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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-04-1244.
NEOPLASIA
From the Laboratory of Molecular Genetics of Stem
Cells, Clinical Research Institute of Montreal; the Department of
Medicine, University of Montreal; and the Division of Hematology,
Maisonneuve-Rosemont Hospital; Montreal, QC, Canada.
Chronic myelogenous leukemia (CML) is a clonal stem cell disease
caused by the BCR-ABL oncoprotein and is characterized, in its early
phase, by excessive accumulation of mature myeloid cells, which
eventually leads to acute leukemia. The genetic events involved in
CML's progression to acute leukemia remain largely unknown. Recent
studies have detected the presence of the
NUP98-HOXA9 fusion oncogene in acute leukemia
derived from CML patients, which suggests that these 2 oncoproteins may
interact and influence CML disease progression. Using in vitro purging
of BCR-ABL-transduced mouse bone marrow cells, we can now
report that recipients of bone marrow cells engineered to coexpress
BCR-ABL with NUP98-HOXA9 develop acute leukemia
within 7 to 10 days after transplantation. However, no disease is
detected for more than 2 months in mice receiving bone marrow
cells expressing either BCR-ABL or
NUP98-HOXA9. We also provide evidence of high levels of
HOXA9 expressed in leukemic blasts from acute-phase CML
patients and that it interacts significantly on a genetic level with
BCR-ABL in our in vivo CML model. Together, these studies
support a causative, as opposed to a consequential, role for
NUP98-HOXA9 (and possibly HOXA9) in CML disease progression.
(Blood. 2002;100:4177-4184) During the last 3 decades, cytogenetic studies have
revealed recurrent chromosomal translocations in human
leukemias.1,2 These translocations result in
fusion genes that frequently encode for chimeric proteins that
participate in leukemic transformation of bone marrow-derived cells.
Past and ongoing studies from many laboratories, including our own,
have shown that these chimeric oncoproteins are incapable of inducing
complete transformation of myeloid precursors and that they
unquestionably require coactivation of other oncogenes and/or
inactivation of tumor suppressor genes3 in order to do
so.4-15 Defining the full complement of oncoproteins that
are necessary for leukemic transformation is relevant to our
understanding of leukemogenesis and might offer new molecular targets
for the development of more specific antileukemic
drugs.16-18 Thus, it is both fundamentally and
clinically relevant to identify sets of oncogenes that
participate in human leukemias, in particular in the progression of
chronic myelogenous leukemia (CML) from the chronic to
the acute phase.
As an initial step in defining oncogenes involved in CML progression,
we focused on recent cytogenetic studies that describe additional
chromosomal translocations in acute leukemia derived from CML patients,
suggesting that these translocations potentially collaborated with
BCR-ABL in disease progression. Specifically, the
NUP98-HOXA9 fusion gene was detected in blast
cells in 3 patients with typical Philadelphia-positive
(Ph+) CML.19,20 However, there have
also been patients who developed NUP98-HOXA9-induced
myeloproliferation and subsequently acquired a Ph chromosome in
their leukemic cells when acute leukemia was diagnosed,21
suggesting that NUP98-HOXA9 and BCR-ABL may
genetically interact in human leukemia.
There have also been blast crisis specimens from CML patients that
express high levels of the HOXA9 oncogene when compared with
cells isolated from chronic phase patients, raising the possibility that HOXA9 may also directly interact with BCR-ABL
in the transformation of bone marrow cells.22 These
results were extended in our current studies, where we found that cells
from blast crisis patients express higher levels of HOXA9
than those found in mononuclear cells from accelerated phase CML
patients (Figure 2).
Several other additional translocations, typically observed in acute
human leukemia, have been manifested by CML cells undergoing blast
transformation (ie, at diagnosis of acute leukemia in patients previously in chronic phase CML). These include the t(11;17), which
involves the MLL/ALL/HRX gene23; the
t(8;21) with the AML1 gene24; the t(3;21)
translocation, which results in a fusion between the AML1,
MDS1, and EVI1 genes25; and the
t(9;16)(q34;p11) found in one case in blast crisis and severe
disseminated intravascular coagulopathy.26 We also
recently found a patient with acute leukemia with a typical t(1;19)
involving the E2a-PBX1 fusion oncoprotein and a Philadelphia
chromosome, which also raises the possibility that these 2 elements
might interact in leukemic transformation (G.S. and A. Hendrick,
unpublished observation, 1999).
Together, these results suggest that several leukemic
oncoproteins in the family of Hox genes and their cofactors
(eg, PBX1), including NUP98-HOXA9, HOXA9, and
E2a-PBX1, might genetically interact with BCR-ABL
and lead to acute leukemic transformation of human bone marrow cells.
