[CANCER RESEARCH (SUPPL.) 59, 1794s-1798s, April 1, 1999]

The Effect of Chromosomal Translocations in Acute Leukemias: The LMO2

Paradigm in Transcription and Development I

T e r e n c e H . R a b b i t t s , 2 K a t h a r i n a B u c h e r , G r a c e C h u n g , G e r a l d G r u t z , A l a n W a r r e n , a n d Y o s h i Y a m a d a

Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 2QH United Kingdom

Abstract

Two general features have emerged about genes that are activated after chromosomal translocations in acute forms of cancer. The protein prod- ucts of these genes are transcription regulators and are involved in developmental processes, and it seems that the subversion of these normal functions accounts for their role in tumorigenesis. The features of the LMO family of genes, which encode LIM-domain proteins involved in T-cell acute leukemia through chromosomal translocations, typify these abnormal functions in tumorigenesis. For example, the LMO2 protein is involved in the formation of multimeric DNA-binding complexes, which may vary in composition at different stages of hematopoiesis and function to control differentiation of specific lineages. In T cells, enforced expres- sion of Lmo2 causes aberrant protein complex formation that primarily seems to hinder the T-ceU differentiation program. These observations underscore the conclusion that protein-protein interaction (in this case, through the LIM domain) is a key determinant in tumorigenesis. Further- more, the study of chromosomal translocations as naturally occurring mutations has been informative about mechanisms in hematopoiesis as well as in tumor etiology.

Introduction

The cytogenetic analysis of tumors, particularly those of hemato- poietic origin, have revealed that reciprocal chromosomal transloca- tions are recurring features of these tumors (reviewed in Ref. (1). Furthermore, it has become clear that particular chromosomal trans- locations are consistently found in specific tumor subtypes. Through the cloning of the chromosomal breakpoints and identification of oncogenes at many different breakpoints, followed by transgenic (2) and homologous recombination knock-in analysis (3), it has become clear that these abnormal tumor-associated chromosomes are impor- tant in the etiology of tumors. The scientific challenge of the last decade has been to define the contribution of the genes activated by translocations to the course of tumor development and to ascertain whether any general principles can be discerned about these "trans- location" genes.

There are a variety of chromosomal translocations in leukemias and in solid tumors of mesenchymal origin (sarcomas), and two main outcomes are apparent (1). One of these is confined to lymphoid tumors in which the process of antigen receptor rearrangement (im- munoglobulin and T-cell receptor) occurs and which occasionally aberrantly mediates chromosomal translocation. This type of chromo- somal translocation causes oncogene activation resulting from the new chromosomal environment of the rearranged gene, and, in gen- eral, this means inappropriate gene expression. In this first category of chromosomal translocation, the B- and T-cell tumors exemplify many consistently occurring features. For instance, the cloning of the CMYC

Received 11/11/98; accepted 2/4/99. 1 Presented at the "General Motors Cancer Research Foundation Twentieth Annual

Scientific Conference: Developmental Biology and Cancer," June 9-10, 1998, Bethesda, MD. This work was supported by the Medical Research Council and by Grants from the Leukaemia Research Fund (United Kingdom) and the National Foundation for Cancer Research (United States). K.B. is supported by the Roche Foundation; G.G. was supported by an EMBO Fellowship, and Y. Y. by Kyoto University.

2 To whom requests for reprints should be addressed, at MRC Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 2QH United Kingdom.

proto-oncogene from a Burkitt's lymphoma translocation breakpoint t(8;14)(q24;q32) revealed that, while the translocated gene was intact, there were many mutations in both the noncoding first exon and within the coding region itself (4-11). In T-cell acute leukemias, there are several different chromosomal translocations found in individual tumors, and the analysis of these has led to the discovery of many different, novel genes that can contribute to T-cell tumorigenesis (1).

These and many other studies have led to two conclusions: (a) many of the chromosomal translocation-activated oncogenes are

transcription regulators in their normal sites of expression, and it is this property that is instrumental in their involvement in tumor etiol- ogy after the chromosomal translocation. This was suggested previ- ously from the early studies on hematopoietic tumor translocations and later, in addition, from the very large range of changes found in hematopoietic and mesenchymal tumors (reviewed in Ref. 1); and

(b) the biological role of many chromosomal translocation-acti- vated genes is normally in developmental processes leading to the notion that subversion of development may be their crucial biological role in tumorigenesis, perhaps explaining why so few of the chromo- somal translocation-activated genes were previously identified as on- cogenes from other experimental approaches. The LMO 1 and LMO2 LIM-only proteins are involved in T-cell leukemia because of their ability to interact with other transcription factors. This enhances the conclusion that protein-protein interaction is a common component of oncogene function.

