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Leukemia (2007) 21, 462–471
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ORIGINAL ARTICLE
NOTCH1-induced T-cell leukemia in transgenic zebrafish
J Chen1, C Jette2, JP Kanki2, JC Aster3, AT Look2 and JD Griffin1
Department of Medical Oncology, Dana-Farber Cancer Institute of Harvard Medical School, Boston, MA, USA; 2Department of
Pediatric Oncology, Dana-Farber Cancer Institute of Harvard Medical School, Boston, MA, USA and 3Department of Pathology,
Brigham and Women’s Hospital, Boston, MA, USA
1
Activating mutations in the NOTCH1 gene have been found in
about 60% of patients with T-cell acute lymphoblastic leukemia
(T-ALL). In order to study the molecular mechanisms by which
altered Notch signaling induces leukemia, a zebrafish model of
human NOTCH1-induced T-cell leukemia was generated.
Seven of sixteen mosaic fish developed a T-cell lymphoproliferative disease at about 5 months. These neoplastic cells
extensively invaded tissues throughout the fish and caused
an aggressive and lethal leukemia when transplanted into
irradiated recipient fish. However, stable transgenic fish
exhibited a longer latency for leukemia onset. When the stable
transgenic line was crossed with another line overexpressing
the zebrafish bcl2 gene, the leukemia onset was dramatically
accelerated, indicating synergy between the Notch pathway
and the bcl2-mediated antiapoptotic pathway. Reverse transcription-polymerase chain reaction analysis showed that
Notch target genes such as her6 and her9 were highly
expressed in NOTCH1-induced leukemias. The ability of this
model to detect a strong interaction between NOTCH1 and bcl2
suggests that genetic modifier screens have a high likelihood
of revealing other genes that can cooperate with NOTCH1 to
induce T-ALL.
Leukemia (2007) 21, 462–471. doi:10.1038/sj.leu.2404546;
published online 25 January 2007
Keywords: Notch; bcl2; T-ALL; zebrafish
Introduction
T-cell acute lymphoblastic leukemia (T-ALL) comprises 10–25%
of all pediatric and adult ALL. Unlike the more common B-cell
lineage ALL, T-ALL is characterized by the expression of stagerelated T-cell surface antigens, higher leukocyte counts, older
age at presentation and a male predominance. Even though cure
rates for adult and children with T-ALL have reached 50 and
65–75%, respectively,1–5 the prospects for additional improvements in therapy are dependent upon a more complete
understanding of the molecular aspects of T-ALL pathogenesis
and how they influence prognosis.
Notch receptors are a family of cell surface proteins that
regulate cell fate decisions and growth control.6,7 Dysregulated
Notch signal transduction can contribute to several types of
cancers, in addition to other human diseases. Notch signaling
was first linked to tumorigenesis through identification of a
recurrent t(7;9)(q34;q34.3) chromosomal translocation that is
found in an uncommon subset of human T-ALL.8 This
Correspondence: Dr JD Griffin, Department of Medical Oncology,
Dana-Farber Cancer Institute of Harvard Medical School, 44 Binney
Street, Boston, MA 02115, USA.
E-mail: [email protected]
Received 11 September 2006; revised 13 November 2006; accepted
21 November 2006; published online 25 January 2007
chromosome translocation juxtaposes the NOTCH1 intracellular domain (ICN1) with the T-cell receptor-b chain
promoter/enhancer region, resulting in the constitutive activation of Notch signaling in the T cells. Mice reconstituted with
hematopoietic progenitor cells expressing the truncated human
NOTCH1 gene develop T-cell leukemia.9,10 Immature, doublepositive T cells accumulate in the bone marrow with simultaneous inhibition of B-cell development, indicating that Notch1
signaling drives common lymphoid progenitor cells into the
T-cell lineage. T-cell neoplasia can also be caused by proviral
integration into the Notch gene, which leads to aberrant
Notch signaling. Proviral integration of the Moloney murine
leukemia virus or feline leukemia virus into Notch1 or
Notch2, respectively, causes T-cell leukemia.11,12 Although
the t(7;9)(q34;q34.3) chromosomal translocation is uncommon,
it was recently found that about 60% of adult and childhood
T-ALL patients carry deletions and/or mutations in the NOTCH1
gene.13–16 In vitro Notch reporter assays indicated that these
mutations and/or deletions lead to aberrant NOTCH1 activation.
Both g-secretase inhibitors and dominant-negative Mastermind
(MAML1) induce G1 cell-cycle arrest of these T-ALL cell lines,
indicating that activity of the Notch1-MAML1-CSL transcription
complex is essential to maintain the transformed phenotype of
these T-ALLs. Although it is known that Notch1 can collaborate
with c-Myc,11 E2A-PBX117 and Ikaros18 to induce T-ALL, the
in vivo molecular mechanisms by which altered Notch signaling
contributes to T-cell leukemia are not known.
