Oncogene (2001) 20, 5763 ± 5777 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Chromosomal translocation master , mouse models and experimental therapeutics

Terence H Rabbitts*,1

1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

Molecular biologists have elucidated general principles but we used the technique to map the human K light about chromosomal translocations by cloning oncogenes chain genes to 2, band p12 (Malcolm et or fusion genes at chromosomal translocation junctions. al., 1982), using a variable region probe (which is These genes invariably encode intracellular and e€ectively multi-copy due to cross hybridization in acute cancers, often involve transcription and devel- between di€erent V genes (Bentley and Rabbitts, opmental regulators, which are master regulators of cell 1981)). The presence of antibody genes on 14q and fate (e.g. LMO2 which is involved in acute leukaemia). 2p coincided with positions of chromosomal transloca- Chromosomal translocations are usually associated with tion breakpoints in the Burkitt's lymphoma B cell speci®c cell types. The reason for this close association is tumour (Bernheim et al., 1981; Klein, 1981) suggesting under investigation using mouse models. We are trying to that rearrangement of Ig genes might be associated emulate the cell-speci®c consequences of chromosomal with these aberrant (Rabbitts, 1983). The translocations in mice using homologous recombination in demonstration that rearrangement of genomic frag- embryonic stem cells to generate de novo chromosomal ments of the CMYC oncogene coincide with Ig translocations or to mimic the consequence of these rearrangement clinched this proposal (Dalla-Favera et translocations. In addition, chromosomal translocation al., 1982; Taub et al., 1982) and subsequent cloning of genes and their products are important targets for translocated CMYC revealed many features of the therapy. We have designed new therapeutic strategies mechanism and consequences of chromosomal translo- which include antigen-speci®c recruitment of endogenous cations (reviewed in Rabbitts, 1994). The association of cellular pathways to a€ect cellular viability and a novel the rearranging antigen receptor genes with chromoso- structured form of antisense to ablate the function of mal translocations in leukaemia was taken further fusion mRNAs. We will evaluate these procedures in the when the T cell receptor (TCR) b chain was mouse models of chromosomal translocations and the mapped to chromosome 7, band q35 (Rabbitts et al., long term aim is to perfect rapid procedures for 1985), suggesting that chromosomal translocations in T characterizing patient-speci®c chromosomal transloca- cell tumours are mediated by the intrinsic chromoso- tions to tailor therapy to individual patients. Oncogene mal instability of TCR genes, which undergo rearran- (2001) 20, 5763 ± 5777. gement in normal lymphoid lineage development. This was con®rmed by cloning of LMO1 and LMO2 at Keywords: cancer; therapy; chromosomal transloca- chromosomal translocation junctions with TCRd and tion; cytogenetics; leukaemia; sarcoma TCRa (Boehm et al., 1988a, 1991; McGuire et al., 1989; Royer-Pokora et al., 1991) and of HOX11 and LMO2 at chromosomal translocations with TCRb (Boehm et al., 1991; Dube et al., 1991; Hatano et al., Introduction 1991; Kennedy et al., 1991; Lu et al., 1991). Cloning genes at chromosomal translocation breakpoints Lymphoid chromosomal translocations are linked to The molecular cloning of chromosomal translocation mechanisms of antigen receptor diversity junctions and of cDNA copies of genes a€ected by the chromosomal translocations resulted in a plethora of In the lymphoid tumours, the breakpoints of chromo- di€erent translocations being studied (reviewed in somal translocations occur within the Ig or TCR genes, Rabbitts, 1994). For lymphoid tumours, one of the exactly at the junctions of diversity (D) or joining (J) ®rst clues came from the mapping of the human segments, which is where the RAG-recombinase joins immunoglobulin (Ig) heavy chain locus to the long arm V genes during intra-chromosomal antigen receptor of chromosome 14 (Croce et al., 1979; Hobart et al., gene joining. Thus the VDJ recombinase enzyme is 1981). At the time, gene mapping by chromosome in most likely involved in creating these lymphoid-speci®c situ hybridization was restricted to multi-copy genes chromosomal translocations. The sequence speci®city on the reciprocal chromosome is less clear, although certainly some junctions do carry the hallmark of *Correspondence: TH Rabbitts; E-mail: [email protected] recombinase recognition signals and in those cases Chromosomal translocations and therapeutic strategies TH Rabbitts 5764 recombinase must be involved on both chromosomes. These questions will be analysed in this article. Further, in Burkitt's lymphoma the translocated CMYC gene shows somatic mutations (Rabbitts et al., 1983, 1984) in a manner analogous to mutations of Chromosomal translocation master gene model: 10 years V genes further suggesting that lymphoid