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NEOPLASIA
From the Department of Pathology, Stanford University
Medical Center, Stanford, CA.
PBX1 is a proto-oncogene that plays important roles in
pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a
subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins
display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be
impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice.
Retroviral insertions in the Notch1 gene were observed in
88% of tumors arising with a shortened latency. Among these,
approximately half created a NotchIC allele,
encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involving Notch1 occurred in a more 3' exon, resulting in truncating
mutations that deleted the carboxy-terminal region of
Notch1 containing negative regulatory sequences
(Notch1 A number of genes whose products regulate animal
development are also targets for acquired mutations in human
cancers.1 The characterization of these so-called
developmental oncogenes has contributed to our current understanding
that neoplastic transformation can result from the misregulation of
molecular mechanisms governing cell fate determination and cellular
identity. However, the pathogenetic connections between disrupted
development and cancer are poorly understood at a molecular level.
PBX1 is a proto-oncogene that plays an important role in
pattern formation during development. It codes for a
homeodomain protein that is the mammalian homolog of
Drosophila extradenticle (exd).2 Pbx and exd
proteins serve as dimerization partners for a wide variety of Hox
proteins3-7 and make critical contributions to the
segment-specific execution of Hox programs in arthropods and
vertebrates.8-10 In a subset of pediatric acute
lymphoblastic leukemias, chromosomal translocations fuse the
PBX1 and E2A genes, resulting in the production
of chimeric E2a-Pbx1 proteins.11,12 E2a proteins, members
of the bHLH family of master developmental regulators, contribute to
tissue-specific regulation of the IG genes and are essential for
B-lymphoid lineage development.13 Fusion of the strong
transcriptional activation domains of E2a onto the DNA-binding domain
of Pbx1 results in a profound disruption of Pbx1 transcriptional
regulatory properties as evaluated using in vitro
assays.14-16 Furthermore, E2a-Pbx1 displays potent
oncogenic properties, in a variety of cell types, that appear to
require dimerization with Hox DNA-binding partners.17,18
Taken together, the available data strongly suggest that
E2a-Pbx1-mediated oncogenesis results from perturbations of
Hox-dependent transcriptional pathways that normally orchestrate the
differentiation programs of lymphoid progenitors. However, the
subordinate genes and transcriptional pathways that are subject to
Hox-Pbx regulation in normal and neoplastic lymphocytes remain undefined.
Notch1, like Pbx1, plays important roles in cell fate
determination and lymphoid oncogenesis. It codes for a highly conserved transmembrane protein whose extracellular portion contains a
ligand-binding domain, composed of 36 EGF repeats, as well as 3 lin12/Notch repeats of uncertain function.19-21
The ligands for Notch1 are also cell surface proteins whose
interactions induce the release and nuclear migration of the
intracellular portion (NotchIC) of
Notch1.22-24 Forced expression of
NotchIC, which lacks the extracellular
ligand-binding domain, circumvents the need for ligand to activate the
Notch1 pathway thereby creating a constitutive
Notch1 signal. This has been shown to suppress myogenesis
and neurogenesis and results in profound alterations on the fate
decisions of maturing thymocytes.25-28
NotchIC was originally isolated as a lymphoid
oncogene (TAN1) in a subset of T-cell acute lymphoblastic
leukemias that harbor chromosomal translocations that truncate and
ectopically express the signaling form of Notch1.29
Neoplastic transformation of various cell types by NotchIC
has subsequently been reported.30-32
In the studies reported here, we used a genetic screen to identify
genes that accelerate development of T-cell tumors in
E2A-PBX1 transgenic mice. Mutations of the Notch1
gene were observed in 88% of tumors arising with a shortened latency.
Although a significant number induced the intracellular signaling form
of Notch1, more than 50% of the cases harbored activating mutations
that preserved the ligand-binding domain of Notch1. These observations
indicate that Notch1 collaborates with E2a-Pbx1 in lymphoid oncogenesis and reveal a novel mechanism for oncogenic activation of Notch1.