The zebrafish is a valuable vertebrate model in which to
elucidate novel molecular mechanisms of tumorigenesis. In
particular, this model organism can be induced to develop
T-cell leukemias resembling those in humans and is amenable to
large-scale forward-genetic screens that can be used to uncover
novel cancer pathways.19 Comparisons of zebrafish and
mammalian hematopoiesis and lymphopoiesis indicate that
the genetic programs underlying vertebrate blood development
have been highly conserved through evolution.20 The zebrafish
exhibit thymic architecture similar to mammals, with a cortical
and medullary region compartmentalizing T cells as they
mature.21 T- and B-cell development in the zebrafish relies on
many of the same molecules and pathways used in mammalian
lymphopoiesis.22,23 Similarly, lymphoid-specific genes, including ikaros, rag-1, rag-2 and lck, have also been identified in
the zebrafish, and these genes exhibit appropriate spatiotemporal expression patterns. Therefore, knowledge gathered
from zebrafish oncogenesis studies should provide direct
insights into the molecular pathogenesis of human neoplasia.
Recently, a rag2-EGFP-mMyc transgenic zebrafish line has
been created that developed T-ALL.19 These T-cell leukemias
express the zebrafish orthologues of the human T-ALL oncogenes scl and lmo2, thus providing an animal model for a
subgroup of human T-ALL.24
NOTCH1-induced zebrafish T-ALLJ
Chen et al
In this study, we generated a transgenic zebrafish model of
human NOTCH1-induced T-cell leukemia. The leukemias were
T cell in origin, oligoclonal and transplantable, and the Notch
target genes such as her6 and her9 were highly expressed in
NOTCH1-transformed T cells. Although mosaic F0 fish developed leukemia at about 5 months, stable F1 transgenic fish
exhibit a longer latency for leukemia onset. When the stable
transgenic line was crossed with another fish line overexpressing
the zebrafish bcl2 gene, the leukemia onset was greatly
accelerated, indicating the synergy between the Notch pathway
and bcl2-mediated antiapoptotic pathway. This zebrafish model
is important because genetic modifier screens using transgenic
zebrafish prone to NOTCH1-induced T-cell leukemia may
ultimately reveal mutant genes that can either promote specific
aspects of the malignant phenotype, or delay or totally suppress
the onset of leukemia. The proteins encoded by these genes will
serve as candidate targets for the development of new therapies
for more effective treatment of human T-ALL.
Materials and methods
Vector production and microinjection of DNA construct
into zebrafish embryos
Human NOTCH1 intracellular domain (amino acid 1762–2555)
was fused at its C-terminus with an enhanced green fluorescent
protein (EGFP) tag and cloned downstream of the zebrafish rag2
promoter in the pBluescript KS( þ ) vector. The rag2-ICN1-EGFP
construct was linearized and resuspended in 0.5 Tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) þ 100 mM
KCl to a concentration of 50 ng/ml and was injected into
zebrafish embryos at the one- or two-cell stage of development.
rag2-ICN1-EGFP transgenic founder lines were identified on the
basis of GFP expression in the thymus at about 30 days
postfertilization (dpf).
Histology
Wild-type and leukemic zebrafish were fixed in 4% paraformaldehyde at 41C for 4 days and were then transferred to
0.25 M EDTA (pH 8.0) for no less than 2 days. The fish were then
dehydrated in alcohol, cleared in xylene, and infiltrated with
paraffin. Tissue sections (4 mm thick) from paraffin-embedded
tissue blocks were placed on charged slides, deparaffinized in
xylene, rehydrated through graded alcohol solutions, and
stained with hematoxylin/eosin.
Immunohistochemistry
Paraffin sections were used for immunohistochemical detection
of human NOTCH1 intracellular domain or EGFP protein
expression as described.10 EGFP antibody dilution was 1:1500
(Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the
antibody to the human NOTCH1 intracellular domain was
diluted at 1:2000.10
Fluorescence-activated cell sorting analysis
Kidney and spleen isolated from wild-type fish or leukemic rag2ICN1-EGFP fish were homogenized in ice-cold 0.9 phosphatebuffered saline (PBS) þ 5% fetal bovine serum (FBS) and passed
through a 40 mm filter. Cells were washed once with the same
solution, stained with propidium iodide and analyzed by
fluorescence-activated cell sorting (FACS) to quantify the extent
of leukemic cell infiltration into these organs.19,25
Southern blot analysis
463
Genomic DNAs were isolated from FACS-sorted GFP þ thymocytes from rag2-GFP control fish or GFP þ leukemic cells from
rag2-ICN1-EGFP fish, digested with BglII, and hybridized with
T-cell receptor (TCR-a) probe as described.19,24
RT-PCR
FACS sorted GFP-positive leukemic cells were extracted for
RNA (Trizol, Invitrogen, Carlsbad, CA, USA) and used for
reverse transcription-polymerase chain reaction (RT-PCR). RNA
was treated with DNAseI before RT and PCR primers were
designed to span introns. Those primers and reaction conditions
used for RT-PCR for scl, lmo1, lmo2, hox11, tlx3a, tlx3b, lck,
rag2 and b-actin were reported previously.24 The primers for
her1, her4, her6, her9, myc-a and myc-b are: (her1, forward,
50 -TGGCAAGTCGAAGACCAACCAAACG-30 ; reverse, 50 -TTGGA
TTGCTGGAAACTCTGCCGTG-30 ), (her4, forward, 50 -CTCCTA
CAATCACTGGATCAATCAGC-30 ; reverse, 50 -ACTGCGGGCT
GGAGTGTGTTGTTTG-30 ), (her6, forward, 50 -CGGCTTCGGAA
CACAGAAAGTCT-30 ; reverse, 50 -CGTCAGAAGTCAAGTTGG
AGGAT-30 ), (her9, forward, 50 -AGAATGCCAGCGAGCATAG
AAAG-30 ; reverse, 50 -AGGCTGACCCAATTGAGCCTGTT-30 ),
(myc-a, forward, 50 -GGCTAGCAACAATCACAGCA-30 ; reverse,
50 -ATGCACTCTGTCGCCTCCTT-30 ), (myc-b, forward, 50 -GGTG
TTTCCCTTTCCACTGA-30 ; reverse, 50 -TTCTCTTTTCCACCGTG
ACC-30 ).