chromosomal translocations involve natural genetic instability of Ig Early analysis of gene structure after chromosomal genes. Indeed some Burkitt's lymphoma cell lines have translocations had occurred showed that either gene been identi®ed in which the process of somatic V gene fusion or oncogene activation (enforced expression or CMYC mutation can still be observed (Bemark and due to the new chromosomal environment) had Neuberger, 2000; Sale and Neuberger, 1998). We resulted (reviewed in Rabbitts, 1994). Further, it conclude that chromosomal translocations into Ig/ became clear in the early 1990s that transcription TCR genes occur by mistakes of mechanisms evolved factors were a frequent target of chromosomal for diversi®cation of the immune system. translocations (Cleary, 1991; Rabbitts, 1991) but that a clear distinction could be drawn between chromo- somal translocations in chronic and acute leukaemias Chromosomal translocations and sarcomas (Rabbitts, 1991) and only the latter category involved Are chromosomal translocations involving speci®c transcription factors. This led to the chromosomal oncogenes restricted to tumours of haematopoietic translocation-master gene model (Rabbitts, 1991), origin? Whilst epithelial tumours (which comprise which postulated that transcription regulators and 495% of human tumours) have few recorded developmental regulators were the major target of recurring, balanced chromosomal translocations (see chromosomal translocations, and that their enforced http://cgap.nci.nih.gov/Chromosomes/Mitelman), sar- expression or gene fusion, altered the balance of comas (mesenchymal tumours) have well characterized transcription thereby a€ecting the control of cell fate abnormalities (see Rabbitts, 1994). We set out to clone in the a‚icted cell. The chromosomal translocation- an example of this group, namely the chromosomal genes in acute cancers are upstream master genes translocation t(12;16)(q13;p11) from malignant myxoid whose products control downstream pathways. There- liposarcoma and found that the chromosomal translo- fore, a major part of the tropism of chromosomal cation-junctions occurs in a gene on chromosome 16 translocations is due to the speci®city of transcription which we called FUS (Rabbitts et al., 1993) (also called factor interactions which occur after the chromosomal TLS; Crozat et al., 1993) and the CHOP gene on translocation and the consequent e€ect on transcrip- . This resulted in a fusion gene, tion pathways. The essential role of chromosomal analogous to the BCR ± ABL gene which had ®rst translocation genes in acute cancer is in cell fate been described in chronic myelogenous leukaemia (de decisions, perhaps o€ering an explanation for the Klein et al., 1982), encoding an FUS ± CHOP . scarcity of speci®c chromosomal translocations in This observation con®rmed that chromosomal translo- epithelial tumours, which may not be in¯uenced by cations in mesenchymal tumours cause fusion proteins master regulators in this fashion. and functional studies of the FUS ± CHOP protein suggested that a DNA-binding transcriptional activator The LMO2 gene and its protein had been created out of a repressor (Sanchez-Garcia and Rabbitts, 1994), adding verisimilitude to the The LMO2 gene in T cell acute leukaemias (T-ALL) is notion that transcription factors are a major target of a paradigm of a chromosomal translocation-master chromosomal translocations. gene (reviewed in Rabbitts, 1998). LMO2 belongs to a family of four genes encoding small LIM-only proteins, two of which are involved in independent chromoso- Questions mal translocations causing T-ALL (Table 1). LMO2 is The consistent occurrence of chromosomal transloca- located on , band p13 and is activated tions in human tumours has been established from by chromosomal translocations with 14q11 or 7q35 many studies. Several important questions were raised (Figure 1a), speci®cally in T cells. The breakpoints of by this earlier work: these chromosomal translocations occur upstream of the natural LMO2 promoter(s) causing enforced . Can any general principles be enumerated about the expression of LMO2 protein in cells with the type of gene activated by chromosomal transloca- translocation (the protein product per se is not a€ected tions? by this). The three exon gene is transcribed and . Why are speci®c chromosomal translocations found translated into a 156 aa protein, comprising two zinc- in speci®c cell types (tumour tropism)? binding LIM domains, each with two LIM ®ngers . Can mouse models of chromosomal translocations (Figure 1b). While the LIM domain is structurally be developed? related to the GATA Zn-®nger, the characteristic of a . Since chromosomal translocations are somatically LIM domain is the separation of each LIM Zn-®nger created, and thus are tumour-speci®c within an indi- by 2 aa only. The function of the LIM domain is in vidual patient, can they or their products be used as protein-protein interactions (Schmeichel and Beckerle, therapeutic targets? 1994; Valge-Archer et al., 1994; Wadman et al., 1994)