Transgenic mice and retroviral infections
Southern and Western blotting
Fresh or snap-frozen tissues were lysed in SDS gel-loading buffer lacking bromophenol blue.36 Proteins (50 µg) were fractionated through either 6% or 10% SDS-polyacrylamide denaturing gels, transferred to nitrocellulose by electroblotting, and subjected to Western blot analysis.36 Primary antibodies were reactive with the FLAG epitope and secondary antibodies consisted of peroxidase-conjugated polyclonal goat antirabbit IgG. All antibodies were used at a dilution of 1:5000, and immune complexes were visualized by chemiluminescence (Amersham) performed according to the manufacturer's recommendations. Characterization of proviral integration sites Proviral insertion sites were characterized in tumors from MMuLV-injected E2A-PBX1 mice at 2 months of age before outward signs of disease to favor isolation of proviral integrations that occurred early in the tumor process. Seminested PCR was used in combination with Southern blot enrichment to isolate sites of MMuLV proviral insertions. DNA was purified from tumors, digested with the restriction enzymes EcoRI and NheI, and then subjected to electrophoresis through a 0.8% agarose gel. Portions of the agarose gel containing DNA fragments in the size range corresponding to the integration of interest were excised and DNA was purified from the agarose matrix using glass bead absorption (Gene Clean, Bio 101). The purified DNA fragments were then ligated to pUC19 DNA that had been predigested with EcoRI and XbaI. Seminested PCR was performed directly on 10% of the ligation reaction mix using Expand High Fidelity polymerases (Boehringer Mannheim, Mannheim, Germany) and primers designed by Natarajan et al37 in the reverse orientation. The identity of mouse genomic DNA flanking the proviral LTR was determined by nucleotide sequencing of the resulting PCR products using an automated sequencer (model 373; Applied Biosystems, Foster City, CA) and the manufacturer's recommended protocols. The resulting nucleotide sequences of flanking Notch1 exonic DNA were used to design oligonucleotide primers for use in PCR to screen for proviral integrations into the Notch1 gene in additional tumors. PCR was performed on tumor DNA using primers homologous to the MMuLV LTR (5'-CCCGTGTATCCAATAAACCCTCTTG-3') and Notch1 (5'-GCCGTAGTGGGTTGTACTGGC-3'). Amplified DNA fragments were gel purified and used as templates for automated sequence analyses.Notch1-MLV fusion RNA transcripts were detected and analyzed by RT-PCR. Polyadenylated RNA was isolated from snap frozen tumor tissues using Fastrack 2.0 (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Complementary DNA was synthesized using oligo (dT)12-18 and Superscript II reverse transcriptase (Gibco BRL) according to the manufacturer's protocol. PCR was performed using primers homologous to the U3 portion of the MMuLV LTR (5'-CCATCTGTTCCTGACCTTGATCTG-3' or 5'-CAAGAGGGTTTATTGGATACACGGG-3') and Notch1 (5'-CACCTGCCTGGTATGCCTGACA-3' or 5'-CCTGGGCATCAGCCACTTGAATG-3') with Expand High Fidelity polymerases. The resulting DNA fragments were subjected to automated nucleotide sequencing. Tumor analyses Freshly dissected tissues were fixed overnight in buffered formalin and then embedded in paraffin using standard techniques. Paraffin-embedded tissues were sectioned at 5 to 6 µm thickness, mounted on slides, deparaffinized, and stained with hematoxylin and eosin. For FACS analyses, cells freshly isolated from the thymus were stained, and the fluorescence was analyzed using a dual-laser FACS Vantage (Becton Dickinson Immunocytometry Systems, Mountain View, CA) with 4 decade logarithmic amplifier. Dead cells were detected by propidium iodide staining (1 µg/mL) and gated out electronically. Residual red blood cells were also gated out electronically. All antibodies were purchased from Pharmingen Research Products (San Diego, CA). Specificities of antibodies were as follows: FITC-conjugated 145-2C11 (anti-CD3 ); phycoerythrin (PE)-conjugated RM4-5 (anti-CD4);
and biotinylated 53-6.7 (anti-CD8 ).
