal illness
developed (Figure 3, open squares). At necropsy, these mice were found
to have enlarged, reddened, and distended small intestine; a pale
liver; and increased prominence of the vasculature (Figure
4). In contrast to mice with CML-like
disease, these mice had only slightly elevated PB leukocyte counts and
their spleens were minimally enlarged (Figure 4 and Table 1). We named
this distinct disease process small-bowel syndrome (SBS). Mice in which
SBS developed tended to die earlier, between 23 and 28 days after
transplantation, than mice with CML-like disease. Some animals (2 of
22) that were premorbid slightly later clearly died of SBS but had
evidence of CML-like disease, with marked splenomegaly and leukocytosis
(Figure 3, mixed symbols). This suggests that the 2 diseases
can occur independently and that mice that survive the rapid onset of
SBS may subsequently have CML-like disease.
Histopathological analysis of the mice with SBS (Figure
5) showed extensive myeloid cell
infiltration of villi in the small bowel, with prominent villous
necrosis (Figure 5H). Myeloid cells were also found adjacent to Peyer
patches, which are lymphoid nodules normally present in the gut. Some
sections showed evidence of transmural necrosis and possible
perforation. Livers from these mice had a distinctive histopathological
feature: a striking microvesicular change in the cytoplasm of
hepatocytes (Figure 5I), an appearance that can be caused by an
accumulation of either glycogen or neutral lipids such as
triglycerides. To distinguish between these, liver sections were
stained with periodic acid-Schiff (PAS), which stains glycogen, and
with oil red O (ORO), which stains neutral lipids. Livers from mice
with SBS were stained strongly by ORO (Figure 5J) but not by PAS (data
not shown). In contrast, livers from healthy mice or mice with CML-like
disease induced by BCR-ABL or TEL-ABL showed only
minimal ORO staining (Figure 5O,T), demonstrating that this fatty
change was unique to SBS.
Livers from mice with SBS also showed marked hepatocyte degeneration suggestive of apoptosis (Figure 5I) but lacked the myeloid and erythroid cell infiltrates and periportal macrophage aggregates that were uniform features of CML-like disease (Figure 5N,S). Spleens from mice with SBS had partial disruption of follicular architecture with mild infiltration of neutrophils and erythroid cells (Figure 5G). Kidneys from these animals had evidence of acute tubular necrosis, with degeneration of proximal tubular cells and proteinaceous material in the tubular lumen (data not shown). Finally, lungs from mice with SBS had minimal focal myeloid infiltrates without hemorrhages (data not shown). In contrast, mice with CML-like disease had extensive pulmonary myeloid cell infiltration and hemorrhages that caused morbidity or death.2 Endotoxemia is a possible pathophysiologic mechanism of TEL-ABL-induced SBS The histopathological findings in mice with SBS, including fatty liver and acute tubular necrosis in the kidneys, were suggestive of endotoxin-induced injury. A likely source of endotoxin is the small bowel, in which necrosis of villi might lead to increased vascular permeability and endotoxemia from gut bacteria. The pathophysiologic response to endotoxin challenge in mice, which has been studied extensively,24 is followed by an elevation in levels of circulating tumor necrosis factor (TNF) within 2 hours and an
elevation in interleukin 1 levels within 6 hours. Liver damage,
including acute fatty liver and hepatocyte apoptosis, is induced
directly by TNF.25 Secondary effects include
hypotension and renal acute tubular necrosis.