Leukemic cell transplantation
Leukemic fish were killed, diced in 0.9 PBS þ 5% FBS, and
homogenized. The resulting cell suspension was filtered over a
40 mm filter, washed with 0.9 PBS þ 5% FBS and resuspended
at a concentration of 1 million cells per 5 ml. Two days after
receiving a sublethal dose of radiation (23 Gy from a 137Cs
source), irradiated AB wild-type recipient zebrafish were
injected intraperitoneally with 1 million leukemic cells. The
homing of leukemic cells to the thymus and their infiltration into
adjacent regions were frequently monitored by fluorescent
microscopy.
Stable transgenic lines
Pair-wise mating of about 300 mosaic rag2-ICN1-EGFP F0 fish
was performed and the genomic DNAs of the F1 offspring
were purified and screened by PCR for transgene expression.
The forward primer was within the human NOTCH1
coding sequence (50 -CACTATTCTGCCCCAGGAGA-30 ), and
the reverse primer lay within the EGFP coding sequence
(50 -CGTCGCCGTCCAGCTCGACCAG-30 ). Control primers (forward, 50 -ATGCTAATTTGAAGCACTAGCA-30 ; reverse, 50 -AGT
GAGCTATCGCAGGACAT-30 ) amplified a 515-bp genomic
fragment comprising a portion of zebrafish rag2 promoter and
rag2 open reading frame. One mosaic transgenic founder was
found to have the transgene transmitted to its F1 progeny with a
positive PCR band. This mosaic fish was mated again and its
transgenic F1 offspring were identified based on EGFP expression in the thymus when they were about 1 month old.
TUNEL assay
Chromogenic terminal deoxynucleotidyl transferase mediated
nick end labeling staining using the ApopTag Peroxidase In Situ
Apoptosis detection Kit (Chemicon International, Temecula, CA,
USA) as per the manufacturer’s instructions.
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464
A zebrafish model of human NOTCH1-induced T-cell
leukemia
We generated transgenic zebrafish that express a fusion protein
containing the EGFP and the truncated human NOTCH1 protein
(ICN1-EGFP) targeted to developing lymphocytes through use of
the zebrafish rag2 promoter. Zebrafish rag2 is transcribed by
T-cell precursors of the developing thymus starting at about
72 hpf and its promoter has been shown to target transgene
expression to the immature T-cell compartment.26–28 In vitro
Notch reporter assays showed that fusion of EGFP to the Cterminus of truncated human NOTCH1 protein did not affect
NOTCH1 activity (data not shown).
Sixteen of 747 (2.1%) mosaic F0 zebrafish embryos injected
with the transgene were found to have GFP expression in the
A a
b
C
OB
E
T
OB
T
d
E
E
PF
B
Percentage
PF
c
thymus. At approximately 5 months, seven fish began to show
visible infiltration of GFP þ cells locally adjacent to the thymus,
followed by development of a distended abdomen and splayed
eyes due to retro-orbital infiltration by GFP þ cells. Some fish
also had growths protruding from under the operculum, whereas
some had tumors at the base of the pectoral fin (Figure 1A). The
remaining nine mosaic fish appeared healthy, without evidence
of GFP þ cell expansion. In retrospect, the strength of the GFP þ
signal in the thymus was higher in the seven fish ultimately
developing extra-thymic proliferation of GFP þ cells than in the
nine fish that remained healthy, suggesting that expression levels
of the transgene may be critical for clonal expansion.