Neonatal retroviral infection accelerates development of thymic lymphomas in E2A-PBX1 transgenic mice Transgenic mice expressing the E2A-PBX1 chimeric oncogene in the T-lymphoid compartment are highly susceptible to development of malignant lymphomas that arise with an average latency of more than 150 days.33 The long latency and clonal composition of the lymphomas indicated that forced expression of E2A-PBX1 alone was not sufficient for induction of the malignant phenotype, which likely required one or more secondary events in addition to the E2A-PBX1 transgene. We used retroviral mutagenesis38,39 in a genetic screen to identify genes that cooperate with E2A-PBX1 to accelerate the development of tumors and shorten the survival of E2A-PBX1 mice. Newborn mice resulting from backcrosses of E2A-PBX1 transgenic mice were injected with MMuLV. Malignant lymphomas developed with a mean latency of 75 days in transgenic mice that were infected neonatally with virus (Figure 1A), which was significantly shorter than the time required for lymphoma development in transgenic littermates that had not been injected with virus (mean = 180 days). In addition, it was significantly different from that displayed by virus-injected, nontransgenic littermates that exhibited a reduced survival compared with nontransgenic, noninjected mice, consistent with previous observations.40
Tumors that arose in MMuLV-infected E2A-PBX1 mice primarily involved the thymus and were essentially indistinguishable from those in E2A-PBX1 mice (data not shown). They comprised clonal populations of T-lineage progenitors expressing surface antigens CD3, 4, and 8 characteristic of mid-thymocytes. Histologic features were those of diffuse high-grade lymphomas that were usually metastatic to the spleen, lymph nodes, liver, and lungs. Proviral integration patterns were evaluated by Southern blot analysis using a probe specific for the U3 region of the viral LTR. In addition to endogenous proviral genomes detected with the U3 probe, tumors characteristically displayed 1 to 5 unique bands corresponding to acquired proviral integrations (Figure 1B). Therefore, neonatal MMuLV injection of E2A-PBX1 mice resulted in a substantially accelerated development of thymic lymphomas that harbored clonal integrations of proviral DNA. Notch1 is frequently targeted by MMuLV proviral insertions in E2A-PBX1 mice To characterize the sites of MMuLV insertion, genomic DNA fragments that hybridized with the U3 probe were cloned and sequenced. Tumors with the least number of proviral insertions were used in an effort to isolate those that may be specifically cooperating with the transgene. Hybridizing DNA fragments of interest were purified by agarose gel electrophoresis, ligated into pUC19, and used as templates for seminested PCR. Resulting DNA fragments were subjected to nucleotide sequence analyses. Using this approach, one of several proviral insertions analyzed was found to involve the Notch1 gene.To establish the prevalence of proviral insertions in
Notch1, DNA from accelerated tumors was examined by Southern
blot analysis using probes specific for Notch1. A frequent
site (cluster region I) for proviral insertions into Notch1
was identified by previous studies of malignant lymphomas induced by
MMuLV neonatal infections in MMTVD/myc
transgenic mice.41 Using a probe for this central region of the Notch1 gene that spans exons encoding the LNR and a
portion of the EGFR domains, approximately 50% of accelerated tumors
from E2A-PBX1 mice displayed Notch1 gene
rearrangements (Figure 2A). Thus, a
substantial proportion of the accelerated tumors contained proviral-induced mutations of the Notch1 gene, thereby
implicating it as a frequent collaborating gene in
E2A-PBX1-initiated lymphomagenesis.