Transduction of mouse bone marrow cells by BCR-ABL
retroviral vectors was recently demonstrated to accurately
reproduce human CML with the caveat that mice die of acute
myeloproliferation with pulmonary hemorrhage within 2 to 3 weeks
following reconstitution.27-29 This early death
from myeloproliferation precluded any studies aimed at defining genetic
interactions that are mostly based on shortening the time to acute
leukemia produced by the collaborating oncogenes.
In the studies reported here, we have improved upon the mouse model of
BCR-ABL-induced chronic leukemia by circumventing
BCR-ABL-induced early lethality. With our mouse model, it
was possible to identify 2 oncogenes that genetically interact with
BCR-ABL to generate acute leukemia. The system described
should open avenues for testing collaborator oncogenes to
BCR-ABL using mouse bone marrow (BM) cells.
Animals
Recombinant retroviral vectors
Retroviral generation, infection, and transplantation of primary bone marrow cells High-titer helper-free recombinant retroviruses were generated and tested as previously described.5 Bone marrow cells were obtained from C57BL/6Ly-Pep3b or (PepC3)F1 mice injected 4 days earlier with 5-fluorouracil (5-FU) (150 mg/kg body weight), prestimulated, and cocultivated with irradiated viral producer cells, and then loosely adherent and nonadherent cells were recovered and injected immediately (t = 0), or following a 7-day culture (Figure 1), into sublethally irradiated (850 cGy) 7- to 12-week-old (B6C3)F1 recipient mice, as previously described.6 Gene transfer efficiencies to donor BM cells (BMCs) were determined by flow cytometry 24 hours following harvesting from viral producers at between 32% to 77% (highest, BCR-ABL) or by G418 resistance as reported.7
In vitro cultures Bone marrow cells harvested from coculture on viral producers were seeded in liquid cultures at 1.2 × 105 cells per milliliter and expanded in vitro for 7 days in Iscove modified Dulbecco medium (IMDM) containing 10% fetal calf serum (FCS), 5 ng/mL murine interleukin-3 (IL-3), 10 ng/mL human IL-6, 25 ng/mL murine Steel factor and 3 U/mL erythropoietin, 192 ng/mL transferrin, 2% deionized bovine serum albumin (BSA), 2 mM glutamine, and 5 × 10 5 M  -mercaptoethanol.
RT-PCR studies Semiquantitative studies for detecting the expression of HOXA9 were essentially performed as previously reported.31 Briefly, total cDNA was amplified with the use of an oligo deoxythymidine (dT)-based oligonucleotide global polymerase chain reaction (PCR) amplification method. Amplified cDNA was transferred to nylon membranes, which were later hybridized to a probe specific to HOXA9 or-actin. Semiquantitative BCR-ABL expression studies were
done as previously described,32 with the following
modifications: single-step reverse transcriptase (RT) and
first PCR amplification and primers for internal control: ABL Fc1
external (ext) (5'-TTCAGCGGCCA-GTAGCATCTGACT-3', sense) and
Fc3 ext (5'-GCAGTGTTGATCCTGTAATGG-3', antisense). Nested PCR was then
performed with one fifth of the first RT-PCR reaction with ABL internal
primers: Fc2 internal (int) 5'-TTGTGGCCA-GTGGAGATAACA-3'sense, and Fc3 int 5'-TATCTCAGCGA-GATGGACCT-3' antisense. Amplification was
carried out for 35 cycles.
Clinical specimens Leukemia samples representing greater than 80% infiltration by leukemia cells (all confirmed by D.-C.R. and/or G.S.) were collected in preservative-free heparin, and mononuclear cells were separated by ficoll-hypaque density gradient centrifugation. Samples were obtained with the informed consent of the patients under protocols approved by the Human Subjects Protection Committee of the Maisonneuve-Rosemont Hospital (Montreal, QC, Canada) and the internal review board of Institut de recherches cliniques de Montréal (IRCM). All cell samples were cryopreserved in 10% dimethylsulfoxide (DMSO) by means of standard techniques and stored in the vapor phase of liquid nitrogen until used as described.33DNA and RNA analysis Southern blot analysis was performed as described previously.5 Expression of appropriate proviral mRNAs was confirmed by Northern blot analysis. The probes used for RNA and DNA analyses were random primer 32P-labeled fragments of HOXA9 (HindIII fragment of pBS NUP98-HOXA9, no. 727); BCR (BamHI fragment of MSCV-BCR-ABL-ires-EGFP, no. 802); EGFP (NcoI/ClaI fragment of MSCV-pgk-EGFP, no. 652); RFP (HpaI/NheI fragment of pDsRed-CI, from Clontech); and actin (PstI fragment as described31). Northern blots were performed as follows: membranes were stripped and rehybridized by means of an end-labeled oligonucleotide, 5'-ACG GTA TCT GAT CGT CCT CGA ACC-3', specific for 18S rRNA to evaluate the relative amounts of total RNA loaded in each lane.