Histological analysis showed infiltration of cells with the
morphology of lymphoblasts to most tissues and organs of the
seven mosaic fish (Figure 1B). Immunostaining with antibodies
against EGFP or the intracellular domain of human NOTCH1
granulocytes+
monocytes
Kidney
100
90
90
80
80
70
70
60
50
40
erythrocytes
Spleen
100
60
50
40
30
30
20
20
10
10
0
PF
lymphocytes
Percentage
Results
0
WT
ICN1
WT
a
b
c
d
i
e
f
g
h
j
ICN1
Figure 1 External and histological features of leukemic rag2-ICN1-EGFP F0 mosaic fish. (A) Wild-type fish and rag2-ICN1-EGFP fish with
infiltration of leukemic cells into the retro-orbital soft tissues, olfactory region and pectoral fin. (a) Wild-type fish (3 month) under ultraviolet (UV)
light with the normal green thymus. (b) Seven-month-old rag2-ICN1-EGFP F0 mosaic fish under UV light with local infiltration of the leukemic
cells around the gill arch and in the head region. (c) Eleven-month-old rag2-ICN1-EGFP F0 mosaic fish under visible light with massive
dissemination of the leukemic cells into the retro-orbital soft tissues, olfactory region and pectoral fin. (d) The same 11-month-old rag2-ICN1-EGFP
F0 mosaic fish under UV light. (B) Infiltration of leukemic cells into muscles, gills, adipocytes and under the skin (red arrows) of leukemic fish
as shown by histological analysis. (a–d) tissues from wild-type fish. (e–h) tissues from rag2-ICN1-EGFP leukemic fish. (a), (e) muscle; (b, f) gills;
(c, g) adipocytes; (d, h) skin. Expression of ICN1-EGFP fusion protein in leukemic cells (red arrows) in gills (i) and muscles (j) as shown by immunostaining with antibody to human NOTCH1 intracellular domain. T, thymus; OB, olfactory bulb; PF, pectoral fin; E, eye. (C) Infiltration of leukemic
cells into kidney and spleen of the diseased fish. FACS analysis of cells isolated from kidney and spleen from wild-type fish and rag2-ICN1-EGFP
leukemic fish. The leukemia blasts comprised about 85% of the kidney marrow and 70% of the spleen with a granularity similar to that of normal
lymphocytes. Populations of cells within each category are described as mean percentage of total cells.
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demonstrated that these cells express the fusion protein encoded
by the transgene (Figure 1B and data not shown). Kidney
‘marrow’ and spleen cells were isolated from rag2-GFP control
fish or rag2-ICN1-EGFP fish and were analyzed by FACS to
quantify the extent of lymphoid cell infiltration into these
organs. In wild-type zebrafish, the kidney contained about
(6.372.5) 105 blood cells, and the spleen contained
(1.170.6) 105 blood cells. In fish with NOTCH1-associated
disease, blood cells were increased sevenfold in the kidney to
(4.6771.4) 106 and fivefold in the spleen to (5.372.1) 105.
The lymphoid cells comprised about 85% of the kidney marrow
and 70% of the spleen, and the leukemic lymphoblasts had a
FACS scatter profile similar to that of lymphocytes (Figure 1C).
The finding that all fish developed disease initially in the
thymus suggested that the lymphoid cells were likely to be of
T-cell derivation. RT-PCR analysis of RNA isolated from GFPpositive cells showed that genes that are expressed in pre-T cells
such as lck and rag2 are highly expressed in the human
NOTCH1 transformed zebrafish cells (Figure 4), confirming that
these lymphoid cells are T cells in origin.
NOTCH1-induced zebrafish leukemias are oligoclonal
To study the clonality of NOTCH1-induced zebrafish leukemias,
Southern blot analysis was performed with genomic DNAs
isolated from normal thymocytes of control fish or from
leukemic cells of rag2-ICN1-EGFP fish, using radiolabeled
TCR-a probe. NOTCH1-induced zebrafish leukemias contained
oligoclonal TCR-a gene rearrangements (Figure 2).
ICNI
1
2
WT
3
4
5
6
- 8.0
- 6.0
- 5.0
- 4.0
Figure 2 NOTCH1-induced leukemias have oligoclonal TCR-a gene
rearrangements. Southern blot analysis of genomic DNAs digested
with BglII and hybridized with the TCR-a probe. Genomic DNAs were
isolated from either leukemic cells of rag2-ICN1-EGFP fish or from
normal thymocytes of rag2-GFP control fish (numbered). Sizes of DNA
standards in kb are noted to the right of blots. The nonrearranged BglII
digested fragment is around 8.0 kb.
a
NOTCH1-induced zebrafish leukemias are
transplantable
Transplantation and propagation of disease into secondary
recipients is an important assay for clonal expansion and is a
hallmark of cancer. To assess the transplantability of NOTCH1transformed zebrafish T cells, mononuclear cells were harvested
from an 11-month-old F0 mosaic fish and then transplanted
intraperitoneally into 12 irradiated wild-type adult zebrafish.