Results and Discussion

The LMO Family Genes. The LMO family of genes was uncov- ered (Fig. 1) by the association of LM01 (previously called RBTN1 or TTG1) with the chromosomal translocation t(11;14)(p15;ql 1). The transcription unit was first observed in a T-cell line (12), and the mRNA sequence was obtained from its cDNA sequence (13, 14) and shown to encode a protein essentially consisting of two zinc-binding LIM domains (15). Using an LMO1 probe, the two related genes LM02 and LM03 were isolated (previously called RBTN2 or TTG2 and RBTN3, respectively; Refs. (16, and 17), of which LM02 was found located at the junction of the chromosomal translocation t(11; 14)(p13;ql 1) in T-ALL 3 (16, 18). The LMO-associated chromosomal translocations seem to have occurred by an error of the usual RAG- mediated variable diversity joining recombinase process inasmuch as sequence analysis of the breakpoints on chromosome 1 lp13 detected recombinase signal sequences at the junctions and because the joins on chromosome 14 in the TCR~ locus or on chromosome 7 in the TCR~ locus occur precisely at the end of D-segments (19).

The unique feature of the LMO-derived protein sequences is that they are small proteins comprising two tandem LIM domains. These zinc-binding finger-like structures have structural similarities to the DNA-binding GATA fingers (20, 21) but as yet no case of a specific LIM-DNA interaction has been reported; rather the function of this domain seems to be restricted to protein-protein interaction (see "LMO2 Functions by Protein Interaction").

3 The abbreviations used are: T-ALL, T-cell acute lymphocytic leukemia; bHLH, basic-helix-loop-helix; DN, double negative; Tall , Tall/Scl.

1794s

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

LMO GENES IN LEUKEMIA

LMO2 Is a Regulator of Mouse Hematopoiesis. To gain insights into the function of the LMO genes in tumorigenesis, an integrated approach has been adopted to attempt to understand the normal function at the biological level as well as the function of LMO proteins and to use this information to explore the function in tumors. As a first step, gene targeting was used to introduce null mutations in mouse Lmo2 gene, which showed that Lmo2 is necessary for yolk sac erythropoiesis in mouse embryogenesis (22). Furthermore, the use of embryonic stem cells with null mutations of both alleles of Lmo2 in chimeric mice has shown that adult hematopoiesis, including lympho- poiesis and myelopoiesis, fails completely in the absence of Lmo2 (23). These data show that Lmo2 must function at early stages of hematopoiesis (Fig. 2) either at the level of the pluripotent stem cell or even perhaps before this when ventral mesoderm gives rise to these precursor cells. Remarkably similar results have been shown for another T-cell oncogene, Tall/Scl (herein called Tall; Refs. (24-27). The notion of genes that function in developmental processes is clearly delineated by the Lmo2 gene.

LMO2 Functions by Protein Interaction: The Role of the LIM Domain. The concept that oncogenes can interfere with developmen- tal processes because they are themselves normally performing this function has been suggested to explain their role in tumor etiology. The finding that genes that encode proteins such as Lmo2 control development of hematopoietic lineages led to clear tests of molecular function in both normal and tumor contexts. A particularly relevant observation was that the T-cell oncogenes Lmo2 and Tall were coexpressed in erythrocytes (22), and this suggested a functional synergy between the two proteins. This turned out to be even more germane because it was shown that the Lmo2 and Tal 1 proteins--the latter is a bHLH protein that is activated in T-cell leukemias by chromosomal translocations or promoter deletions (28-33) and which can also interact with the bHLH E47 protein in a DNA-binding complex (34, 35)--could interact directly with each other (36, 37), and this was through the LIM domains. This observation and those made with isolated LIM fragments (38) establish a role for the LIM domain in protein-protein interaction. The LIM domain does not seem to directly bind DNA despite the similarity to the GATA DNA- binding zinc finger.

The ability of the LIM protein interaction domain to bind various proteins was also shown in the ability of Lmo2 to bind to Gata-1 (39) and to bind to the Ldbl/NLI protein (40). In addition, LMO1 and

LMO family Human

Gene Chromosome Translocation Man Mouse

LMO1 11p15 7 t(11;14)(p15;q11)

LMO2 11p13 2 t(11;14)(p13;q11)

LMO3 12 p12-13 6 ND

Fig. 1. LMO family of genes and oncogenes. The LMO genes (so designated as LIM-Only genes and previously known as RBTN or TTG genes) have three known members. LM01 (previously RBTN1 or TTG1) was identified first and then, LM02 (previously RBTN2 or TTG2) and LM03 (previously RBTN3). LM01 and LM02 are both located on the short arm of chromosome 11 and are both involved in independent chromosomal translocations in T-ALL. As yet, LMO3 has not been found in association with any chromosomal translocations.

Pluri-potent cell Myeloid

M od~erm- '~~ lineage es

Lymphoid Self-renewa/j lineage

Lmo2 null defect Fig. 2. Lmo2 is necessary for adult hematopoiesis in mice. The tissue contribution of

embryonic stem cells with homozygous null mutation of Lmo2 showed that hematopoiesis in adult mice is dependent on this gene (23). Thus, the gene product is required for all of the stages of adult hematopoiesis, functioning at least before the bifurcation of myeloid and lymphoid lineages. The function may be restricted to the bone marrow stem cell (either at the self-renewal stage or at the proliferative stages that produce committed progenitors) or in the ventral mesoderm bone marrow precursor cells.