To investigate the hypothesis that endotoxin was involved, serum from
premorbid mice with TEL-ABL-induced SBS and CML-like disease was assayed for endotoxin and TNF
Myeloid cells from mice with TEL-ABL-induced SBS and CML-like disease express Tel-Abl protein PB myeloid cells and spleen cells from mice with TEL-ABL-induced CML-like disease expressed the Tel-Abl fusion protein at levels several-fold greater than did endogenous c-Abl (Figure 6). Importantly, both splenic and PB myeloid cells from mice with TEL-ABL-induced SBS also expressed Tel-Abl protein at similar levels. Because of the difficulty in recovering sufficient numbers of neutrophils from diseased intestine and the lack of a reporter gene such are green fluorescent protein or-galactosidase in our viral vector, direct demonstration of the
presence of Tel-Abl in myeloid cells infiltrating the small bowel in
these mice was not possible. However, because of the expression of
Tel-Abl in circulating neutrophils, it is very likely that Tel-Abl was
also expressed in the myeloid cells infiltrating the intestinal villi in mice with SBS.
The TEL-ABL-transduced bone marrow cell that initiates CML-like leukemia is an early multipotential progenitor Analysis of genomic DNA from spleen cells from mice given transplants of TEL-ABL-transduced bone marrow demonstrated that many distinct proviral clones contributed to the SBS and CML-like diseases (Figure 7A). Polyclonal disease is also characteristic of BCR-ABL-induced CML-like leukemia,2 and this suggests that TEL-ABL is both necessary and sufficient to induce the diseases observed in mice. In BCR-ABL-induced CML-like disease, the same spectrum of proviral clones is found in neutrophils, macrophages, erythroid cells, B-lymphoid cells, and sometimes T lymphocytes, demonstrating that the cells initiating the myeloproliferative disease have multilineage repopulating ability.2 To characterize the cells initiating TEL-ABL-induced myeloproliferative disease in this study, we purified cells of different hematopoietic lineages from selected mice. Genomic DNA from PB neutrophils, peritoneal macrophages, splenic erythroid progenitors, splenic B lymphocytes, thymocytes, and peripheral node lymphocytes from a mouse with TEL-ABL-induced CML-like disease carried the same spectrum of proviral clones, at about one proviral copy per cell, whereas splenic erythroid progenitors and B lymphocytes from another mouse shared the same proviral clones as the spleen tissue and PB neutrophils (Figure 7B). Similar patterns were observed in 4 other mice (data not shown). These results show that the cells initiating CML-like leukemia after transduction with TEL-ABL can repopulate multiple myeloid and lymphoid lineages in transplant recipients.
To characterize further the cells initiating TEL-ABL-induced CML-like disease, we tested the ability of these cells to generate day-12 spleen colonies in a secondary transplant. Such colonies are clonal and are initiated by an early multipotential myeloid progenitor cell, day-12 spleen colony-forming unit (CFU-S12). Previous studies demonstrated that a subset of BCR-ABL-transduced clones contributing to the leukemia in a primary animal with CML-like disease generate day-12 spleen colonies in secondary recipients.2 We observed a similar phenomenon with TEL-ABL-induced CML-like disease (Figure 7C). Most day-12 spleen colonies in secondary recipients of bone marrow from 2 different primary mice with TEL-ABL-induced myeloproliferative disease contained TEL-ABL provirus. Interestingly, as with BCR-ABL, only a minor subset of the clones contributing to the leukemia in the primary animal generated day-12 spleen colonies, and these were often not the most abundant clones in the primary mouse. These results indicate that only some of the cells initiating TEL-ABL-induced CML-like disease are early hematopoietic progenitors that contain or can give rise to CFU-S12. TEL-ABL-induced SBS and CML-like disease cannot be transplanted to secondary recipients We attempted to adoptively transfer TEL-ABL-induced hematologic diseases by transplanting large amounts of bone marrow and spleen from diseased primary mice into lethally irradiated syngeneic Balb/c secondary recipient mice. Cells from 17 primary mice (5 with SBS, 10 with CML-like disease, and 2 with mixed SBS and CML-like disease) were transplanted into pairs of recipients. None of the recipients developed either SBS or myeloproliferative disease; instead, several different outcomes were observed (Table 3). The most frequent outcome of secondary transplantation was failure to engraft, with the recipients dying between 11 and 23 days after transplantation with pancytopenia, hypocellular bone marrow, and often with signs of disseminated bacterial infection. Two other mice appeared to have delayed graft failure, with pancytopenia developing 84 and 99 days after transplantation.