GFP-positive cells were apparent at the site of injection after 7
days of transplantation and had begun to spread throughout the
peritoneal cavity within 14 days (Figure 3). Lymphoid cells
homing to the thymus was observed by 21 days posttransplantation. Histological analysis showed that nearly all
the recipient’s organs and tissues exhibited extensive lymphoid
cell infiltration (Supplementary Figure S1). These transplantation
experiments establish that the NOTCH1-induced lymphoproliferative disorder in zebrafish is a highly malignant leukemia/
lymphoma.
Expression of Notch target genes in NOTCH1-induced
T-cell leukemias
Among the best characterized effectors of Notch signaling in
vertebrates are the basic helix-loop-helix (bHLH) transcription
factors of the Hairy/Enhancer-of-split (E(spl)) family. her family
members are Notch-dependent zebrafish hairy/E(spl)-related
genes. Semiquantitative RT-PCR analysis showed that her6,
the zebrafish orthologue of human HES1, is expressed at
low levels in normal zebrafish thymus (Figure 4). Although
the expression of her6 is induced in NOTCH1-induced
leukemias, it is not expressed in mMyc-induced zebrafish
leukemias (Figure 4). her9, another Hes-related gene, is
expressed at low level in normal thymus of rag2-GFP fish. The
expression of her9 is not induced in mMyc-induced leukemias.
However, each of the zebrafish human NOTCH1-induced TALLs expressed higher levels of her9 (Figure 4). Previous studies
showed that her9 is mainly expressed in the zebrafish central
nervous system.29 her9 expression appears not to require Notch
signaling in the central nervous system. Instead, her9 is a
downstream effector of Nodal or Bmp signaling.30,31 Here, we
show that her9 is expressed at low levels in normal zebrafish
thymus and its expression is induced by constitutively active
Notch signaling. The expression of her6 and her9 by NOTCH1induced T-cell leukemias provide evidence for Notch pathway
activation. her1 is the zebrafish orthologue of human HES7. It is
not expressed by the normal thymus of control fish, nor is it
expressed in either NOTCH1- or mMyc-induced T-cell leukemias (Figure 4). Interestingly, although her4 (the zebrafish
orthologue of human HES5) is not expressed in NOTCH1mediated leukemias, it is highly expressed in mMyc-induced
leukemias (Figure 4).
c E
b
E
T
PF
injection site
AF
465
PF
PF
Figure 3 Transplantability of zebrafish rag2-ICN1-EGFP leukemias. GFP-positive leukemic cells transplanted into sublethally irradiated adult fish
were detected at (a) the site of injection into the peritoneal cavity at 7 days after injection; (b) homing of leukemic cells to the thymus of the
recipient fish at 21 days after injection and (c) massive dissemination of leukemic cells in the head region at 21 days after injection. T, thymus;
PF, pectoral fin; AF, anus fin; E, eye.
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rag2GFP
466
2
3
1.0
mMyc
ICN1
4
5
29
30
31
32
0.8
scl
lmo1
hox11
tlx3a
tlx3b
her1
her4
her6
her9
myc-a
myc-b
lck
rag2
-actin
Figure 4 Leukemic lymphoblasts express T-cell markers, T-ALL
oncogenes and Notch pathway components. Semiquantitative RTPCR analysis of FACS-sorted GFP-positive thymocytes from 90-day-old
rag2-GFP control transgenic fish (rag2GFP) or leukemic lymphoblasts
isolated from rag2-EGFP-mMyc or rag2-ICN1-EGFP diseased fish
(denoted by numbers).
c-Myc was recently shown to be the direct target of Notch1 in
Notch-dependent T-ALL cell lines.32,33 Inhibition of c-Myc
interferes with the pro-growth effects of activated Notch1.
Enforced expression of c-Myc rescues multiple Notch1-dependent T-ALL cell lines from Notch inhibition.32,33 myc-a and
myc-b are the zebrafish orthologues of mammalian c-Myc. RTPCR analysis showed that both myc-a and myc-b are expressed
in the normal thymus of the control fish (Figure 4). Similar
expression levels of myc-a and myc-b were also detected in both
NOTCH1- and mMyc-induced zebrafish leukemias (Figure 4),
indicating that myc-a and myc-b may not be the Notch targets in
NOTCH1 transformed zebrafish T cells.
Expression of other known T-ALL oncogenes in
NOTCH1-induced T-cell leukemia
The upregulation of the T-ALL oncogenic transcription factors
such as TAL1/SCL, LMO1, LMO2, HOX11 can result from
chromosomal translocation into the TCR locus.34–44 However, it
has recently been shown that aberrant overexpression of T-ALL
oncogenes can occur in the absence of chromosomal translocation.45 In order to characterize the expression of T-ALL
oncogenes, such as scl, lmo1, lmo2, hox11, tlx3a and tlx3b in
NOTCH1-induced T-cell leukemias, RT-PCR analysis was
performed using oncogene-specific primers with RNA from
FACS-sorted GFP-positive leukemic cells. In zebrafish mMycinduced leukemias, there is overexpression of tal1/scl and lmo2
(Figure 4). However, in human NOTCH1-induced T-cell
leukemias, there was undetectable expression of scl, lmo1,
hox11, tlx3a and tlx3b oncogenes, and only a small amount of
lmo2 expression (Figure 4), implicating a mechanism for human
NOTCH1-induced T-cell leukemias that is distinct from mMycinduced T-ALLs.