LDB1 interact in vivo in T-ALL and in neuroblastoma cell lines (41). This array of interactions led to the observation that Lmo2 is found in an oligomeric complex in erythroid cells that involves--in addition to Lmo2--Tal l , E47, Ldbl and Gata-I (40). This complex is able to bind DNA at least in vitro and in reporter assays, with the Lmo2 and Ldbl components seeming to bridge a bipartite DNA-binding com- plex (Fig. 3A). These findings suggest that Lmo2 is part of a tran- scription complex in hematopoiesis. The involvement of Lmo2, Tall, and Gatal in a common DNA-binding complex suggests that this complex regulates downstream target genes, perhaps explaining why the null mutation of these genes leads to the lack of primitive eryth- ropoiesis (22, 25, 42, 43). Conversely, there are differences in some specific aspects of hematopoiesis related to the individual null muta- tions, which suggests that each of them can act in separate complexes at different stages of hematopoiesis.

The distinct roles of Lmo2 in primitive and definitive hematopoi- esis, uncovered by the analysis of null mutations of Lmo2 (22, 23), suggested that the molecular complexes in which Lmo2 is involved may also differ at different stages (40). In erythroid cells, we observed a complex of Lmo2 with GATA-1, E47, Tall and Ldbl (40) that could bind to a unique bipartite E box-GATA motif (Fig. 4B). This suggested that earlier stages of hematopoiesis may have different complexes; for instance, GATA-1 is absent from early hematopoietic progenitors; therefore, it may be replaced by GATA-2 (39). Indeed, there may also be a variation in the bHLH pair present, although phenotypes of Tal 1/Scl null mutations (26, 27) parallel those of Lmo2 (22, 23). In our model, variations in the composition of Lmo2- containing complexes may influence lineage differentiation (40, 23), presumably by controlling distinct sets of target genes (Fig. 4).

The Role of LMO2 in T-ALL. The existence of an oligomeric complex in which Lmo2 protein is apparently a linking molecule in RBCs suggests that the function of the Lmo2 protein in T-cell acute leukemias may be to participate in an analogous but aberrant complex after the chromosomal translocation has enforced expression of the Lmo2 gene in T cells. The emulation of the human T-cell LMO2-

enforced expression after chromosomal translocation has been achieved using transgenic expression of Lmo2 in the T-cell lineage (44-47). This results in clonal T-cell leukemia arising in the trans- genic mice with a long latency, on average about 9 months. This latency period indicates that the transgene is necessary but not suffi- cient to cause tumors in this model, as is the case for many transgenic oncogene models, and that mutations in other oncogenes must be occurring to allow development of overt disease.

The long latency period facilitated the detailed study of possible effects in the asymptomatic thymuses of transgenic mice (45, 47). An outline of normal T-cell differentiation is shown in Fig. 4, which

1795s

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

Fig. 3. Lmo2 lbrms part of a complex that can bind a bipartite DNA site. A, the ability of the Lmo2 protein to interact with Tall and with GATA1 appears to facilitate the formation of a complex in which two DNA contacting regions comprise a Tal 1-E47 dimer [binding an E box (CANNTG)] and a GATA1 molecule [binding a GATA site] as part of an erythroid complex. In model experiments (40), the two parts of the bipartite recognition sequence are separated by approximately one turn of the DNA helix. This erythroid complex can specifically bind a unique bipartite DNA site. Thus, the probable function of the complex is to bind to the DNA of chromosomal target genes and to regulate their expression (positively or negatively; Ref. 40). B, an analogous DNA-binding complex has been identified in T-cell of Lmo2-expressing transgenic mice (48). In this case, however, a novel complex is formed which can recognize a dual E-box motif, appar- ently via two bHLH dimers linked by Lmo2 and Ldbl proteins. Therefore, enforced Lmo2 expression by transgenesis or by chromo- somal translocations may cause formation of an aberrant T-cell com- plex that binds to and controls the expression of target genes (48). These may well be different from those genes normally the focus of Lmo2 function.

LMO GENES IN LEUKEMIA

Molecular consequences of enforced LMO2 expression I

AI Erythroid progenitor complex

S m

Aberrant T cell complex

Q ~ A : N N T G ............... G'A~T~A .................. CAt',~'TC, CAt'~'TC

serves to illustrate the individual points in T-cell development. There was a marked accumulation of immature CD4-, CD8-, CD25 +, CD44 + T cells (herein referred to as DN T cells) in transgenic thymuses compared with nontransgenic litter mates, an effect that was exacerbated in mice transgenic for both Lmo2 and Tall (47). Thus, the role of the transgene products is to cause an inhibition of T-cell differentiation that appears reversible, presumably by antigenic stim- ulation occurring after birth because different transgenic mice exhibit different levels of DN cell accumulation.

It is of note that the DN T-cell population is RAG variable diversity joining recombinase-positive, and thus is a population where, in humans, the recombinase-associated translocations may occur. This suggests that the T-cell acute leukemia precursors in humans acquire the chromosomal translocations within the DN T-cell population, and that this produces a cell with inhibited differentiation analogous to that of the transgenic mouse model (Fig. 5). It is proposed that the overt tumor eventually arises in the targeted, predisposed population of T cells that emerges from the event of chromosomal translocation by the accumulation of secondary mutations in other genes. Therefore, these tumors can display any other stage of T-cell differentiation.