In 11 secondary recipients, hematologic malignant diseases developed after long latent periods (85-194 days). In 6 mice, T-cell leukemia-lymphoma developed, characterized by thymic and abdominal tumors of Thy-1-positive lymphoblasts. Five mice had onset of a histiocytic malignant disease of macrophage-monocyte origin that was characterized by tumors in the liver, mesentery, bone marrow, myocardium, and lungs and frequently accompanied by hemiparesis. Both these malignant diseases are observed in recipients of BCR-ABL-transduced marrow under certain circumstances; similar T lymphomas develop in recipients of marrow transduced with a mutant of BCR-ABL (Y177F) that is deficient in binding to the Grb2 adapter molecule,5 whereas monocyte-macrophage tumors unaccompanied by myeloproliferative disease can occur in recipients of BCR-ABL-transduced marrow from donors not treated with 5-fluororuracil.2 Finally, 4 mice remained healthy and were killed 270 to 300 days after transplantation; their blood counts were normal and they had no evidence of disease. In contrast to these findings, BCR-ABL-induced CML-like disease was successfully transplanted to secondary recipient mice in contemporaneous experiments more than 80% of the time by using identical techniques (data not shown). We analyzed genomic DNA from tissues of different secondary recipients
for the presence of TEL-ABL provirus (Figure
8). Significant levels of
provirus-positive cells were found in hematopoietic tissues of mice
with graft failure, suggesting that TEL-ABL-transduced cells from the primary mouse were engrafted in these recipients but
lacked radioprotective and long-term repopulating activity. T lymphomas
and macrophage tumors that developed in secondary recipients also
uniformly contained TEL-ABL provirus. In some cases, the T
lymphomas and macrophage tumors shared proviral integration sites with
myeloid cells from the donor (Figure 8), suggesting that these diseases
arose from the clones that were responsible for the myeloproliferative
disease in the primary mouse. In other cases, the macrophage tumors and
T lymphomas arose from novel clones not highly represented in the
donor.
Although Tel-Abl and Bcr-Abl have essentially identical transforming properties in cultured cells, a direct comparison of the leukemogenic properties of the 2 kinases in mice revealed important differences between them. In some recipients of TEL-ABL- transduced marrow, a CML-like myeloproliferative disease developed that was very similar to that induced by BCR-ABL, but the latency of the disease was significantly delayed. This was not a consequence of a lower or weaker intrinsic tyrosine kinase activity of Tel-Abl because direct comparison of in vitro and in vivo kinase activity reproducibly found Tel-Abl to be significantly more active than p210 Bcr-Abl (Figure 2). Tel-Abl and Bcr-Abl may differ in their ability to phosphorylate particular substrates, particularly those that might be recruited by specific interactions with the Tel or Bcr polypeptides. However, 2 studies failed to detect significant differences in tyrosine-phosphorylated proteins in cultured cells expressing the 2 kinases.22,23 Alternatively, other functions of the Abl fusion partner in addition to oligomerization may influence disease latency. Oligomerization of c-Abl by fusion of just the Bcr coiled-coil domain induces myeloproliferative disease in mice only after a long latent period,26 suggesting that other Bcr domains contribute to the efficiency of disease induction. One critical function is direct binding of the Grb2 adapter protein by Bcr, which is required for induction of fatal myeloproliferative disease in mice by p210 Bcr-Abl.5,26 However, the Tel-Abl fusion protein studied here also directly binds Grb2, and a site-specific mutation that blocks Grb2 binding attenuates induction of the CML-like disease by Tel-Abl (R.P.M. and R.A.V., unpublished data, January 2002). Additional studies are necessary to define the biochemical basis of the differences in leukemogenic activity of Tel-Abl and Bcr-Abl. Our results confirm that Tel-Abl is the direct cause of the myeloproliferative disease in patients with typical and atypical CML and TEL-ABL fusion genes. Clinical studies have suggested that the reciprocal ABL-TEL fusion gene is not present in most patients with leukemia and TEL-ABL,12 perhaps because of the complex translocations involved, whereas loss of the second TEL allele, a frequent finding in TEL-AML1-induced B-ALL,27 does not occur in TEL-ABL-associated disease.12,28 Our studies demonstrated directly that the leukemogenic action of TEL-ABL is independent of ABL-TEL expression or loss of TEL function. Because the kinase activity of Tel-Abl is inhibited29 by the Abl kinase inhibitor STI-571 (imatinib mesylate [Gleevec; Novartis Pharmaceuticals, East Hanover, NJ]), this drug should be of value in treating TEL-ABL-associated myeloproliferative disease. Most recipients of TEL-ABL-transduced marrow in this study