Leukemia
0.7
Survival Probability
lmo2
wild type
0.9
0.6
ICN1-EGFP
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Months
Figure 5 Kaplan–Meier plot of survival for wild type and rag2-ICN1EGFP F1 transgenic fish (n ¼ 53).
Germline transmission of ICN1-EGFP
The power of the zebrafish model lies in the ability to use
forward-genetic screens to identify modifier genes that influence
the development of NOTCH1-induced leukemias, an application that requires a stable transgenic zebrafish line. Approximately 300 mosaic rag2-ICN1-EGFP F0 fish were mated and the
F1 offspring were screened for germline transmission and
expression of the chimeric transgene. One mosaic transgenic
founder was isolated that demonstrated transmission of the
transgene to its F1 progeny. Twenty-one of 53 (40%) stable F1
transgenic fish developed leukemia starting at about 11 months
with growth of leukemic cells from the thymus to the areas
above the eyes and around the olfactory region (Figure 5).
Cooperation between NOTCH1 and bcl2 in the
induction of leukemia in transgenic zebrafish
Previous studies showed that dominant phenotype associated
with NOTCH1 inhibition in the human T-ALL cell lines is G0/
G1 cell cycle arrest without apoptosis.46 This implies that prosurvival signals may be mediated through non-Notch mechanisms such as overexpression of antiapoptotic proteins. In order
to study whether the failure of lymphoid cells to undergo cell
death contributes to NOTCH1-mediated oncogenic transformation, the rag2-ICN1-EGFP stable transgenic line was crossed
with a line overexpressing the zebrafish bcl2 gene under the
control of a rag2 promoter. Sixty to 80% of F1 progeny from this
cross had an enlarged thymus at about 40 dpf (Figure 6). Local
extra-thymic infiltration was detected in tissues of the head of all
the double transgenic fish at about 2 months (Figure 6). After 3
months, the double transgenic fish had infiltration of GFP þ cells
into the base of the pectoral fins and to distal regions of the fish
(Figure 6). These studies indicate cooperation between the
Notch pathway and the bcl2-mediated anti-apoptotic pathway.
TUNEL experiments showed that in the thymus of 2-month-old
rag2-GFP and rag2-ICN1-EGFP fish, there are similar amount of
T-cell apoptosis (Figure 7). However, less apoptosis was
detected in the thymus of rag2-ICN1-EGFP/rag2-EGFP-bcl2
double transgenic lines (Figure 7). Cells collected from double
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A 120
100
Percentage
80
ICN1
ICN1+bcl2 line1
ICN1+bcl2 line2
ICN1+bcl2 line3
60
40
20
0
0 month
B a
1 month
2 months
d
g
T
40 dpf
T
T
b
e
h
c
f
i
60 dpf
100 dpf
Figure 6 Zebrafish bcl2 facilitates human NOTCH1-mediated leukemia onset in transgenic zebrafish. (A) Sixty to 80% of NOTCH1/bcl2 double
transgenic fish had an enlarged thymus at about 40 dpf, whereas no NOTCH1 or bcl2 single transgenic fish have this phenotype. Local extrathymic infiltration was detected in tissues of the head of all the double transgenic fish at about 2 months. (B) Earlier leukemia onset in NOTCH1/
bcl2 double transgenic fish. (a–c) rag2-ICN1-EGFP stable transgenic fish; (d–f) rag2-EGFP-bcl2 stable transgenic fish; (g–i) rag2-ICN1-EGFP/rag2EGFP-bcl2 double transgenic fish. (a, d, g) 40 dpf; (b, e, h) 60 dpf; (c, f, i) 100 dpf. Thymus enlargement was found in the double transgenic
zebrafish with both ICN1 and bcl2 at 40 dpf (g), which was not found in transgenic fish line with only ICN1 (a) or bcl2 (d). At 60 dpf, local
infiltration of leukemic cells along the gill arch was found in double transgenic fish (h). Infiltration of leukemic cells to the head region and the base
of pectoral fin in double transgenic fish were found at 100 dpf (i).
transgenic fish were transplanted into irradiated wild-type adult
zebrafish to study the transplantability of this line. Two weeks
after transplantation, homing of GFP þ leukemic cells to the
thymus was found in the recipient fish. Some recipients also had
GFP þ cells in the olfactory bulb, head region as well as other
part of the fish (Supplementary Figure S2), indicating that the
leukemia in the double transgenic fish is highly malignant. RTPCR analysis showed that myc-a and myc-b were expressed at
similar levels in rag2-ICN1-EGFP/rag2-EGFP-bcl2 double transgenic fish to those in rag2-ICN1-EGFP or rag2-EGFP-mMyc fish
(Supplementary Figure S3). Finally, bcl2 expression blocks
irradiation sensitivity of NOTCH1-induced T-cell leukemias. In
response to 23 Gy g-irradiation, leukemias were ablated by 3
days after treatment in the stable rag2-ICN1-EGFP fish (Figure 8).