However, a Notch1 DNA rearrangement was not detected in cluster region I for the tumor from which we originally cloned a Notch1 proviral insertion. Therefore, Southern blot analyses were also conducted using an additional Notch1 probe flanking the site of this proviral insertion. Notch1 gene rearrangements were detected in more than 50% of cases with this second probe. Most of the cases detected with this probe lacked rearrangements in cluster region I (Figure 2A). This probe therefore defined a second cluster region for proviral insertions that appeared to be distant from cluster region I by mapping and sequencing analyses (see below). Rare tumors contained DNA rearrangements in both regions of Notch1 (Figure 2A, lane 4). Different hybridization intensities of rearranged bands for cluster region I versus II in cases with double insertions suggested that they were present in different subpopulations within the same tumor mass. Overall, 88% of the accelerated tumors that arose in E2A-PBX1 mice contained DNA rearrangements of the Notch1 gene, indicating that it was targeted at high frequency by proviral insertions, which clustered in 2 different regions of the gene. A second cluster site for proviral insertions in E2A-PBX1 mice targets Notch1 3' exonic sequences To further characterize the proviral insertions in cluster region II of the Notch1 gene, PCR was performed on tumor DNAs using primers homologous to the proviral LTR and Notch1 exonic sequences from cluster region II. Amplification products were observed in several tumors from E2A-PBX1 transgenic mice injected with MMuLV but not in tumors from noninjected mice. Nucleotide sequence analyses of the amplification products showed similar configurations of U5 LTR sequences fused to Notch1 (Figure 2B). The precise point of fusion with Notch1 differed in each of the analyzed tumors but occurred in a relatively short segment of the gene-encoding nucleotides 6832-7225 of the Notch1 cDNA and contained in a single exon. In 2 of the tumors, nucleotide insertions were observed between LTR and Notch1 sequences. In one case, the inserted nucleotides were of unknown origin (case 5), in the other they originated from further 3' in Notch1 (case 6). The fusion of LTR and Notch1 coding sequences indicated that proviral integrations had occurred into Notch1 exonic sequences (schematically shown in Figure 3A). The exon that was targeted by these events constituted a 3' portion of the Notch1 gene coding for amino acids between the ankyrin repeats and the C-terminal PEST domain.
Lymphomas arising with shortened latencies express mutated Notch1 gene products Proviral insertions into exonic regions of Notch1 were predicted to disrupt the production of appropriately processed Notch1 transcripts. Because proviral LTRs contain transcriptional initiation and termination signals, they can induce production of prematurely truncated 5' and/or ectopically initiated 3' transcripts fused at one end to retroviral LTR sequences (shown schematically in Figure 3A). Potential synthesis of Notch1-LTR fusion transcripts was evaluated by RT-PCR analysis of RNA using primers homologous to Notch1 and the U3 segment of the LTR. Under these conditions, specific amplification products were detected in accelerated tumors harboring 3' Notch1 proviral insertions (Figure 4A). Nucleotide sequence analyses of the PCR products showed that 5' Notch1 sequences were fused directly to U3 sequences of the MMuLV LTR (Figure 4B). Translation of the sequences demonstrated that the Notch1 open-reading frame prematurely terminated downstream of the fusion site because of in-frame stop codons in the U3 portion of the chimeric transcripts. As a result, the transcripts were predicted to encode mutated Notch1 proteins lacking C-terminal amino acids that encode the PEST domain (hereafter referred to as Notch1 C and schematically
illustrated in Figure 3B) and a portion of the transactivation
domain.42
Mice transgenic for both Notch1 C, we constructed transgenic mice expressing this
protein in the thymic compartment. A Notch1 cDNA lacking 3'
sequences encoding the 238 C-terminal amino acids (all of PEST domain
and most of the transactivation domain) was expressed under control of
the LCK proximal promoter that provides high-level
expression in the thymic compartment (Figure
5A). Two independent lines of
Notch1C mice were evaluated and found to have no
detectable alterations in their thymocyte subset ratios compared with
normal mice by FACS analysis at 12 weeks of age (Figure 5E). This
contrasts with the substantial elevations in CD8+ T cells
observed in Notch1ICC transgenic
mice28 and with the disorganized thymocyte
differentiation observed in E2A-PBX1 mice33
(Figure 5E).
Notch1 All Notch1
These studies demonstrate that E2A-PBX1 and
Notch1 collaborate in lymphoid oncogenesis and suggest a
possible mechanistic link in their mode of collaboration. When forcibly
expressed in the lymphoid compartment of transgenic mice,
E2A-PBX1 results in the development of T-lineage
lymphoblastic lymphomas with a mean latency of 5 to 6 months.33 However, the latency for tumor induction by
E2A-PBX1 is substantially shortened by neonatal MMuLV infection (Feldman et al43; this study). As demonstrated
here, this acceleration in tumor development is associated with a high frequency (88%) of proviral insertions in the Notch1 gene.
