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Proviral Insertion Indicates a Dominant Oncogenic Role for Runx1/AML-1 in T-Cell Lymphoma¹

Molecular Oncology Laboratory, Institute of Comparative Medicine, University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom

	ABSTRACT

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The RUNX1/AML1 gene is a frequent target for chromosomal translocations in human leukemia. The biological properties of the resulting fusion products and the finding that haploinsufficiency increases the risk of developing leukemia (W-J. Song et al., Nat. Genet., 23: 166–175, 1999; M. Osata et al., Blood, 93: 1817–1824, 1999) have led to the widely held view that RUNX1 loss-of-function is a key event. However, we now report that the gene is a target for insertional mutagenesis in T-cell lymphomas of mice carrying a MYC oncogene, where promoter insertion results in overexpression without affecting the integrity of the coding sequence. Moreover, Runx1 haploinsufficiency does not accelerate lymphoma development in MYC/Runx2 transgenic or murine leukemia virus-infected mice. These findings reveal that the Runx1 gene can also act as a dominant oncogene and suggest that the involvement of the Runx gene family in human leukemia may be more widespread and complex than previously realized.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
RUNX1 (AML1, CBFA2) encodes a DNA-binding subunit of the heterodimeric core-binding factor and is expressed in a variety of myeloid and lymphoid lineages. Although Runx1 is essential for definitive hematopoiesis (1, 2, 3, 4) , the presence of CBF-binding sites in many hematopoietic-specific target genes indicates an important role at subsequent stages of development. Indeed, the RUNX1 gene is a frequent mutational target in human leukemias, where a wide spectrum of chromosomal translocations result in truncation and fusion of RUNX1 to heterologous proteins (5) . Expression of these abnormal fusion products is believed to inhibit normal RUNX1 function, perturb lineage differentiation, and predispose to leukemia. We have shown recently that Runx2, a close relative of Runx1, can function as a dominant oncogene in T-cell lymphoma (6 , 7) . The oncogenic potential of Runx2 was discovered first in a screen for Myc-collaborating oncogenes by retroviral gene tagging in CD2-MYC mice, where over 30% of tumors displayed insertions at the distal (P1) promoter of Runx2. Runx3 has recently been shown to act as an alternative target for insertional activation in the same system, although insertions at this gene were rare (8) . Proviral insertions have been reported at the Runx1 gene in a number of MLV³ -induced myeloid leukemias from a high-throughput screen of MLV insertion sites, although the consequences for gene expression were not examined (9) . These observations led us to consider the alternative hypotheses that (a) Runx1 can act as a dominant oncogene or a tumor suppressor according to context and lineage or (b) Runx2 and Runx3 act as antagonists of the Runx1 tumor suppressor. Our results support the first hypothesis.

	Materials and Methods

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Transgenic Mice and Cell Lines.
The CD2-MYC and CD2-Runx2 transgenic mice were generated and identified by Southern blot analysis on tail biopsy DNA, as described previously (7) . Runx1^(+/-) mice have been previously described (4) . Neonatal Runx1^(+/-) mice and littermate controls were infected with MoMLV within 24 h of birth. Animals heterozygote for both CD2-MYC and CD2-Runx2 were bred onto a Runx1^(+/-) background that also generated CD2-MYC/Runx2 littermate controls wild type for Runx1. Animals showing signs of lymphoma development were sacrificed and necropsied. The lymphoma cell lines T1i, T6i, and T47i were established from thymic lymphomas induced after neonatal MLV infection of CD2-MYC mice. The murine T-cell lines EL4 and BW5147, and the P19 murine embryonal carcinoma cell line, were obtained from the European Collection of Animal Cell Cultures (Salisbury, England).

cDNA Cloning.
cDNA was prepared from 5 µg of poly(A)⁺ RNA from the T6i lymphoma cell line using the ZAP-cDNA synthesis kit (Stratagene). The cDNAs were size fractionated, and the largest fraction was cloned into Uni-ZAP XR vector arms. An unamplified phage library was screened using a Runx1 exon 6-specific probe, and 17 positive recombinants were plaque purified and excised as pBluescript phagemids.