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5765 which is a consistent feature of the chromosomal Table 1 LMO family of LIM-only genes translocation-master gene products. Chromosome Chromosomal Leukaemia LMO2 can bind to TAL1/SCL (another T-ALL Gene Man Mouse translocation type translocation associated protein), LDB1 and GATA-1 LMO1 11p15 7 t(11;14)(p15;q11) T-ALL in a DNA-binding complex found in erythroid cells LMO2 11p13 2 t(11;14)(p13;q11) T-ALL (Wadman et al., 1997). This complex can bind to a t(7;11)(q35;p13) bipartite DNA site, comprising an E box separated LMO3 12p12-13 6 none described / from a GATA site by about 12 base pairs (Figure 1c). LMO4 1p22.3 3 none described By studying T cell tumours arising in an Lmo2-gain-of- The LMO gene family (LiM-Only genes and previously called RBTN function transgenic mouse model, we have been able to and TTG genes) has four known members. LMO1 (previously detect an analogous Lmo2-associated complex, in this RBTN1/TTG1) was identi®ed ®rst and then LMO2 (previously case which binds a bipartite sequence comprising RBTN2/TTG2) and LMO3 (previously RBTN3 (Rabbitts, 1998) and LMO4. LMO1 and LMO2 are both located on the short arm of tandem E boxes. The putative complex binding to this chromosome 11 and are both involved in independent chromosomal site is shown in Figure 1c. These observations lead to translocations in human T cell acute leukaemia. LMO2 is the most two conclusions. Firstly, as Lmo2 appears to be a commonly involved in chromosomal translocations and can be bridging component of the protein complex detected in activated by association with TCRg, TCRa (14q11) or TCRb erythroid cells, it seems possible that the components (7q35). As yet, LMO3 or LMO4 have not been found in association with any chromosomal translocations of the Lmo2-complex might vary through-out haema- topoietic di€erentiation, controlling various, stage- speci®c functions by regulating sets of genes. A