Two types of insertions were observed and resulted in either ectopic expression of NotchIC or deletion of the carboxy-terminal
portion of Notch1 containing its PEST domain (Notch1 Half of the Notch1 insertions we observed created a mutant NotchIC gene, which is known to contribute to thymocyte transformation in several different settings. It was originally discovered as an oncogene (TAN1) at the sites of chromosomal translocations in a subset of human lymphoblastic lymphomas.29 These translocations bisect the Notch1 gene, resulting in constitutive expression of NotchIC, which lacks the extracellular ligand-binding domain. A pathogenetic role for NotchIC was conclusively demonstrated experimentally by retroviral gene transfer into primary mouse hematopoietic cells, which resulted in the preferential induction of acute T-lineage lymphomas.32 In studies similar to ours, NotchIC was found to be ectopically expressed after proviral insertions in a subset of thymic lymphomas arising in MMTVD/myc transgenic mice infected neonatally with MMuLV.41 Therefore, in both humans and mice, inappropriate signaling through the Notch1 pathway renders developing thymocytes highly susceptible to oncogenic conversion. Frequent ectopic NotchIC expression in accelerated tumors from E2A-PBX1 mice establishes that events downstream in the Notch1 signaling pathway synergize with E2A-PBX1 in thymocyte oncogenesis. Although Notch1 signaling pathways have not been fully characterized, some of their downstream effects appear to abrogate bHLH protein expression and/or function through at least 2 distinct pathways. One pathway involves CBF-1/Su(H)44; whereas a second appears to be CBF-1 independent45,46 and impinges on E47 through Ras and Deltex.47 Perturbations of bHLH protein function through ectopic expression48-52 and/or loss of function53-57 may be a final common pathway in thymocyte neoplasia. Notch1 expression is modulated during thymocyte development58 and forced expression of NotchIC perturbs the fate decisions of differentiating thymocytes.28,59 Notch1 is also capable of protecting T cells from apoptosis by a pathway that involves interactions with Nur77.60 Thus, it is possible that Notch1 collaborates in thymic lymphomagenesis by suppressing the apoptosis associated with forced expression of E2A-PBX133,61 in addition to perturbing bHLH-mediated developmental programs. Many of the Notch1 retroviral insertions in accelerated
tumors of E2A-PBX1 mice, however, were further 3' in the
gene and resulted in carboxy-terminal Notch1 truncations.
Carboxy-terminal deletions mediated by retroviral insertions constitute
a potent mechanism for activation of cellular oncogenes as reported for c-myb62 and Tpl-2.63 The
C-termini of these proteins negatively regulate their respective
functions in transcription and signal transduction.63,64
In regards to Notch1, the deleted C-terminal region contains a PEST
domain involved in protein turnover by targeting of proteins to the
ubiquitin-proteosome complex for subsequent degradation.65
Loss of the PEST domain would be expected to render
Notch1 Unlike the more conventional NotchIC, which is a ligand
independent oncogenic mutant of the Notch receptor,
Notch1
We thank Gerry Weinmaster for Notch cDNAs and helpful discussions. We thank David Feldman, Cathy Schnabel, and Jorge DiMartino for comments on the manuscript. We acknowledge Yelena Marchuk and Yanru Chen for microinjections, Kathie Jones for histotechnology support, and Phil Verzola for photography.
Submitted January 21, 2000; accepted April 28, 2000.
Supported by grants CA34233, AI-07290, and CA42971 from the National Institutes of Health. B.J.F. was supported by funds from the Lucille P. Markey Charitable Trust.
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: Michael L. Cleary, Department of Pathology, Stanford University Medical Center, Stanford, CA 94305; e-mail: michael.cleary{at}stanford.edu.
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Top
Abstract
Introduction
2-specific
probe on EcoRI-digested DNA.
C
Notch1 transgenes were expressed at comparable levels in the
thymi of transgenic mice as determined by quantitative RT-PCR (Figure
B triggers Notch pathway
signaling by inducing expression of the Notch ligand Jagged1.