Sequence Analysis.
DNA sequence was determined using a Thermosequenase primer cycle sequencing kit (Amersham Pharmacia Biotech) with the aid of infra-red dye-labeled primers (MWG Biotech, Ebersberg, Germany) and a LiCor 4000 automated sequencer.

PCR.
Amplification was carried out on 1-µg aliquots of genomic DNA with 100 pmol of primer pairs MLV-U5-LTR (5'-GCAGTTGCATCCGACTTGTGG) and Runx1 exon 1 (5'-CTCATGAAGCACTGTGGATATG) or MLV-U3-LTR (5'-CCACCTGTAGGTTTGGCAAGC) and Runx1 exon1. Amplification was performed in 1.1x Reddy Mix (ABgene) at 95°C for 5 min, 95°C for 1 min, 60°C for 1 min (U5-LTR/exon 1), 62°C for 1 min (U3-LTR/exon 1), and 72°C for 1 min for 35 cycles. Amplification products were cloned using the TA cloning kit (Invitrogen) and sequenced as described above.

RT-PCR.
Five micrograms of total RNA were reverse transcribed using a First Strand cDNA synthesis kit (Amersham Pharmacia Biotech) and NotI-dT primer. Amplification was carried out on aliquots of one-fifth of the sample with 30 pmol each of primer pair MLV-U5-LTR and Runx1 exon 2 (5'-AAGCGGCGGCTCGTGCTGGC). Amplification was performed in 1.1x Reddy Mix (ABgene) at 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s for 30 cycles. Five-microliter aliquots of each reaction were separated on a 4% polyacrylamide gel and visualized by ethidium bromide staining and UV photography.

Southern and Northern Analysis.
Preparation of high molecular weight DNA and total RNA from primary tumors and tumor-derived cell lines and subsequent DNA and RNA hybridization analysis were as described previously (7) .

Western Analysis.
Whole cell extracts were prepared as described by Chan et al. (10) . Ten micrograms of protein extract were examined by SDS-PAGE, transferred to Hybond-ECL nitrocellulose, and incubated with the appropriate antibody. The antibodies used were -AML1 (rabbit polyclonal antibody; Geneka 1610022), -Runx2 (mouse monoclonal antibody; a gift from Prof. Yoshiaki Ito, Institute for Virus Research, Kyoto University, Kyoto, Japan), and -Actin (goat polyclonal antibody; Santa Cruz sc-1616).

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Overexpression of Runx1 Is a Key Event in T-Cell Lymphomagenesis.
To investigate the possibility that Runx1 can act as a dominant oncogene, we looked for evidence that this gene functions as a target for insertional mutagenesis in MYC-induced murine lymphomas, in a manner similar to that described for Runx2 and Runx3 (6 , 8) . We screened a panel of T-cell lymphomas (n = 124) from MoMLV-infected CD2-MYC mice for rearrangements by Southern blot hybridization analysis using a probe specific for 5' UTRs of Runx1 exon 1. Somatic rearrangements were detected in four of the primary tumors (T6i, 8si, 9Vb16i, 9Vb31i) with the restriction enzymes EcoRI and KpnI (Fig. 1a) . Although these rearrangements were not compatible with the insertion of full-length MLV genomes, the possible presence of truncated proviral elements was explored further by PCR amplification of genomic DNA using a 5' primer based on a MLV U5-LTR sequence and a 3' Runx1 exon 1-specific primer. This analysis demonstrated that the rearrangements in the T6i cell line and the 8si primary tumor were because of exogenous MLV insertion into the 5' UTRs of Runx1 (Fig. 1a) . This interpretation was supported by RT-PCR analysis of T6i and 8si tumor RNA using a 5' U5-LTR-specific primer and a 3' Runx1 exon 2-specific primer that confirmed the presence of hybrid MLV-Runx1 transcripts in both primary tumors (Fig. 1a) . The sequence of the murine Runx1 P1 region (supplied by the United Kingdom Mouse Genome Sequencing Project) is aligned with the human RUNX1 genomic sequence (accession no. AJ229043 and AF15262) to show the extent of homology and the conservation of consensus splice sites within exon 1 (Fig. 1b) . Use of these sites removes one or more untranslated exons from human RUNX1 mRNA (exons 1b and 1c), and an analogous splicing event is evident in the murine T6i RNA. Because the insertion site for 8si is downstream of these splice sites, the corresponding RNA is not spliced within the UTR.