The development of a mouse model to study oncogenes that interact with BCR-ABL in leukemic transformation Retroviral-mediated expression of BCR-ABL in primary mouse bone marrow (BM) cells previously exposed to the cytotoxic drug 5-FU induces an aggressive CML-like disease that kills mice in less than 2 to 3 weeks after bone marrow transplantation. This early demise precluded analysis of genetic interaction between BCR-ABL and other oncogenes chosen for these studies (Figure 1A-F; description of experimental protocols, and of the early myeloproliferative disease occurring in recipients of BCR-ABL-transduced BM cells). Since in vitro cultures appear to selectively deplete BCR-ABL-expressing primitive bone marrow progenitors in humans,34 we reasoned that it might similarly purge BCR-ABL-transduced cells in mice and potentially eliminate this acute myeloproliferative disease, thereby allowing us to study in vivo genetic interactions between BCR-ABL and the other oncogenes.The culture conditions chosen were the same as those used for
retroviral gene transfer, except that cells were maintained for 7 days
in vitro following retroviral gene transfer (Figure 1B). The effect of
this in vitro culture on the leukemogenic potential of transduced bone
marrow cells was determined by comparing the time to leukemia occurence
in recipients of 2 × 105 BM cells transplanted
immediately following retroviral gene transfer with recipients of the
same number of day-0 cells grown in vitro for 7 days. This
comparison was done for all oncogenes used in these studies, that is,
BCR-ABL, NUP98-HOXA9, and HOXA9. While the 7-day
culture period had little impact on the occurrence of leukemia onset
for recipients of NUP98-HOXA9 or
HOXA9-transduced cells (52-223 days after transplantation,
n = 7), it completely abrogated the acute myeloproliferative disease
in recipients of BCR-ABL-transduced cells (Figure 1C,G-H;
Table 1).
As previously reported, recipients of BCR-ABL-transduced BM cells transplanted immediately after viral transduction developed acute myeloproliferation within 11 ± 1 days after transplantation (dotted line, Figure 1C, shows survival; Figure 1D-F describes the myeloproliferative disorder). A description of each mouse dying from acute myeloproliferation is provided in Table 1 (mice 1 to 11). Note that these myeloproliferative disorders were characterized by pulmonary hemorrhage, splenomegaly, and BM infiltration by immature myeloid cells, but low white blood cell (WBC) counts (Table 1; Figure 1D). This acute disease was highly polyclonal, as indicated by the analysis of proviral integration sites into DNA isolated from hemopoietic organs of these animals (data not shown). In contrast, recipients of cells maintained in culture for 7 days thrive normally (solid line, Figure 1C) and were killed at various times for analysis. At 52 days after transplantation, 3 mice were analyzed (mice 5A, 6A, and 7A; Table 1; Figure 1G-H); all mice were either normal (n = 2) or showed signs of a mild chronic myeloproliferative disorder characterized by slight increase in mature neutrophils in the bone marrow and spleen and by the presence of a megakaryocyte in the lungs (data not shown). The 3 mice analyzed were reconstituted with low levels of BCR-ABL-transduced cells in their bone marrow and spleens (mice 5A to 7A), but thymic reconstitution was high for 2 of the 3 mice (mice 5A and 7A; Table 1, Figure 1G). BCR-ABL was expressed at very low levels in these cells, as shown by Northern blot analysis (Figure 1H; exposure time was 8 days, compared with 12 hours for the blot shown in Figure 1F). Clonal analysis indicated that in contrast to the highly polyclonal nature of the acute myeloproliferative disease, recipients of cells grown in vitro for 7 days were reconstituted with very few clones, none of which had lymphoid and myeloid potential (data not shown). The low level of repopulation by BCR-ABL-transduced cells in the 3 mice analyzed at 52 days after transplantation suggested that these cells died in the culture conditions since, according to fluorescence-assisted cell sorter (FACS) analysis of EGFP expression, they represented 70% of the cells that initiated these cultures. A series of limiting dilution experiments were performed to determine
the range of depletion of BCR-ABL-transduced long-term repopulating cells (detected by either GFP positivity [Table 1] or by
Southern and Northern blot analyses [Figure 1G-H]) in these cultures
versus that of untransduced cells (detected as
Ly5.1+GFP While the reconstituting ability of BCR-ABL-transduced