Protein Complexes Involving LMO2 in T-ALL. The work on Lmo2 gene targeting clearly showed that the gene is essential for both yolk sac erythropoiesis and for adult definitive hematopoiesis. This information indicated that erythroid cells might possess a functional Lmo2 protein and made possible the discovery that the Lmo2 protein is part of a DNA-binding oligomeric protein complex in which Lmo2, rather than participating in direct DNA contacts, is a connecting or

bridging molecule between the two DNA-binding arms of a complex. The ability of Lmo2 to interact with other proteins to perhaps mediate the formation of a multiprotein DNA binding complex suggested a similar molecular mechanism for LMO2 protein in T-cell tumors. After enforced expression of LM02, either by chromosomal translo- cation or by transgenesis, the presence of the protein could, therefore, mediate the formation of an aberrant protein complex. This aberrant complex may contain DNA-binding factors that can recognize target genes (perhaps with altered specificity) either activating or repressing gene expression, or the aberrant complex could simply inhibit normal binding of components of the complex to their cognate DNA sites, thereby effectively repressing gene activity.

LMO2 expression in T cells may result in a complex with other proteins that would not normally form. In addition, it might be that, although a complex of proteins does form, the crucial interaction is with only one of the interacting partners, sequestering it from per- forming its normal function, which might not involve DNA binding. For instance, the binding of LDB 1 in the T cell by enforced LMO2 expression might simply remove LDB 1 from its normal function.

In a search for evidence of oligomeric Lmo2-complexes, T-cell lines were derived from CD2-Lmo2 transgenic mice (48) and used as a source of Lmo2 protein complexes for in vitro random site selection CASTing experiments (48). This work resulted in the detection of a Lmo2-containing complex, which, like its analogue in erythroid cells, binds to a bipartite recognition site (Fig. 3B) but, in the T-cell context, recognizes a dual E-box motif, in which the two E-box sequences are separated by about one DNA helical turn. Analysis of the components

Fig. 4. Variation of LMO2 complexes may oc- cur at different hematopoietic stages We have found that the LMO2 protein is part of a multimeric complex in erythroid cells (40), which suggests that it is involved in erythropoiesis. Our data on Lmo2 null mutations in mice suggest possible distinct roles in primitive and definitive hematopoiesis (22, 23). Therefore, distinct Lmo2-multimeric com- plexes may have distinct roles in different hemato- poietic lineages (40), regulating different sets of target genes. The finding of a novel LMO2- containing multimeric complex in T-cells after en- forced Lmo2 expression (48) supports the notion of the flexibility of Lmo2 to be involved in different complexes in different environments. Thus, in erythroid cells (B), Lmo2 is involved in a complex with a number of proteins, and it was suggested that in early hematopoiesis (A) it may be in complexes with a different composition (40). Variations in these complexes may function in lineage differen- tiation programs as suggested by mutational studies of Lmo2 (22, 23).

Ao

Early haematopoiesis

B. T

Erythropoiesis

1796s

. . . . ~NL I

. . . . 'NL I

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

LMO GENES IN LEUKEMIA

Differentiation block . ITransge~

Pre-T CD4- CO8- cell CD25+ CD44+

| | | BM ~ --~ " ~ ~ CD3+ CD8+ OR CD4+ T C R ~ -

RAG+

Onset of TCR Chromosomal ] :Chiiaho0d [ -"- translocation - - ~ i T!ALL,,,,,, ' I rearrangement during TCR joining

Fig. 5. Model of Lmo2 function in T-ALL by inhibition of T-cell differentiation. Bone marrow (BM) produces pre-T cells (designated as TN because these cells do not yet express typical T-cell markers such as CD3, CD4 and CD8). These pre-T cells become DN cells (having a CD4- , CD8 , CD25 +, CD44 + marker phenotype) which do not yet express T-cell receptor (TCR) but begin to express the RAG recombinase proteins. Further differentiation of this immature DN T-cells subset results after TCR rearrangement, resulting in mature functional TCR-bearing T cells. The Lmo2 transgenic mice (45, 46) and double Lmo2-Tall transgenics (45, 47) accumulate the DN cells, apparently as a result of differentiation inhibition caused by the transgene. Because the LMOI- and LMO2-associated chromosomal translocations seem to result from RAG-mediated recom- binase errors, it is proposed that this same target population of DN T cells is affected in humans with the chromosomal translocation. The enforced LMO2 expression affects multimeric protein complex formation, which seems to function by alteration of the T-cell differentiation program. Thus, altered T-cell clones provide the precursors from which the overt tumor arises after secondary mutations.

of this complex showed that E47-Tall bHLH heterodimeric elements were present as well as Lmo2 and the Ldbl protein (illustrated in Fig. 3B). The function of this complex may, therefore, be to bind to unique sites in chromosomal target genes to control their expression (posi- tively or negatively). The formation of an aberrant Lmo2-containing complex suggests that the target genes are affecting T-lineage devel- opment, providing a cell population in which secondary mutations occur preceding overt tumors.