However, GFP þ -bcl2-expressing leukemic cells were retained
within the rag2-ICN1-EGFP/rag2-EGFP-bcl2 double transgenic
fish by 3 days after treatment, indicating that bcl2 expression in
NOTCH1-induced leukemias confer resistant to irradiationinduced cell death (Figure 8).
Discussion
The t(7;9)(q34;q34.3) chromosomal translocation juxtaposing
the human NOTCH1 gene with the T-cell receptor-b chain
promoter/enhancer has been found in a subset of human
T-ALLs.8 Human NOTCH1 gain-of-function mutations/deletions
have also been found in about 60% of T-ALL patients.13 These
make NOTCH1 the most commonly mutated oncogene in this
disease. Therefore, understanding the detailed molecular
Leukemia
NOTCH1-induced zebrafish T-ALLJ
Chen et al
468
a
c
b
Figure 7 Reduced apoptosis in the thymus of rag2-ICN1-EGFP/rag2-EGFP-bcl2 double transgenic fish. T-cell apoptosis was detected in the
thymus of 2-month-old rag2-GFP fish (a), and 2-month-old rag2-ICN1-EGFP fish (b). However, less apoptotic cells were detected in the thymus of
2-month-old rag2-ICN1-EGFP/rag2-EGFP-bcl2 double transgenic fish (c).
a
c
b
T
T
d
T
Figure 8 bcl2 expression blocks irradiation sensitivity of NOTCH1-induced T-cell leukemias. (a) A stable transgenic rag2-ICN1-EGFP leukemic
fish before g-irradiation. (b) The same rag2-ICN1-EGFP leukemic fish treated with 23 Gy g-irradiation (3 days after treatment). (c) A rag2-ICN1EGFP/rag2-EGFP-bcl2 double transgenic fish before g-irradiation. (d) The same double transgenic fish treated with 23 Gy g-irradiation (3 days after
treatment).
mechanism by which altered Notch signaling induces T-cell
leukemia is of importance and may reveal target genes for the
development of new therapies. In order to evaluate potential
genetic interactions between NOTCH1 and other T-ALL
oncogenes and to do the genetic modifier screens for enhancers
or suppressors of Notch pathways in T-ALLs, we developed a
zebrafish model of NOTCH1-induced T-cell leukemia.
In contrast to F0 mosaic fish, the stable transgenic rag2-ICN1EGFP fish examined developed leukemias with a longer latency.
This could be due to a lower level of transgene expression in
stable transgenic lines. In mosaic fish, the higher expression of
the transgene as shown by strong green fluorescence in the
thymus is likely from the integration of multiple copies of
the transgene into the fish genome, or the integration of the
transgene into some enhancer regions that increases the
transgene expression. In the stable fish lines, the transgene
expression appears to be quite low as shown by detection of
only faint green fluorescence in the thymus. Furthermore,
Leukemia
immunostaining with EGFP antibody or human NOTCH1
intracellular domain antibody showed that the ICN1-EGFP
fusion protein is expressed at a very low level in the thymus of
2-month-old rag2-ICN1-EGFP stable fish (data not shown). The
low transgene expression could result from the integration of
fewer copies of the transgene into the fish genome or the
integration of the transgene into some locus with an inhibitory
sequence nearby. Another possibility arises because of the
previous observation that the rag2 promoter is tightly regulated
during T-cell development and is only induced in cells
undergoing active TCR-a and TCR-b gene rearrangement.47,48
This would provide only two waves of NOTCH1 expression
during thymocyte development. In mosaic fish with multiple
copies of the ICN1-EGFP transgene or for a potent oncogene
such as mMyc, these two waves of expression may be strong
enough for earlier leukemia induction. However, in stable rag2ICN1-EGFP fish, these two waves of transgene expression might
not be sufficient. Weak gain of function Notch1 alleles induce
NOTCH1-induced zebrafish T-ALLJ
Chen et al
T-ALL in mouse models with a longer latency (JC Aster,
unpublished data), suggesting that high level of expression is
needed for robust induction of leukemias.
TUNEL experiments showed that in the thymus of rag2-GFP
and rag2-ICN1-EGFP fish, there is certain amount of T-cell
apoptosis. In order to study the oncogene-cooperativity in the
induction of T-ALL and whether failure of lymphoid cells to
undergo cell death can contribute to NOTCH1-mediated
oncogenic transformation, rag2-ICN1-EGFP fish were crossed
to another fish line overexpressing zebrafish bcl2. Previous
studies showed that thymocytes from rag2-EGFP-bcl2 transgenic
fish are resistant to radiation- and dexamethasone-induced
apoptosis.49 However, no rag2-EGFP-bcl2 transgenic fish have
developed lymphoma or leukemia. Our studies showed that
expression of bcl2 dramatically facilitated NOTCH1-mediated
leukemia onset. It is possible that in rag2-ICN1-EGFP stable line,
as the transgene expression is not strong enough to transform all
the T cells, many cells die over the time as the thymus evolve.