Figure 1 LMO2-associated chromosomal translocations, gene and protein. The LMO2 gene was discovered by cloning the breakpoints of chromosomal translocations t(11;14)(p13;q11) and t(7;11)(q35;p13) (Rabbitts, 1998). (a) The breakpoints of the chromosomal translocations t(11;14)(p13;q11) occur within the TCRd and TCRa genes on chromosome 14 and upstream (5')of LMO2 on chromosome 11. In the variant chromosomal translocation t(7;11), the chromosome 7 break occurs within the TCRb gene. All known breakpoints occur upstream of one of the two promoters (Royer-Pokora et al., 1995) controlling the LMO2 gene. The three exon gene codes for a protein of 156 amino-acids. (b) The LMO2 protein comprises two LIM domains with small upstream and downstream segments (Boehm et al., 1990, 1991). Each LIM domain has two LIM ®ngers, each with a zinc atom co- ordinated between cys and his residues. The characteristic feature of the LIM domain is two amino acids separating the LIM ®ngers. (c) The role of the LIM domain is in protein-protein interaction. LMO2 binds to TAL1/SCL (a helix ± loop ± helix protein, which in turn binds to the helix ± loop ± helix protein E47), to LDB1 and to GATA-1 in a multi-protein complex able to bind a bipartite DNA sequence comprising an E box (CANNTG), and a GATA site, (Wadman et al., 1997) in erythroid cells. The LMO2 and LDB1 molecules appear to bind as bridging components, although the exact nature and stoichiometry of this complex has not be elucidated. The ability of the LMO2-containing complex to bind DNA indicates a role in control (positive or negative) of target . If other complexes, involving Lmo2 occur at other stages of , these might bind and control distinct sets of target genes. An analogous complex was found in T cell tumours arising in CD2-Lmo2 transgenic mice (Grutz et al., 1998). The complex binds to a bipartite DNA site but di€erent from the one found in erythroid cells (in this case, two adjacent E boxes) and composed of di€erent proteins. The T cell tumour complex appears to be involved in control of gene expression, which has a primary e€ect on T cell di€erentiation (see Figure 4)