cells harvested from our 7-day culture was poor, untransduced cells (Ly5.1+GFP Our culture conditions thus significantly and preferentially depleted BCR-ABL-transduced repopulating cells, which possibly explains the lack of acute myeloproliferation in the subject mice. Interestingly, however, limiting dilution analysis also showed that our culture conditions greatly supported the ex vivo expansion of leukemia-repopulating cells (the myeloid leukemia was generated from BM cells overexpressing HOXA9 plus Meis1 as described5,30). These results suggested that our 7-day culture system was purging BCR-ABL-transduced long-term repopulating cells while only mildly influencing the untransduced cells, but at the same time providing a good environment to expand "fully transformed" myeloid cells. As tested below, this provided an opportunity to evaluate possible collaboration between BCR-ABL and the NUP98-HOXA9 or the HOXA9 oncogenes. NUP98-HOXA9 and HOXA9 genetically interact with BCR-ABL to generate acute myeloid leukemia (AML) in vivo As previously mentioned, cytogenetic studies have reported the association between NUP98-HOXA9 and BCR-ABL in myeloid leukemic blasts, suggesting that both oncoproteins genetically interact in human leukemias. In addition, blast phase CML cells were previously reported to express higher levels of HOXA9 than cells isolated from chronic phase patients, again suggesting that HOXA9 might also interact with BCR-ABL to transform BM cells. These observations were confirmed by semiquantitative RT-PCR studies using mononuclear cells from patients in blast (n = 3) versus accelerated phase CML (n = 2) (Figure 2A).
Recipients of cells transduced with BCR-ABL plus NUP98-HOXA9 or BCR-ABL plus HOXA9 that were not grown in vitro for 7 days died within 12 days of an aggressive myeloproliferative disease that was very difficult to distinguish from acute myeloproliferation as seen in recipients of cells engineered to express only BCR-ABL. Taking advantage of the in vitro "purging culture" described in the previous section, the interaction between the NUP98-HOXA9 (or HOXA9) and BCR-ABL oncoproteins was tested as illustrated in Figure 1B. As expected from our previous studies,5,6,30 recipients of HOXA9-transduced BM cells started to die of AML at approximately 3 months after transplantation, and recipients of NUP98-HOXA9-transduced cells at 11/2 to 2 months (Figure 2B).7 As detailed in Table 1, recipients of
BCR-ABL-transduced cells killed at 52 to 175 days after
transplantation showed no signs of acute leukemia. In contrast, mice
that received BM cells infected with BCR-ABL plus
HOXA9 or BCR-ABL plus NUP98-HOXA9
retroviruses and grown in vitro for 7 days died within 9 days of acute
leukemia (see B plus H and B plus N in Figure 2B). In all cases, the
leukemias were myeloid as evaluated by morphological criteria (Figure
3E-H shows NUP98-HOXA9
plus BCR-ABL, and Figure 3I-L shows HOXA9
plus BCR-ABL). High proportions of the BM cells in
these mice were myeloid blasts, and the remaining cells were myeloid
precursors at various stages (Figures 2C,3E). All leukemic animals had
very elevated WBC counts (estimated by blood-smear evaluation at
greater than 50 000/µL; G.S.); had enlarged spleens (Figure
2C); and presented liver, lung, and kidney infiltration by leukemic
blasts (Figures 2C,3E-L). In contrast to the acute myeloproliferative
disease, no evidence of pulmonary hemorrhage was detected in these
leukemic animals.
FACS analysis showed that the majority of the leukemic blasts were
GFP+ (from BCR-ABL provirus) but did not express
(fewer than 1%) the red fluorescent protein (RFP) (from either the
NUP98-HOXA9 or the HOXA9 proviruses) although
both of these proviruses were easily detected in the leukemic cells by
Southern blot analysis (Figure 4A, second
panel from top), and expression of NUP98-HOXA9 or
HOXA9 was very high in the leukemic blasts as indicated in
Figure 4A (lower panels; see RNA). This suggests that the pgk-RFP
cassette was inactive in our leukemic cells but that the promoter and
enhancer elements in the MSCV long terminal repeat
(LTR) were active and driving the expression of the oncogenes.
More detailed phenotypical analysis of these leukemias could not be
performed with these cells, but when the analysis was repeated
with leukemic cells from another experiment, in which the RFP
selector gene was replaced by neor,
leukemic blasts transduced with NUP98-HOXA9 (or
HOXA9) plus BCR-ABL expressed Mac1 and to a
lesser extent Gr1 (Figure 5). The
presence of a functional neor gene in the
NUP98-HOXA9 or in the HOXA9 integrated provirus
and of EGFP in the BCR-ABL provirus made it
possible to demonstrate the nature of the interaction between
BCR-ABL and NUP98-HOXA9 or HOXA9.
