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Leukemia (2007) 21, 462–471 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu 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. Leukemia NOTCH1-induced zebrafish T-ALLJ Chen et al 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. Leukemia NOTCH1-induced zebrafish T-ALLJ Chen et al 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. Leukemia NOTCH1-induced zebrafish T-ALLJ Chen et al 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 NOTCH1-induced zebrafish T-ALLJ Chen et al 467 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). 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