died of a distinct illness characterized by extensive infiltration of
small-bowel villi and submucosa with neutrophils, accompanied by
hepatic and renal failure. The clinical picture was suggestive of
endotoxin-mediated injury, and this idea was supported by the findings
of an analysis of circulating endotoxin and TNF Some additional factor, however, must account for the shift in
migration of Tel-Abl-expressing neutrophils from small bowel early
after transplantation to spleen, liver, and lungs later on. A plausible
candidate is altered expression of chemokine or adhesion molecule in
the gut resulting from ionizing radiation used to prepare the
recipients, which persists for 1 to 2 weeks and then resolves. The same
frequency of SBS was observed in mouse recipients of
TEL-ABL-transduced marrow that received only 450 cGy
total-body irradiation as conditioning and in those given larger doses
(data not shown), but it would be interesting to test the efficiency of
induction of SBS with further deceases in radiation dose. An
alternative possibility is that TNF The most dramatic difference between TEL-ABL-induced and BCR-ABL-induced myeloproliferative diseases was the absolute failure of the former to be adoptively transplanted into secondary recipients. Thirty-four lethally irradiated secondary recipient mice given transplants of large doses of viable marrow and spleen cells from primary mice with TEL-ABL-induced SBS or CML-like disease failed to have development of either illness. In contrast, in contemporaneous experiments with BCR-ABL-induced CML-like disease, the myeloproliferative disease could be adoptively transferred, with use of identical techniques, more than 80% of the time. The failure of transplantability of TEL-ABL-induced leukemia therefore represents a very striking biologic difference. It is possible that more efficient TEL-ABL transduction or transplantation of larger numbers of cells might permit adoptive transfer of TEL-ABL-induced myeloproliferative disease, but the fact that CFU-S12 cells were effectively transduced and transplanted to secondary recipients suggests that this would not be the case. Its lack of transplantability does not necessarily imply that the TEL-ABL-induced myeloproliferative disease is not a malignant disease. The use of transplantability as a criterion for malignancy originated from studies of solid tumors, in which acquisition of the ability to proliferate autonomously in a secondary host is an important functional test for distinguishing hyperplasia and benign tumors from true neoplastic lesions. Transplantability is less relevant to leukemias, which can kill the host without spreading outside the bone marrow and blood system. Retroviral gene-marking studies in patients given autografts30 found that at least some cases of BCR-ABL-induced CML are transplantable into irradiated syngeneic recipients (ie, the patients themselves), as is the case with murine CML-like disease induced by BCR-ABL. However, it is not known whether human TEL-ABL-associated myeloproliferative disease can likewise be transplanted. What is the explanation for the lack of transplantability of TEL-ABL-induced myeloproliferative disease? It can be assumed that the BCR-ABL and TEL-ABL retroviruses transduce an identical spectrum of hematopoietic progenitors in the marrow of donors given 5-fluorouracil, including hematopoietic stem cells. In both cases, the cells initiating the CML-like leukemia can repopulate multiple hematopoietic lineages in the primary recipient. However, the cells initiating myeloproliferative disease in the primary recipient of TEL-ABL-transduced marrow lack self-renewal capacity, as measured by secondary transplantation. Only a subset of the many BCR-ABL-positive clones contributing to the