Conclusions. The extensive work that has been carried out on the cloning and characterization of chromosomal translocation break- points, coupled with functional studies of the oncogenes found to be associated with these breakpoints, has allowed some general conclu- sions to be made, which are typified by the studies discussed in this paper on the LM02 gene.

Clearly, chromosomal translocations are important for the etiology of the tumors that contain them. It seems likely that in many cases, the translocation event occurs early in tumor formation and involves the alteration of developmental processes in some way. In terms of the formation of the chromosomal translocation, there are two crucial points:

(a) the mechanism of the formation of the aberrant chromosome itself. In the lymphoid tumors, this can be through mistakes of T-cell receptor rearrangement creating an interchromosomal event rather than the intrachromosomal VDJ join, whereas in cases where the fusion of genes occurs, it is far from clear what the mechanism is; and

(b) in either situation, the outcome is a cell with some growth advantage over its partners, and this cell may eventually appear as an overt tumor after accumulation of other mutations.

The discovery of multimeric complexes involving Lmo2 in normal, erythroid cells (40) and in T-cell tumors (48) suggests that different chromosomal target genes are controlled in the different settings. The study of these genes will prove interesting in the future. The combined study of the etiology and function of the naturally occurring chromo- somal mutations (i.e., translocations) and the function of genes found at their junctions has been informative about general mechanisms. The data suggest that variation of Lmo2-complexes may influence hema-

1797s

topoietic lineages (40). The nature of Lmo2 protein-protein interac- tions in early hematopoiesis will guide additional investigations. It is also very evident from the study of LM02 and of other translocation- activated oncogenes, that transcription regulation is a key target within the tumor precursor. Finally, as the LIM domain interactions demonstrate, protein-protein associations are crucial molecular events in tumor formation.

Note Added in Proof

A fourth m e m b e r o f the L M O family (LMO4) has recent ly been identified

(Gmtz, G., Forster, A., and Rabbitts, T. H., Identif icat ion o f the L M 0 4 gene

encoding an interact ion partner o f the LIM-b ind ing protein L D B 1 / N L I I : a

candidate for d isp lacement by L M O proteins in T cell acute leukaemia.

Oncogene , 17: 2 7 9 9 - 2 8 0 3 , 1998).

References

1. Rabbitts, T. H. Chromosomal translocations in human cancer. Natm-e (Lond.), 372: 143-149, 1994.

2. Adams, J. M., and Cory, S. Transgenic models of tumor development. Science (Washington DC), 254:1161-1167, 1991.

3. CoiTal, J., Lavenir, I., lmpey, H., Warren, A. J., Forster, A., Larson, T. A., Bell, S., McKenzie, A. N. J., King, G., and Rabbitts, T. H. An Mll-Af9 fusion gene made by homologous recombination causes acute leukemia in chimeric mice: a method to create fusion oncogenes. Cell, 85: 853-861, 1996.

4. Rabbitts, T. H., Hamlyn, P. H., and Baer, R. Altered nucleotide sequences of a translocated c-myc gene in Burkitt lymphoma. Nature (Lond.), 306: 760-765, 1983.

5. Rabbitts, T. H., Forster, A., Hamlyn, P. H., and Baer, R. Effect of somatic mutation within translocated c-myc genes in Burkitt's lymphoma. Nature (Lond.), 309: 592- 597, 1984.

6. Showe, L. C., Ballantine, M., Nishikura, K., Erikson, J., Kaji, H., and Croce, C. M. Cloning and sequencing of a c-myc oncogene in a Burkitt's lymphoma cell line that is translocated to a germ line a switch region. Mol. Cell. Biol., 5: 501-509, 1985.

7. Murphy, W., Sarid, J., Taub, R., Vasicek, T., Battey, J., Lenoir, G., and Leder, P. Translocated human c-myc oncogene is altered in a conserved coding sequence. Proc. Natl. Acad. Sci. USA, 83: 2939-2943, 1986.

8. Cesarman, E., Dalla-Favera, R., Bentley, D., and Groudine, M. Mutations in the first exon are associated with altered transcription of c-myc in Burkitt lymphoma. Science (Washington DC), 238:1272-1275, 1987.

9. Morse, B., South, V. J., Rothberg, P. G., and Astrin, S. M. Somatic mutation and transcriptional deregulation of myc in endemic Burkitt's lymphoma disease: hep- tamer-nonamer recognition mistakes'? Mol. Cell. Biol., 9: 74-82, 1989.

10. Bhatia, K., Huppi, K., Spangler, G., Siwarski, D., Iyer, R., and Magrath, I. Point mutations in the c-Myc transactivation domain are common in Burkitt's lymphoma and mouse plasmacytomas. Nat. Genct., 5: 56-61, 1993.

11. Yano, T., Sander, C. A., Clark, H. M., Dolezal, M. V., Jaffe, E. S., and Raffeld, M. Clustered mutations in the second exon of the MYC gene in sporadic Burkitt's lymphoma. Oncogene, 8: 2741-2748, 1993.