Overexpression of bcl2 may allow a subgroup of NOTCH1transformed T cells to survive longer enough to acquire a
necessary second mutation for earlier leukemia onset. Microarray analysis showed that in a subset of human T-ALL patients,
there is overexpression of the BCL2 gene.45 It will be interesting
to find out whether these patients also carry mutations and/or
deletions in their NOTCH1 gene. Certain patients with both
overexpression of BCL2 gene and mutations and/or deletions in
the NOTCH1 gene may have the leukemia with earlier onset.
bHLH transcription factors Hes1 and Hes5 are Notch target
genes that encode transcriptional repressors. Hes1 is required at
the earliest stage of T-cell development.50 Overexpression of
Hes1 or Hes5 is sufficient to block B cell generation by bone
marrow lymphoid precursors.51 Our studies showed that her6,
the zebrafish orthorogus of Hes1, and her9 are expressed at low
level in normal thymus of rag2-GFP fish. Their expression is
highly induced in NOTCH1-induced leukemias. Recent studies
showed that c-Myc is another direct downstream target of
Notch1 and is required for pro-growth effects of activated
Notch1.32,33 Retroviral expression of c-Myc rescues the growth
arrest associated with Notch1 inhibition. However, c-Myc fails
to rescue all Notch-dependent cell lines from Notch withdrawal,
indicating the existence of other Notch1 target genes.32 Our
studies showed that the expression levels of myc-a and myc-b
in NOTCH1-transformed zebrafish T cells were similar to those
in normal thymocytes of control fish, indicating myc-a and mycb may not be the Notch targets in this context. Therefore,
NOTCH1-induced zebrafish T-cell leukemia is likely mediated
by her6 and her9 or by other downstream target genes that
contribute to T-ALL cell growth and survival.
Extensive phenotyping studies of human T-ALL have identified
several recurring patterns. Among the genes implicated in the
malignant transformation of T-cell precursors are those encoding bHLH transcription factors, including TAL1/SCL, TAL2, LYL1
and BHLHB1, oncoproteins such as MYC, other nuclear
regulatory proteins such as LMO1 and LMO2, the orphan
homeobox proteins such as HOX11 and HOX11L2, and
NOTCH1.52–54 In a comprehensive microarray and RT-PCR
analysis of primary T-ALL tumor samples, Ferrando, et al.45,55
observed at least five molecular signatures of human T-ALL –
TAL1/SCL plus LMO2 or LMO1; HOX11; HOX11L2; LYL1 plus
LMO2 and MLL-ENL. Recently, Weng et al.13 identified
activating mutations in NOTCH1 in about 60% of human
T-ALLs. Mutations were seen in tumors associated with
misexpression of HOX11, HOX11L2, TAL1, LYL1 and MLLENL.13 In zebrafish mMyc-induced leukemias, there is overexpression of tal1/scl and lmo2, resembling a subgroup of
human T-ALLs at the molecular level.24 Our studies showed that
the expression of T-ALL oncogenes such as scl, lmo1, lmo2,
hox11, tlx3a and tlx3b are not induced in NOTCH1-transformed
T cells. Interestingly, recent studies showed that in tumors from
either Tal1/ þ , Tal1/ þ HEB þ / and Tal1/ þ Ink4a/Arf þ /
transgenic mice56 or from SCL/LMO1 transgenic mice,57 there
are mutations in Notch1 heterodimerization domain or PEST
domain. No mutations or deletions were found in Notch2,
Notch3 and Notch4 genes in these models.56 These studies
suggested that NOTCH1 mutations are likely to complement the
overexpression of these T-ALL oncogenes.
Like other T-ALL oncogenes, Notch receptors appear to need
additional mutations to cause transformation. This is consistent
with the previous finding that constitutively active Notch1
transforms rat kidney cells in conjunction with the viral
oncogene E1A.58 Transformation can also be induced in various
cell types in vitro when constitutively active Notch1 is
expressed with human papillomavirus E6 and E7,59,60 Ras,61
Myc11 or simian virus 40 large T.62 Genetic modifier screens
using transgenic zebrafish prone to NOTCH1-induced T-cell
leukemia may ultimately reveal suppressors or enhancers of
Notch pathway in T-ALLs. These modifier genes could be the
targets for the development of new therapies.
469
Acknowledgements
We thank J Kutok, J Williams, D Skinner and V Nguyen for
histology services; G Kourkoulis, J Kilgore, A Hagen and B Baker
for excellent fish care; D Zahrieh for Kaplan–Meier survival
analysis; A Letai and D Langenau for reagents and discussions.
Supported by grants from the National Institute of Health CA36167 (JDG), CA-68484 (ATL). JC is a fellow of Leukemia and
Lymphoma Society. CJ is supported by a National Institute of
Health Program Training Grant in Molecular Hematology.
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Leukemia