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5766 corollary is that Lmo2 has distinct roles in control of promoter, CD2 (Fisch et al., 1992) (Figure 4a). This haematopoietic cell fate. Secondly, the role of LMO2 resulted in transgenic mice which develop clonal T cell in T-ALL is probably by enforced formation of protein tumours with a long latency (average 9 months) but in complexes (the direct result of enforced LMO2 large proportion of mice (Figure 4b) (Larson et al., expression after the chromosomal translocation). Thus 1994, 1995, 1996). The long latency implies that the gene regulation via LMO2-complexes may be a€ected; is necessary but not sucient for overt this may occur by direct DNA-binding complexes or tumour development and that mutations in other genes by sequestration of proteins from their normal roles must occur to compliment the e€ect of the transgene. (Rabbitts, 1998). The nature of these secondary mutations is obscure at present but one source of the changes has been ruled out, viz. RAG recombinase-mediated mutations. It has The LMO2 master gene: a regulator of cell fate in been shown that RAG can cause transposition as well haematopoiesis and vascular formation as intra-chromosomal rearrangements (Agrawal et al., The role of Lmo2 in controlling cell fate in normal 1998; Hiom et al., 1998) raising the possibility that it haematopoiesis has been documented mainly by gene could also make mutations. If so, these mutations do targeting experiments (summarized in Figure 2). The not appear to be the ones needed in the transgenic generation of null mutation of the Lmo2 gene mouse model because the rate and incidence of demonstrated that the gene is necessary for primitive tumours in the transgenic mice is identical in the erythropoiesis in mouse embryogenesis (Figure 2a) presence or absence of the RAG1 gene (Figure 4b). (Warren et al., 1994) and for de®nitive haematopoiesis An important feature of the latency period in the (Yamada et al., 1998). The latter may be at the level of Lmo2-transgenic mouse model is that we observed the the pluripotent stem cell, at the level of the immediate accumulation of CD47, CD87 double negative (DN multi-potential progeny or even perhaps before this, cells) T cell precursors in the thymus of these mice when mesoderm gives rise to these precursors (Figure (Larson et al., 1994, 1995, 1996). These DN cells are 2a). Furthermore, Lmo2 has a speci®c intraembryonic the precursors of the mature double positive (DP cells) expression pattern, in endothelial cells and blood T cells and their developmental is inhibited in the progenitor cells (Figure 3) (Delassus et al., 1999; thymus by enforced expression of Lmo2. The overt T Yamada et al., 2000). In mouse embryogenesis, cell tumours appear to arise from this population of haemangioblasts (putative common precursors of DN cells since we observe comparable T cell tumour endothelium and blood cells) are thought to arise from formation in RAG17/7; CD2-lmo2 (RAG7/7 mice unspeci®ed posterior mesoderm and thus the primary have a complete block at the DN stage; (Fischer and capillary network is made from these haemangioblasts. Malissen, 1998) and RAG+/7 CD2-lmo2 transgenic The ®rst process of vasculature construction, in which mice (Drynan et al., 2001). the capillary network is formed, is called vasculogen- These data suggest a model in which enforced esis. The more mature vascular system is made by the expression of Lmo2 has an e€ect on T cell develop- remodelling of primary capillary network, in the ment within the thymus (Figure 4c) without a process called angiogenesis (Hanahan and Folkman, signi®cant e€ect on thymus cellularity and these DN 1996). Again use of null mutations of the Lmo2 gene T cell precursors are the subject of mutations, which (Yamada et al., 2000), showed that formation of the eventually accumulate in clones to result in the clonal primary capillary network does not require Lmo2 but T cell tumours appearing. This mouse model is highly re-modelling into mature vessels does. Thus Lmo2 is relevant to the mechanism of T-ALL occurrence by necessary for angiogenesis, but not for vasculogenesis. LMO2 enforced expression following chromosomal In summary, Lmo2 is expressed in both endothelial translocation. The site of the chromosomal transloca- cells and blood progenitor cells, and has dual functions tion breakpoints occur within TCR genes and speci®c in controlling cell fate in haematopoiesis and angiogen- VDJ recombinase recognition-like sequences are found esis. These processes seem to be regulated via the at the breakpoints near the LMO2 gene (Boehm et al., ability of the Lmo2 protein to participate in multi- 1988b; Sanchez-Garcia et al., 1991) suggesting that protein complexes a€ecting gene expression. RAG recombinase mediates the chromosomal translo- cations. Furthermore, recombinase is expressed in the human T cell DN precursors analogous to those which The LMO2 master gene: a regulator of cell fate in T cell we observed to accumulate as a result of Lmo2-gain- leukaemogenesis of-function in our transgenic mice (Larson et al., 1995). LMO2 is a master gene in the haematopoietic lineage. It seems very likely that this is the population of T cells A role in cell fate decisions accounts for the function of in which the recombinase-mediated chromosomal LMO2 in T-ALL development after chromosomal translocations occur in humans (Figure 4c). The translocation (Rabbitts, 1998; Rabbitts et al., 1999). chromosomal translocation provides the cells for overt A gain-of-function transgenic mouse model of T cell disease to occur following further mutations in other expression of Lmo2 speci®cally in T cells has been oncogenes or suppressor genes. It is a corollary that in informative. Two features of this mouse model are humans, chromosomal translocations of this type may important (Figure 4). Lmo2 gene expression was not always result in overt cancer, as other mutations enforced in the T cell lineage using a T cell speci®c may not always occur.

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5767

Figure 2 Lmo2 is necessary for adult haematopoiesis and angiogenesis in mice. The LMO2 gene is a master regulator of cell fate in haematopoiesis as shown by mutational analysis in mice. (a) Lmo2 null mutations introduced into the germ-line of mice cause lethality at E9-10 due to a failure of yolk sac erythropoiesis (Warren et al., 1994). In addition, the contribution of embryonic stem cells with homozygous null mutation of Lmo2 to haematopoietic cell populations showed that de®nitive haematopoiesis in adult mice is dependent on Lmo2 (Yamada et al., 1998). Thus the Lmo2 gene product is required for both embryonic and adult haematopoiesis in mice. (b) The formation of haematopoietic cells and vascular endothelium is thought to occur through a putative precursor cell, the haemangioblast, which derives from the posterior mesoderm. Formation of the capillary network de novo (vasculogenesis) precedes a remodelling of the existing endothelium (angiogenesis). Studies with the Lmo2-lacZ null ES cell derivatives in chimaeric mice showed that the Lmo2 gene is necessary only for the latter process of angiogenesis (Yamada et al., 2000). The failure of adult haematopoiesis may be due to the failure of angiogenesis in null Lmo2-cells as haematopoietic cells may arise in large blood vessels which do not form in the absence of Lmo2. An alternative origin for haematopoietic cells is directly from haemangioblasts