12. Boehm, T., Baer, R., Lavenir, I., Forster, A., Waters, J. J., Nacheva, E., and Rabbitts, T. H. The mechanism of chromosomal translocation t(1 t ;14) involving the T-cell receptor C6 locus on human chromosome 14ql I and a transcribed region of chro- mosome 1 lp15. EMBO J., 7." 385-394, 1988.

13. McGuire, E. A., Hockett, R. D., Pollock, K. M., Bartholdi, M. F., O'Brien, S. J., and Korsmeyer, S. J. The t(11; 14)(p 15 ;q 11 ) in a T-cell acute lymphoblastic leukcmia cell line activates multiple transcripts, including Ttg-l, a gene encoding a potential zinc finger protein. Mol. Cell. Biol., 9: 2124-2132, 1989.

14. Boehm, T., Greenberg, J. M., Buluwela, L., Lavenir, I., Forster, A., and Rabbitts, T. H. An unusual structure of a putative T cell oncogene which allows production of similar proteins from distinct mRNAs. EMBO J., 9: 857-868, 1990.

15. Boehm, T., Foroni, L., Kennedy, M., and Rabbitts, T. H. The rhombotin gene belongs to a class of transcriptional regulators with a potential novel protein dimerisation motif. Oncogene, 5:1103-1105, 1990.

16. Boehm, T., Foroni, L., Kaneko, Y., Perutz, M. P., and Rabbitts, T. H. The rhombotin family of cysteine-rich LlM-domain oncogenes: Distinct members are involved in T-cell translocations to human chromosomes 1 lpl 5 and 1 l p13. Proc. Natl. Acad. Sci. USA, 88: 4367-4371, 1991.

17. Foroni, L., Boehm, T., White, L., Forster, A., Sherrington, P., Liao, X. B., Brannan, C. I., Jenkins, N. A., Copeland, N. G., and Rabbitts, T. H. The rhombotin gene family encode related LIM-domain proteins whose differing expression suggests multiple roles in mouse development. J. Mol. Biol., 226: 747-761, 1992.

18. Royer-Pokora, B., Loos, U., and Ludwig, W.-D. TTG-2, a new gene encoding a cysteine-rich protein with the LIM motif, is overexpressed in acute T-cell leukaemia with the t(11 ;14)(p13;ql 1). Oncogene, 6: 1887-1893, 1991.

t9. Sanchez-Garcia, I., Kaneko, Y., Gonzalez-garmiento, R., Campbell. K., White, L., Boehm, T., and Rabbitts, T. H. A study of chromosome l lp13 translocations involving TCR~9 and TCR6 in human T cell leukaemia. Oncogene, 6: 577-582, 1991.

20. Sanchez-Garcia, I., and Rabbitts, T. H. The LIM domain: a new structural motif in zinc-finger-like proteins. Trends Genet., IO: 315-320, 1994.

21. Perez-Alvarado, G. C., Miles, C., Michelsen, J. W., Louis, H. A., Winge, D. R., Beckerle, M. C., and Summers, M. F. Structure of the carboxy-terminal LIM domain from the cysteine rich protein CRP. Nat. Stnlct. Biol., 1: 388-398, 1994.

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

LMO GENES IN LEUKEMIA

22. Warren, A. J., Colledge, W. H., Carlton, M. B. L., Evans, M. J., Smith, A. J. H., and Rabbitts, T. H. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell, 78: 45-58, 1994.

23. Yamada, Y., Warren, A. W., Dobson, C., Forster, A., Pannell, R., and Rabbitts, T. H. The T cell leukaemia LIM protein Lmo2 is necessary for adult mouse haematopoiesis. Proc. Natl. Acad. Sci. USA, 95: 3890-3895, 1998.

24. Robb, L., Lyons, 1., Li, R., Hartley, L., Kontgen, F., Harvey, R. P., Metcalf, D., and Begley, C. G. Absence of yolk sac hematopoiesis from mice with a targeted disrup- tion of the scl gene. Proc. Nail. Acad. Sci. USA, 92: 7075-7079, 1995.

25. Shivdasani, R. A., Mayer, E., and Orkin, S. H. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-I/SCL. Nature (Lond.), 373: 432-434, 1995.

26. Robb, L., Elwood, N. J., Elefanty, A. G., Kontgen, F., Li, R., Bamet, L. D., and Begley, C. G. The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J., 15: 4123-4129, 1996.

27. Porcher, C., Swat, W., Rockwell, K., Fujiwara, Y., Alt, F. W., and Orkin, S. H. The T cell leukemia oncoprotein SC1/tal-1 is essential for development of all hematopoi- etic lineages. Cell, 86: 47-57, 1996.

28. Begley, C. G., Aplan, P. D., Davey, M. P., Nakahara, K., Tchorz, K., Kurtzberg, J., Hershfield, M. S., Haynes, B. F., Cohen, D. 1., Waldmann, T. A., and Kirsch, 1. R. Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor &chain diversity region and results in a previously unreported fusion transcript. Proc. Natl. Acad. Sci. USA, 86: 2031-2035, 1989.