While at the time of BM transplantation (t = 0; Figure 6), 10% to 16% of the colony-forming
cells were resistant to G418 (NUP98-HOXA9 and
HOXA9, respectively) and also expressed EGFP (BCR-ABL), a much higher proportion (43% to 70%) of the
leukemic progenitors derived from the recipients suffering from overt
leukemia were both EGFP+ and neomycin resistant
(t = AML; Figure 6), indicating that cells expressing both oncogenes
were positively selected in vivo.
In agreement with our previous studies, leukemia that developed in recipients of NUP98-HOXA9-transduced (Figure 4B, right lower panel) or HOXA9-transduced (Figure 4B, left lower panel) cells were monoclonal or oligoclonal, clearly indicating the requirement for additional genetic events for leukemic transformation of these cells. In contrast, clonal analysis of leukemias from recipients that received transplants of cells coexpressing BCR-ABL plus NUP98-HOXA9 or HOXA9 exposed the highly polyclonal nature of these leukemic cells for all mice that were studied (presence of smear in Figure 4A, third and fourth panels). This clearly indicated that the combination of these oncogenes was sufficient for the full leukemic transformation of mouse BM cells and, at least for NUP98-HOXA9, strongly suggests its involvement in the progression of CML to AML in selected patients. Together, these studies indicate that our in vitro purging system (1) was effective at eliminating the acute myeloproliferative disease caused by BCR-ABL-transduced 5-FU-treated BM cells even when high cell doses were transplanted; (2) did not accelerate the occurrence of leukemia onset in recipients of NUP98-HOXA9- or HOXA9-transduced cells; (3) allowed the expansion of leukemia-repopulating cells of myeloid origin ex vivo; and (4) was capable of maintaining (and perhaps potentially expanding) BCR-ABL-transduced cells that coexpressed either NUP98-HOXA9 or HOXA9, thus indicating the presence of a strong genetic interaction between BCR-ABL and these genes in AML.
In these studies, we developed an in vitro/in vivo model that allows the purging of BCR-ABL-induced acute myeloproliferative disease. With this model, it was possible to demonstrate a potent genetic interaction between BCR-ABL and 2 oncoproteins: namely, NUP98-HOXA9 and HOXA9. In particular, the interaction between BCR-ABL and NUP98-HOXA9 is potentially relevant to human leukemias as their paired presence has been observed in several leukemic specimens (see "Introduction"). Significantly, the strength of the interaction between BCR-ABL and the other oncoproteins analyzed in our studies was such that, in all cases, a highly polyclonal leukemia occurred in vivo. The polyclonal nature of the leukemias combined with the extremely short time required to develop full-blown AML strongly suggests that these oncoproteins are sufficient to fully transform at least a subset of mouse BM cells. Our findings also demonstrate the importance of the cytogenetic studies describing the presence of more than one recurrent translocation in leukemic cells since, at least for the genes studied here, the correlation between coexpression (detected cytogenetically) and genetic interaction was established. Supporting this, a recent study suggested that BCR-ABL and AML1/MDS1/EVI1 (AME) also genetically interact.37 The availability of additional results, especially with the use of more sensitive tools such as spectral karyotyping (SKY),38 should facilitate the design of functional studies like this one that should help establish the potential number of complementation groups involved in BCR-ABL-induced transformation of BM cells. Since both NUP98-HOXA9 and HOXA9 collaborate with BCR-ABL and with Meis1,7,30 we speculated that NUP98-HOXA9 and HOXA9 belong to the same complementation group. This hypothesis was tested in the course of these studies where mouse BM cells were engineered to coexpress these 2 genes and transplanted into primary recipients together with cells expressing either HOXA9 or NUP98-HOXA9. Leukemia onset was not accelerated in mice that received transplants of cells coexpressing both oncogenes, which indicates the absence of oncogenic collaboration between HOXA9 and NUP98-HOXA9 (data not shown). It remains to be demonstrated whether Meis1 and BCR-ABL belong to the same complementation group. Although the involvement of NUP98-HOXA9 in the progression of CML seems unambiguous, the role of HOXA9 is, in this context, less clear. As shown here, 3 of 3 cases of blast crisis CML expressed high levels of HOXA9 when compared with levels detected in cells from accelerated phase CML. Although we used the mononuclear fractions to eliminate mature cells in our samples, and morphological analysis showed that cells in both groups consisted mainly of blasts and promyelocytes, the possibility of selection of cells that naturally express high levels of HOXA9 in samples from patients in blast phase cannot be eliminated. However, this proves that overexpressed proteins (eg, HOXA9) can reproduce the oncogenic effect of fusion oncoproteins (ie, NUP98-HOXA9). Therefore, it will be important to evaluate misexpression of oncogenes (in addition to fusion oncogenes) when seeking genes involved in the progression of CML to acute leukemia. Recent studies done with human large-cell lymphomas support this argument.39 By exploiting both the results of cytogenetic studies made with human leukemic specimens and our in vitro purging system, we were able to demonstrate the presence of a strong genetic interaction between BCR-ABL and NUP98-HOXA9 or HOXA9. These results also suggest that these oncogenes could change the course of CML from an indolent chronic disorder to an aggressive acute leukemia. It is hoped that the reported system will be effective enough to permit a functional screen of other collaborators to BCR-ABL, a subject of ongoing investigation in our laboratory. Note. While this paper was being revised, the oncogenic collaboration between BCR-ABL and NUP98-HOXA9 was demonstrated by Dash et al.40 Significantly different from our study, these authors used a diluted retroviral preparation to avoid the myeloproliferative disease normally occurring in recipients of BCR-ABL-transduced cells.