Mouse models of chromosomal translocations timing and cell speci®city of the translocation and its biological outcome. The gain-of-function Lmo2- A key issue about the role of chromosomal transloca- mediated model discussed above was informative as tion-genes is their tumour tropism i.e. why are speci®c the outcome was dictated by tissue speci®c expression chromosomal translocations mainly found in one cell (i.e. expression only in T cells). Such speci®city is not type, e.g. t(11;14)(p13;q11) in T-ALL, t(9;11)(q21;q23) always easily achieved. Accordingly, we have evaluated in acute myeloid leukaemia or t(12;16)(q13;p11) in the use of homologous recombination to create the myxoid liposarcoma? The reason may simply be equivalent of chromosomal translocation-genes in ES mechanistic in that speci®c chromosomal transloca- cells followed by generation of mice carrying these tions may only take place in certain cell types where targeted genes. accessibility allows for inter-chromosomal events between genes with `open' chromatin. Alternatively, An Mll-Af9gene fusion knock-in tumorigenesis model the translocation-gene product may exert a critical e€ect on a speci®c cell type by altering cell physiology. The ®rst generation constructs were designed to Given that `master-gene' targets of chromosomal introduce cDNA sequences into a target gene by translocations are transcription and development homologous recombination (the gene fusion knock-in regulators, the latter seems most likely. Mouse models approach; Corral et al., 1996) and we chose to of tumour formation are valuable for investigating this introduce AF9 sequences in the Mll gene (Figure 5a). problem but also in obtaining information about initial The choice of MLL fusion genes as a test system for stages of disease which might ultimately be valuable for the knock-in technology was based on two main early detection or treatment. Ideally, the model should features of MLL-associated chromosomal transloca- emulate as closely as possible the e€ects of the tions in humans. Firstly, MLL is involved in a large equivalent human chromosomal translocation, both in spectrum of di€erent fusions in all types of haemato-

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5768

Figure 3 Lmo2 expression in vascularization of mouse embryos. A targeted ES clone with one null Lmo2 allele, in which the lacZ gene was knocked-in as a gene fusion with Lmo2, was injected into blastocysts and germ-line transmission of the Lmo2 null allele was obtained (Yamada et al., 2000). Heterozygous mice were crossed with C57/B16 mice and embryos obtained at E10.5 and E12.5. These embryos were stained with Xgal as a substrate for b-galactosidase activity. Blue staining denotes areas of b-galactosidase due to Lmo2-lacZ gene expression. (a) E10.5 embryo. X-gal staining was seen on major blood vessel walls and capillaries of whole body. (b) E12.5 embryo. In addition to b-galactosidase staining of vasculature, prominent staining was found in the limb buds and the tip of tail. (c) Histological section of an E12.5 embryo stained for b-galactosidase and counter-stained with eosin. Blood vessel endothelial cells can be observed in a background of eosin stained tissue. (d) Histological section of the limb bud of an E12.5 embryo stained for b-galactosidase and counter stained with eosin. The region beneath the apical ectodermal ridge of a limb bud is b-galactosidase positive