View larger version (73K): [in this window] [in a new window]   Fig. 1. a, virus-induced rearrangements of the Runx1 gene in CD2-MYC lymphomas. Top, tumor (T6i, 8si, 9Vb16i, 9Vb31i) and control (C) DNAs were digested with EcoRI and KpnI, and filters were screened with a Runx1 exon 1 UTR probe. GL, unaffected allele in germline configuration. Middle, diagrammatical representation of integration sites and hybrid MLV-Runx1 transcripts in T-cell lymphomas T6i and 8si identified by DNA PCR and RT-PCR, respectively. PCR primers used were: a, MLV U3-LTR; b, Runx1 exon 1; c, MLV U5-LTR; d, Runx1 exon 2. The numbers are based on the murine Runx1 genomic sequence described in b. Bottom, sequence of MLV-Runx1 virus-host DNA junctions in T-cell lymphomas T6i and 8si. IR, MLV LTR inverted repeat sequence. b, comparison of human (H) and murine (M) genomic sequences at the Runx1 P1 promoter, including 5' untranslated and exon 1 sequences. Boundaries of untranslated exons (1a–1d) are indicated by arrowheads (>) above the nucleotide sequence, at the beginning of each exon. A conserved CCAAT box and putative transcription factor-binding sites (CRE, Myb) are indicated. Sites of proviral integration in T6i and 8si are indicated by vertical arrows. Underlining of the murine sequence indicates nucleotides present in T6i-derived cDNAs. The amino acid sequence of exon 1 is indicated above the human sequence. Numbering of the murine sequence starts from the putative transcriptional initiation site, which is predicted on the basis of an extensive match with the human gene. c, reciprocal expression of Runx1 and Runx2 in lymphoma cell lines T6i and T47i carrying proviral insertions at Runx1 and Runx2, respectively. Left, Northern blot analysis of 20 µg of total RNA from T6i and T47i cells and control EL4 and BW5147 cells was performed using Runx1- and Runx2-specific probes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to control for loading. The blot was hybridized sequentially with the Runx1, Runx2, and GAPDH probes, with stripping of probe between hybridizations. Right, Western blot analysis of protein extracts from T1i, T6i, and T47i cells and control EL4 and BW5147 cells using Runx1- and Runx2-specific antibodies and anti-ß actin to control for protein loading. d, Runx1 isoforms encoded by cDNAs isolated from T6i lymphoma cells. The boxed region indicates the domain encoded by exon 1, whereas the runt domain is shown in bold. An amino acid polymorphism between published sequences (A52) is also shown in bold. Sequences absent from alternatively spliced isoforms are indicated by italics (exon 4.1) or underlined (exon 5).

The insertions at T6i and 8si may be expected to have relatively modest effects on the size of Runx1 P1 transcripts, if the 5' UTR sequences that are removed as a result of insertion (64 bp and 394 bp, respectively) are replaced by the MLV R-U5 leader sequence of 146 bp. Northern blot analysis of the T6i cell line is consistent with this prediction, again favoring activation of the cryptic 3' LTR promoter as the likely mode of retroviral activation. The high expression of Runx1 in T6i contrasts with the lack of expression observed in the lymphoma cell line T47i, which carries an insertion at til-1/Runx2 (Fig. 1c) . This pattern is typical of cell lines bearing insertions at til-1/Runx2, which express high levels of Runx2 RNA and have low or undetectable levels of Runx1 expression. The T6i cell line expresses the reciprocal pattern with high levels of Runx1 but no detectable Runx2 (Fig. 1c) . Unfortunately, expression could not be demonstrated in the other tumors with Runx1 rearrangements because of the lack of primary material.