Dr Neal Copeland and Dr Trang Hoang's critical review of this manuscript is greatly acknowledged. Dr Julie Rousseau is also acknowledged for helping with the identification of leukemic specimens used in this study. We thank Ms Theresa Holst for editing and proofreading of this paper. Ms Marie-Eve Leroux and Mr Stéphane Matte are also thanked for their expertise and help regarding maintenance and manipulation of animals kept at the specific pathogen-free (SPF) facility. The support of Ms Nathalie Tessier and Mr Eric Massicotte is acknowledged for flow cytometric analyses and of Mr Christian Charbonneau for digital imaging services at the IRCM.
Submitted April 26, 2002; accepted July 15, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-04-1244.
Supported by a grant from the National Cancer Institute of Canada (grant no. MOP-15064) to G.S. and a grant from the Research Cancer Society to D.-C.R. D.-C.R is a Scholar of the Fonds de la Recherche en Santé du Québec, and G.S. is a Canadian Institutes of Health Research Clinician-Scientist Scholar.
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: Guy Sauvageau, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, QC, Canada, H2W 1R7; e-mail: sauvagg{at}ircm.qc.ca.
1. Rowley JD. The critical role of chromosome translocations in human leukemias. Annu Rev Genet. 1998;32:495-519[CrossRef][Medline] [Order article via Infotrieve]. 2. Burmeister T, Thiel E. Molecular genetics in acute and chronic leukemias. J Cancer Res Clin Oncol. 2001;127:80-90[CrossRef][Medline] [Order article via Infotrieve].
3.
Slany RK, Lavau C, Cleary ML.
The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX.
Mol Cell Biol.
1998;18:122-129 4. Lavau C, Szilvassy SJ, Slany R, Cleary ML. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 1997;16:4226-4237[CrossRef][Medline] [Order article via Infotrieve].
5.
Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G.
Defining roles for HOX and MEIS1 genes in induction of acute myeloid leukemia.
Mol Cell Biol.
2001;21:224-234
6.
Thorsteinsdottir U, Krosl J, Kroon E, Haman A, Hoang T, Sauvageau G.
The oncoprotein E2A-Pbx1a collaborates with Hoxa9 to acutely transform primary bone marrow cells.
Mol Cell Biol.
1999;19:6355-6366 7. Kroon E, Thorsteinsdottir U, Mayotte N, Nakamura T, Sauvageau G. NUP98-HOXA9 expression in hemopoietic stem cells induces chronic and acute myeloid leukemias in mice. EMBO J. 2001;20:350-361[CrossRef][Medline] [Order article via Infotrieve].
8.
Kamps MP, Baltimore D.
E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice.
Mol Cell Biol.
1993;13:351-357
9.
Zimonjic DB, Pollock JL, Westervelt P, Popescu NC, Ley TJ.
Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice.
Proc Natl Acad Sci U S A.