29. Begley, C. G., Aplan, P. D., Denning, S. M., Haynes, B. F., Waldmann, T. A., and Kirsch, I. R. The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif. Proc. Natl. Acad. Sci. USA, 86:10128- 10132, 1989.

30. Bernard, O., Gugliehni, P., Jonveaux, P., Cherif, D., Gisselbrecht, S., Mauchauffe, M., Berger, R., Larson, C-J,, and Mathieu-Mahul, D. Two distinct mechanisms for the SCL gene activation in the t(1;14) translocation of T-cell leukemias. Genes Chromo- somes Cancer, l: 194-210, 1989.

31. Finger, k. R., Kagan, J., Christopher, G., Kurtzberg, J., Hershfield, M. S., Nowell, P. C., and Croce, C. M. involvement of the TCL5 gene on human chromosome 1 in T-cell leukemia and melanoma. Proc. Natl. Acad. Sci. USA, 86: 5039-5043, 1989.

32. Chen, Q., Cheng, J.-T., Tsai, L-H., Schneider, N., Buchanan, G., Carroll, A., Crist, W., Ozanne, B., Siciliano, M. J., and Baer, R. The tal gene undergoes chromosome translocation in T cell leukaemia and potentially encodes a helix-loop-helix protein. EMBO J., 9: 415-424, 1990.

33, Chen, Q., Yang, C. Y-C., Tsan, J., Xia, Y., Ragab, A. H., Peiper, S. C., Carroll, A., and Baer, R. Coding sequences of the tat-I gene are disrupted by the chromosome translocation in human T cell leukaemia. J. Exp. Med., 172: 1403-1408, 1990.

34. Hsu, H-L., Cheng, J-T., Chen, Q., and Baer, R. Enhancer-binding activity of the ta l l oncoprotein in association with the E47/EI2 helix-loop-helix proteins. Mol. Cell. Biol., 11: 3037-3042, 1991.

35. Hsu, H.-L., Huang, L., Tsan, J. T., Funk, W., Wright, W. E., Hu, J-S., Kingston, R. E, and Baer, R. Preferred sequences for DNA recognition by the TALl helix-loop-helix proteins. Mol. Cell. Biol., 14: 1256-1265, 1994.

36. Valge-Archer, V. E., Osada, H., Warren, A. J., Forster, A., Li, J., Baer, R., and Rabbitts, T. H. The LIM protein RBTN2 and the bHLH protein TALl are present in a complex in erythroid cells. Proc. Natl. Acad. Sci. USA, 91:8617 8621, 1994.

37. Wadman, I., Li, J., Bash, R. O., Forster, A., Osada, H., Rabbitts, T. H., and Baer, R. Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukaemia. EMBO J., 13: 4831-4839, 1994.

38. Schmeichel, K. L., and Beckerle, M. C. The LIM domain is a modular protein-binding interface. Cell, 79: 211-219, 1994.

39. Osada, H., Grutz, G., Axelson, H., Forster, A., and Rabbitts, T. H. Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1. Proc. Natl. Acad. Sci. USA, 92: 9585-9589, 1995.

40. Wadman, 1. A., Osada, H., Grutz, G. G., Agulnick, A. D., Westphal, H., Forster, A., and Rabbitts, T. H. The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TALl, E47, GATA-1 and Ldbl/NLI proteins. EMBO J., 16: 3145-3157, 1997.

41. Valge-Archer, V., Forster, A., and Rabbitts, T. H. The LMO1 and LDBI proteins interact in human T-cell acute leukemia with the chromosomal translocation t(11;14)(p15;ql 1). Oncogene, 17: 3199-3202, 1998.

42. Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S-F., D'Agati, V., Orkin, S. H., and Costantini, F. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature (Lond.), 349: 257-260, 1991.

43. Robb, L., Rasko, J. E. J., Bath, M. L., Strasser, A., and Begley, C. G. scl, a gene frequently activated in human T cell leukaemia, does not induce tymphomas in transgenic mice. Oncogene, 10: 205-209, 1995.

44. Fisch, P., Boehm, T., Lavenir, I., Larson, T., Amo, J,, Forster, A., and Rabbitts, T. H. T-cell acute lymphoblastic lymphonra induced in transgenic mice by the RBTN1 and RBTN2 LIM-domain genes. Oncogene, 7: 2389-2397, 1992.

45. Larson, R. C., Osada, H., Larson, T. A., Lavenir, I., and Rabbitts, T. H. The oncogenic LIM protein Rbtn2 causes thymic developmental abelrations that precede malignancy in transgenic mice. Oncogene, 11: 853-862~ 1995.

46. Neale, G. A., Rehg, J. E., and Goorha, R. M, Ectopic expression of rhombotin-2 causes selective expansion of the thymus and T-cell tumours in transgenic mice. Blood, 86: 3060-3071, 1995.

47. Larson, R. C., Lavenir, I., Larson, T. A., Baer, R., Wan'en, A. J., Wadman, I., Nottage, K., and Rabbitts, T, H. Protein dimerisation between Lmo2 (Rbm2) and Tall alters thymocyte development and potentiates T cell tumorigenesis in transgenic mice. EMBO J., 15: 1021-1027, 1996.