expansion of myeloid cells in a primary animal can generate day-12 spleen colonies and engraft and induce disease in secondary recipients,2 thus suggesting that the cells initiating BCR-ABL-induced CML-like disease are also heterogeneous for self-renewal. Because of these observations, we initially anticipated that cells initiating TEL-ABL-induced myeloproliferative disease would lack the ability to generate day-12 spleen colonies in secondary recipients. Surprisingly, a subset of TEL-ABL-transduced clones from primary mice with CML-like leukemia was found to generate day-12 spleen colonies efficiently, but the same bone marrow used in larger doses was unable to reconstitute the myeloproliferative disease in other recipients. Most of these recipients died of early graft failure but had provirally marked cells in the spleen between 2 and 3 weeks after transplantation, a finding that constitutes direct evidence of a lack of self-renewal of these TEL-ABL-transduced progenitors. Some recipients had engraftment, survived lethal irradiation, and showed no evidence of myeloproliferative disease, but T lymphoma or histiocytic tumors developed after several months. Some of these tumors shared proviral clones with myeloid cells from the primary animal, confirming the multipotential nature of the cells initiating the myeloproliferative disease and representing disease progression to acute leukemia. It is curious that a TEL-ABL-marked clone can be transferred successfully to a secondary recipient and induce T lymphoma or histocytic tumors without causing CML-like disease, but a similar phenomenon was observed in some secondary transplants with BCR-ABL-induced CML-like disease31 and may represent transfer of transformed acute leukemia cells present at low numbers in the primary animal. There are at least 2 possible mechanisms that might account for the
lack of transplantation of TEL-ABL-induced
myeloproliferative diseases. The first is that the bone marrow target
cell that initiates CML-like disease on transduction with
TEL-ABL is a progenitor Defining the mechanistic basis of the difference in secondary transplantation of tyrosine kinase-induced murine myeloproliferative diseases will be valuable for understanding the pathophysiologic characteristics of human myeloproliferative and myelodysplastic syndromes. In addition, our results may have clinical implications, since human TEL-ABL-induced myeloproliferative disease might be treatable by autologous marrow transplantation without the need for graft purging.
Submitted December 18, 2001; accepted February 12, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0244.
Supported in part by National Institutes of Health grants CA90576 (R.A.V.), CA09595 (R.P.M.), DK50654, and CA66996 (D.G.G), and grants from 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.
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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V. P. S. Rawat, M. Cusan, A. Deshpande, W. Hiddemann, L. Quintanilla-Martinez, R. K. Humphries, S. K. Bohlander, M. Feuring-Buske, and C. Buske Ectopic expression of the homeobox gene Cdx2 is the transforming event in a mouse model of t(12;13)(p13;q12) acute myeloid leukemia PNAS, January 20, 2004; 101(3): 817 - 822. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-32P-adenosine triphosphate and GST-Crk
substrate. The top panel is the 35S label, indicating
relative levels of expression of the different Abl proteins; the lower
panel is the 32P label, showing levels of
autophosphorylation of Abl proteins and transphosphorylation of the
GST-Crk substrate. The Crk kinase activity of the different Abl
proteins relative to c-Abl after correction for levels of expression is
shown at the top. (B) In vivo kinase activity. NIH 3T3 fibroblasts were
transduced with empty MSCVneo virus or MSCV virus containing
p210 BCR-ABL or TEL-ABL WT, 
) after long latent periods also
shared proviral clones with myeloid cells from the donor (lanes 9, 10, and 11 and lanes 12 and 13), as did thymus DNA from a secondary
recipient in which T-ALL developed (lanes 14 and 15). However, some
macrophage tumors and cases of T-ALL that arose in secondary recipients
were derived from proviral clones that were not represented at
significant levels in the myeloid cells of the donor (lanes 16, 17, and 18).
similar to those described in
normal mouse bone marrow
, BCR