2000;97:13306-13311 10. He L, Bhaumik M, Tribioli C, et al. Two critical hits for promyelocytic leukemia. Mol Cell. 2000;6:1131-1141[CrossRef][Medline] [Order article via Infotrieve]. 11. Dobson CL, Warren AJ, Pannell R, et al. The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis. EMBO J. 1999;18:3564-3574[CrossRef][Medline] [Order article via Infotrieve]. 12. Valge-Archer V, Forster A, Rabbitts TH. The LMO1 AND LDB1 proteins interact in human T cell acute leukaemia with the chromosomal translocation t(11;14)(p15;q11). Oncogene. 1998;17:3199-3202[CrossRef][Medline] [Order article via Infotrieve]. 13. Corral J, Lavenir I, Impey H, et al. An Mll-AF9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell. 1996;85:853-861[CrossRef][Medline] [Order article via Infotrieve]. 14. Largaespada DA, Brannan CI, Jenkins NA, Copeland NG. Nf1 deficiency causes Ras-mediated granulocyte/macrophage colony stimulating 7factor hypersensitivity and chronic myeloid leukaemia. Nat Genet. 1996;12:137-143[CrossRef][Medline] [Order article via Infotrieve]. 15. Nakamura T, Largaespada DA, Shaughnessy JD Jr, Jenkins NA, Copeland NG. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. Nat Genet. 1996;12:149-153[CrossRef][Medline] [Order article via Infotrieve]. 16. Warrell RP Jr, Frankel SR, Miller WH Jr, et al. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med. 1991;324:1385-1393[Abstract].
17.
Levis M, Tse KF, Smith BD, Garrett E, Small D.
A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations.
Blood.
2001;98:885-887
18.
Mauro MJ, Druker BJ.
STI571: targeting BCR-ABL as therapy for CML.
Oncologist.
2001;6:233-238 19. Ahuja HG, Popplewell L, Tcheurekdjian L, Slovak ML. NUP98 gene rearrangements and the clonal evolution of chronic myelogenous leukemia. Genes Chromosomes Cancer. 2001;30:410-415[CrossRef][Medline] [Order article via Infotrieve]. 20. Yamamoto K, Nakamura Y, Saito K, Furusawa S. Expression of the NUP98/HOXA9 fusion transcript in the blast crisis of Philadelphia chromosome-positive chronic myelogenous leukaemia with t(7;11)(p15;p15). Br J Haematol. 2000;109:423-426[CrossRef][Medline] [Order article via Infotrieve]. 21. Shimamoto T, Ohyashiki K, Ohyashiki JH, et al. Late appearance of a Philadelphia translocation with minor-BCR/ABL transcript in a t(7;11)(p15;p15) acute myeloid leukemia. Leukemia. 1995;9:640-642[Medline] [Order article via Infotrieve]. 22. Celetti A, Barba P, Cillo C, Rotoli B, Boncinelli E, Magli MC. Characteristic patterns of HOX gene expression in different types of human leukemia. Int J Cancer. 1993;53:237-244[Medline] [Order article via Infotrieve]. 23. Nishii K, Usui E, Sakakura M, et al. Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia. Cancer Genet Cytogenet. 2001;126:8-12[CrossRef][Medline] [Order article via Infotrieve]. 24. Kojima K, Yasukawa M, Ishimaru F, et al. Additional translocation (8;21)(q22;q22) in a patient with Philadelphia-positive chronic myelogenous leukaemia in the blastic phase. Br J Haematol. 1999;106:720-722[CrossRef][Medline] [Order article via Infotrieve].
25.
Cuenco GM, Nucifora G, Ren R.
Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: a model for human AML.
Proc Natl Acad Sci U S A.
2000;97:1760-1765 26. Fujiwara H, Takahashi N, Tada J, et al. Blast crisis accompanied by severe DIC of Ph negative chronic myeloid leukemia showing t(9;16) and positive M-BCR/ABL rearrangement [in Japanese]. Rinsho Ketsueki. 1997;38:663-668[Medline] [Order article via Infotrieve].
27.
Pear WS, Miller JP, Xu L, et al.
Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.
Blood.
1998;92:3780-3792
28.
Zhang X, Ren R.
Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia.
Blood.
1998;92:3829-3840 29. Van Etten RA. Retroviral transduction models of Ph(+) leukemia: advantages and limitations for modeling human hematological malignancies in mice. Blood Cells Mol Dis. 2001;27:201-205[CrossRef][Medline] [Order article via Infotrieve]. 30. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. EMBO J. 1998;17:3714-3725[CrossRef][Medline] [Order article via Infotrieve].
31.
Lessard J, Baban S, Sauvageau G.
Stage-specific expression of polycomb group genes in human bone marrow cells.
Blood.
1998;91:1216-1224 32. Pichert G, Roy DC, Gonin R, et al. Distinct patterns of minimal residual disease associated with graft-versus-host disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia. J Clin Oncol. 1995;13:1704-1713[Medline] [Order article via Infotrieve].
33.
Leonard BM, Hetu F, Busque L, et al.
Lymphoma cell burden in progenitor cell grafts measured by competitive polymerase chain reaction: less than one log difference between bone marrow and peripheral blood sources.
Blood.
1998;91:331-339
34.
Udomsakdi C, Eaves CJ, Swolin B, Reid DS, Barnett MJ, Eaves AC.
Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level.
Proc Natl Acad Sci U S A.