These observations are mirrored in the levels of protein expression detected in the respective cell lines (Fig. 1c) . Western blot analysis of the T6i lymphoma cell line using Runx1- and Runx2-specific antibodies showed that the T6i cell line expresses a predominant band of close to 50 kDa, as expected for the P1 product of Runx1 (p50^Runx1). In contrast, the lymphoma cell line T47i, which carries a proviral insertion at Runx2 (6) , displays the expected size protein for the major isoform encoded by Runx2-P1 transcripts (p57^Runx2). The BW5147 and EL4 cell lines have previously been shown to express Runx1 and Runx2 (12 , 13) , although the latter seems to be expressed only from the P2 promoter, which encodes a slightly smaller protein (p56^Runx2; Ref. 6 ).⁴ The lack of Runx protein observed in P19 cells and the T1i lymphoma cell line reflects the lack of functional Runx product in the former (14) ,⁵ whereas the T1i lymphoma cell line carries a proviral insertion at Runx3 (8) and exclusively expresses Runx3 protein, which is not reactive with either of the antibodies used in this analysis. The strong family member-specific expression patterns of Runx in cell lines T6i, T47i, and T1i support the view that the major consequences of these insertions are transcriptional activation and overexpression of the relevant target gene. Furthermore, the failure to observe proviral insertion at more than one family member in a cohort of 120 independently derived lymphomas suggests that the Runx genes may be functionally redundant with respect to their oncogenic activity, in a manner similar to that reported for the Myc and Pim oncogene families (15) .

In view of reports of loss-of-function mutations of RUNX1 in familial platelet disorder (16) and in some cases of acute myelogenous leukemia (1) , it was important to investigate the structural integrity of the overexpressed gene in tumor T6i. A cDNA library was generated from poly-A⁺ RNA of the T6i lymphoma cell line and screened with a probe specific for Runx1 exon 6. Seventeen independent clones were isolated, and a further round of screening identified a subset of 10 clones positive for the 5' UTRs of Runx1 exon 1. Sequence analysis revealed that the majority of these clones were hybrids in which MLV LTR sequences were fused to 5' UTRs of Runx1, as expected from the PCR analyses (Fig. 1a) . No clone was found that extended upstream of the identified MLV-Runx1 junction into Runx1 5' UTRs, suggesting that the majority, if not all, of the Runx1 transcripts in the T6i cell line were derived from the allele affected by retroviral insertion. Consistent with the RT-PCR analysis, all of the cDNAs that were sequenced demonstrated splicing and removal of the 5' untranslated exons 1b and 1c (Fig. 1b) . Interestingly, our sequence analysis confirmed a recent report (17) that Runx1 P1 transcripts lack a minor coding exon (1.1), which is present in the human RUNX1 equivalent (18) and serves as the most 5' RUNX1 exon in ETV6-RUNX1 fusions.

The cDNAs fall into three groups according to their coding potential. Six of 10 encode the full-length MASD p50^Runx1 isoform. The two remaining groups code for alternatively spliced isoforms in which either exon 4.1 (3 of 10) or exon 5 (1 of 10) is lacking (Fig. 1d) . The relative abundance of these exon-skipping variants was similar to previous expression analyses of the unrearranged murine Runx1 gene (19) , suggesting that promoter insertion does not perturb the balance of mRNA splicing. The existence of alternatively spliced isoforms is a well characterized feature of Runx1 and Runx2 (6 , 14) , however, the significance of this phenomenon is still poorly understood although there is some evidence of functional heterogeneity and antagonism between overexpressed isoforms (20) . Notably, analysis of the entire panel of cDNA clones revealed no coding sequence mutations, suggesting that overexpression of Runx1 is the key event in the T6i cell line and that mutations are not required to activate the oncogenic potential of the gene.