48. Grutz, G., Bucher, K., Lavenir, l., Larson, R., Larson, T., and Rabbitts, T. H. The oncogenic T cell LIM-protein Lmo2 forms part of a DNA-binding complex specifi- cally in immature T cells. EMBO J., 17: 4594-4605, 1998.

Discussion

S p e a k e r : C a n y o u tell u s . . . y o u said fo r l ack o f t ime y o u c o u l d n ' t

tell us the o the r c o m p o n e n t s o f the c o m p l e x in the L M O 2 t r ansgen i c

T cel ls . W h a t o the r f a c to r s are p r e s e n t in tha t E - b o x b i n d i n g c o m p l e x ?

Dr. Rabb i t t s : T h e o n l y f ac to r s tha t w e k n o w a b o u t at the p r e s e n t

t ime are L D B , E47 , and T A L l . W e are t a lk ing a b o u t c o m p l e x e s

w i th in the t r ansgen ic m o u s e T -ce l l t umor s . So , o f cou r se , that s i tua-

t ion is s l igh t ly ar t i f ic ia l , b e c a u s e w e a l r e a d y k n o w that the T A L l

p r o t e i n is p r e sen t there b e c a u s e o f the t r ansgene .

Dr. S t a n l e y K o r s m e y e r : I am w o n d e r i n g , do y o u th ink L M O 1 ,

L M O 3 w o u l d all be fri l ly subs t i tu tab le in t e rms o f the m e c h a n i s m ?

H a v e y o u had a c h a n c e to c o m p a r e t h e m wi th L M O 2 ?

Dr. Rabb i t t s : Thi s is a c o m p l e x q u e s t i o n ac tua l ly . W i t h L M O 3 , we

h a v e n ' t r ea l ly d o n e a n y t h i n g at all. T h a t p ro t e in , a l t h o u g h e x t r e m e l y

r e l a t ed to the L M O 1 p ro t e in ( the L I M d o m a i n s t h e m s e l v e s are iden-

t ical) has no t ye t b e e n i den t i f i ed as fa r as I am a w a r e in any c h r o m o -

s o m a l t r ans loca t ions , so w e h a v e r ea l ly i g n o r e d it.

W i t h L M O 1 , s o m e r e c e n t da ta on T - c e l l t u m o r s w h i c h e x p r e s s

L M O 1 h a v e s h o w n two things . O n e is tha t L M O 1 b inds to the

e n d o g e n o u s L D B pro te in . And , two , w e h a v e b e e n u n a b l e to f ind any

D N A b i n d i n g s i te f o r the L M O 1-assoc ia ted c o m p l e x . A n d that is w h y

I am b e g i n n i n g n o w to t h ink in t e r m s o f the s e q u e s t r a t i o n L D B p ro t e in

f u n c t i o n as b e i n g the m e c h a n i s m .

Dr. Ph i l l i p S h a r p : I f I u n d e r s t o o d it co r r ec t l y , y o u b e l i e v e y o u f o r m

an abe r r an t c o m p l e x w h e n L M O 2 is o v e r e x p r e s s e d in the T cel l tha t

m i g h t ac t iva t e genes tha t w o u l d n o r m a l l y no t be a c t i v a t e d u n d e r those

cond i t i ons . T h e d i r ec t p r e d i c t i o n o f tha t w o u l d be tha t y o u c o u l d do

p l u s / m i n u s s c r e e n i n g in t hose ce l l p o p u l a t i o n s and see n o v e l R N A s

that w o u l d a p p e a r o n l y in tha t p o p u l a t i o n c o m p a r e d w i th n o r m a l or

o the r t u m o r t ypes o f the s a m e t y p e bu t no t e x p r e s s i n g tha t c o m p l e x .

Has tha t e v e r b e e n l o o k e d at?

Dr. R a b b i t t s : T h o s e are o n g o i n g e x p e r i m e n t s , and w e h a v e n ' t

ac tua l ly b e e n ab le to i d e n t i f y any d i f f e r e n c e s . W e h a v e t aken a

s l igh t ly c o m p l i c a t e d rou t e by t r y ing to f r a c t i o n a t e the d o u b l e - n e g a t i v e

a s y m p t o m a t i c T ce l l s a w a y f r o m the d o u b l e - p o s i t i v e T cel ls and

c o m p a r i n g those t w o p o p u l a t i o n s . W e th ink the d o u b l e - n e g a t i v e cel ls

are the ones w h e r e the p a t h o g e n i c e f f e c t is o c c u r r i n g .

So, a l t h o u g h t h o s e e x p e r i m e n t s are i m p o r t a n t , w e j u s t h a v e n ' t go t

da ta yet .

1798s

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

1999;59:1794s-1798s. Cancer Res   Terence H. Rabbitts, Katharina Bucher, Grace Chung, et al.   DevelopmentLeukemias: The LMO2 Paradigm in Transcription and The Effect of Chromosomal Translocations in Acute

  Updated version

  http://cancerres.aacrjournals.org/content/59/7_Supplement/1794s

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/59/7_Supplement/1794sTo request permission to re-use all or part of this article, use this link

Research. on March 11, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from