1992;89:6192-6196
35.
Sauvageau G, Thorsteinsdottir U, Eaves CJ, et al.
Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.
Genes Dev.
1995;9:1753-1765 36. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell. 2002;109:39-45[CrossRef][Medline] [Order article via Infotrieve]. 37. Cuenco GM, Ren R. Cooperation of BCR-ABL and AML1/MDS1/EVI1 in blocking myeloid differentiation and rapid induction of an acute myelogenous leukemia. Oncogene. 2001;20:8236-8248[CrossRef][Medline] [Order article via Infotrieve]. 38. Odero MD, Carlson KM, Calasanz MJ, Rowley JD. Further characterization of complex chromosomal rearrangements in myeloid malignancies: spectral karyotyping adds precision in defining abnormalities associated with poor prognosis. Leukemia. 2001;15:1133-1136[CrossRef][Medline] [Order article via Infotrieve]. 39. Pasqualucci L, Neumeister P, Goossens T, et al. Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature. 2001;412:341-346[CrossRef][Medline] [Order article via Infotrieve]. 40. Dash AB, Williams IR, Kutok JL, et al. A murine model of CML blast crisis induced by cooperation between BCR/ABL and NUP98/HOXA9. Proc Natl Acad Sci U S A. 2002;28:7622-7627.
© 2002 by The American Society of Hematology.
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  C. Roche-Lestienne, L. Deluche, S. Corm, I. Tigaud, S. Joha, N. Philippe, S. Geffroy, J.-L. Lai, F.-E. Nicolini, C. Preudhomme, et al. RUNX1 DNA-binding mutations and RUNX1-PRDM16 cryptic fusions in BCR-ABL+ leukemias are frequently associated with secondary trisomy 21 and may contribute to clonal evolution and imatinib resistance Blood, April 1, 2008; 111(7): 3735 - 3741. [Abstract] [Full Text] [PDF]
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S. J. Neering, T. Bushnell, S. Sozer, J. Ashton, R. M. Rossi, P.-Y. Wang, D. R. Bell, D. Heinrich, A. Bottaro, and C. T. Jordan Leukemia stem cells in a genetically defined murine model of blast-crisis CML Blood, October 1, 2007; 110(7): 2578 - 2585. [Abstract] [Full Text] [PDF]
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 |
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 K. Y. Chung, G. Morrone, J. J. Schuringa, M. Plasilova, J.-H. Shieh, Y. Zhang, P. Zhou, and M. A.S. Moore Enforced Expression of NUP98-HOXA9 in Human CD34+ Cells Enhances Stem Cell Proliferation Cancer Res., December 15, 2006; 66(24): 11781 - 11791. [Abstract] [Full Text] [PDF]
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 A. Rizo, E. Vellenga, G. de Haan, and J. J. Schuringa Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R210 - R219. [Abstract] [Full Text] [PDF]
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L. Palmqvist, B. Argiropoulos, N. Pineault, C. Abramovich, L. M. Sly, G. Krystal, A. Wan, and R. K. Humphries The Flt3 receptor tyrosine kinase collaborates with NUP98-HOX fusions in acute myeloid leukemia Blood, August 1, 2006; 108(3): 1030 - 1036. [Abstract] [Full Text] [PDF]
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A. Mamo, J. Krosl, E. Kroon, J. Bijl, A. Thompson, N. Mayotte, S. Girard, R. Bisaillon, N. Beslu, M. Featherstone, et al. Molecular dissection of Meis1 reveals 2 domains required for leukemia induction and a key role for Hoxa gene activation Blood, July 15, 2006; 108(2): 622 - 629. [Abstract] [Full Text] [PDF]
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M. Iwasaki, T. Kuwata, Y. Yamazaki, N. A. Jenkins, N. G. Copeland, M. Osato, Y. Ito, E. Kroon, G. Sauvageau, and T. Nakamura Identification of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model Blood, January 15, 2005; 105(2): 784 - 793. [Abstract] [Full Text] [PDF]
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B. Calabretta and D. Perrotti The biology of CML blast crisis Blood, June 1, 2004; 103(11): 4010 - 4022. [Abstract] [Full Text] [PDF]
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X. Jiang, M. Stuible, Y. Chalandon, A. Li, W. Y. Chan, W. Eisterer, G. Krystal, A. Eaves, and C. Eaves Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia Blood, October 15, 2003; 102(8): 2976 - 2984. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
" lane indicates 10 µg DNA isolated from the spleen of a
syngenic control mouse, and the "+" sign is a positive control for
hybridization consisting of 20 pg KpnI-digested retroviral
plasmid no. 652 ("Materials and methods") generating a 2.7-kb
fragment.