Runx1 Haploinsufficiency in Mice Does Not Promote T-Cell Lymphoma Development.
As a further test of the loss-of-function hypothesis, we examined the effect of Runx1 gene dosage on lymphoma development (1) . We found that the incidence and latency of tumor development in Runx1^(+/-) mice infected with MLV was not significantly different from infected littermate controls (Fig. 2a) . A parallel experiment was performed with transgenic mice that coexpress c-MYC and Runx2 in thymocytes. When the highly tumor-prone CD2-Runx2/MYC transgenic mice are placed on a Runx1^(+/-) background, survival shows a small but significant (P < 0.05) increase rather than a decrease when compared with Runx1^(+/+) littermate controls (Fig. 2b) . In addition, Southern blot hybridization analysis of selected tumors from MLV-infected Runx1^(+/-) or Runx1^(+/-)/Runx2/MYC cross mice revealed no evidence of loss of heterozygosity (data not shown). These findings suggest that the oncogenic action of Runx2 cannot be attributed to inhibition of Runx1 function and argue against a general tumor suppressor role for Runx1. Rather, they support the hypothesis that both Runx1 and Runx2 can act as dominant oncogenes in the T-cell compartment.

View larger version (16K): [in this window] [in a new window]   Fig. 2. a, tumor-free survival of Runx1(+/-) mice infected with MLV. Survival curves are shown for Runx1(+/-) mice (, n = 32) and wild-type littermate controls (, n = 39) infected with MLV. b, tumor-free survival of CD2-MYC/Runx2 transgenic mice on a Runx1(+/-) background. Survival curves are shown for CD2-MYC/Runx2 animals that are either Runx1(+/-) (, n = 24) or Runx1 wild-type littermate controls (, n = 19).

Finally, our results indicate that Runx1 can act as a dominant oncogene and collaborate with Myc in the genesis of murine T-cell lymphoma. This contrasts with the prevailing view that inhibition of Runx1 function is the critical event in perturbing differentiation and predisposing to leukemogenesis. The data we have presented here suggests that overexpression of Runx1 may represent an alternative route to aberrant differentiation and leukemogenesis, a finding that is consistent with recent reports of RUNX1 amplification in leukemic cells (24) . The apparent conundrum of loss-of-function and gain-of-function both contributing to neoplastic transformation may find its solution in the fact that both types of event can perturb differentiation (3 , 7 , 25) . It is conceivable that different stages of development or different lineages are particularly sensitive to lesions that result in either gain, or loss, of Runx1 function. Because recent evidence shows that the paradoxical behavior of Runx1 is mirrored by Runx3, which is mutated or down-regulated in gastric carcinomas (26) but transcriptionally activated in T-cell lymphoma (8) , it seems that prevailing models for the oncogenic action of the Runx family will have to be reexamined.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Nancy Speck (Dartmouth, NH) for provision of the Runx1^(+/-) mice and for helpful comments on the manuscript. We also thank Prof. Yoshiaki Ito (Institute for Virus Research, Kyoto, Japan) for the kind gift of the -Runx2 mouse monoclonal antibody.

The murine Runx1 genomic sequence data were produced by the United Kingdom Medical Research Council mouse genome sequencing program, funded by the United Kingdom Medical Research Council, and can be obtained from ftp://ftp.sanger.ac.uk/pub/mouse/chr_16/.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Cancer Research-United Kingdom and the Leukemia Research Fund provided support for this work.

² To whom requests for reprints should be addressed, at Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom. Phone: 44-141-330-5725; Fax: 44-141-330-4874; E-mail: e.r.cameron{at}vet.gla.ac.uk

³ The abbreviations used are: MLV, murine leukemia virus; MoMLV, Moloney MLV; UTR, untranslated sequence; LTR, long terminal repeat.

⁴ Unpublished data.

⁵ J. Lenz, personal communication.

Received 8/ 5/02. Accepted 10/28/02.

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