poietic cells and a model system for this gene would translocation. At face value, these data argue that help assess the various fusion genes thus obtained. the the Mll-Af9 fusion gene, which is present in all Secondly, the most common MLL-associated chromo- haematopoietic cells, including the stem cells, has a somal translocations involving AF9 and AF4 genes specifying role in the cellular phenotype of the appear in myeloid and lymphoid tumours respectively, leukaemia, in turn implying that the Mll-Af4 fusion thereby predicting clearly distinct tumour phenotypes gene would specify lymphoid leukaemia. However, we in mouse models. Accordingly, in our ®rst approach, cannot exclude a default pathway which leads to we knocked-in AF9 into one allele of Mll in ES cells myeloid leukaemia when damage is done to the Mll and made mice heterozygous for the Mll-Af9 fusion gene. Indeed some support for this proposal was gene (Figure 5b) (Corral et al., 1996). This is an obtained when we observed myeloid leukaemias arising imperfect system because the fusion gene is present in in mice with a knock-in of the lacZ gene into Mll the germ-line of the mice and thus does not give the (Dobson et al., 2000), suggesting loss of the 3' part of speci®c haematopoietic expression found for the Mll or release of the 5' part from the 3' part may be naturally occurring MLL-AF9 fusion after chromoso- crucial. In developing a ®nal model, we must have mal translocation. Although this did not seem to be a results with the Mll-Af4 fusion as this is invariably problem for Mll-Af9, we were unable to use the same found in lymphoblastic leukaemias in humans and it is approach with an Mll-Af4 fusion suggesting that there a crucial test for these mouse models. may be a cell non-autonomous e€ect of Mll-Af4 in embryogenesis. De novo chromosomal translocations in mice using Mll-Af9 knock-in mice developed myeloid leukae- cre-loxP recombination mias (Figure 5c) with a latency of about 6 months (Corral et al., 1996; Dobson et al., 1999). The features An alternative approach to mimicing chromosomal of the disease found in mice paralleled those in translocations is the use of the Cre-loxP recombination humans where the t(9;11) chromosomal translocation system in ES cells to induce de novo chromosomal predominates in myeloid rather than in lymphoid translocations (Smith et al., 1995; van Deursen et al., leukaemia (Dobson et al., 1999). This outcome 1995). This has the advantage that tissue speci®c indicates that the Mll-Af9 fusion gene created by the expression of the Cre recombinase would allow the knock-in approach was mimicing the relevant, key translocation to occur in a tissue speci®c manner and, features of the pathological e€ects of the t(9;11) in addition, the de novo chromosomal translocation

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5769

Figure 4 Tumorigenesis in an Lmo2 gain-of-function transgenic mouse model of T-ALL. (a) A gain-of-function transgenic mouse model creating enforced Lmo2-expression in T cells using the CD2-promoter/enhancer has been described (Fisch et al., 1992; Larson et al., 1994). These mice have thymus expression of Lmo2.(b) Mice carrying the CD2-lmo2 transgene develop clonal T cell tumours with a long latency indicating that mutations in other genes are required before the development of overt disease and therefore that the transgene is necessary but not sucient for tumour formation in this model. During the pre-symptomatic period, transgenic mice displayed an accumulation of immature CD47, CD87 double negative (DN) T cells precursors in the thymus (Larson et al., 1995, 1996). This population of T cells expresses RAG VDJ recombinase genes but these are not responsible for the secondary mutations as CD2-lmo2 mice develop tumours at the same rate whether or not these are null for the RAG1 gene (Drynan, Hamilton and THR, submitted). (c) As the DN T cells are the target cells in the transgenic mouse model and as the chromosomal translocations in humans are mediated by VDJ recombianse, the DN cells are likely to be the target cells in which the chromosomal translocations are e€ective in aetiology of the human disease, and from which the overt leukaemias arise

would yield both reciprocal chromosomes. We have relevant chromosomes in mice behave like their human initiated studies in this area and have described counter-parts and that the organization of Mll and Af9 preliminary results with a mouse model in which de genes on their respective chromosomes is appropriate novo chromosomal translocations are made between for production of the Mll-Af9 fusion mRNA. In order the endogenous Mll and Af9 genes (Collins et al., to see e€ects on tumour development and tropism of 2000). Our strategy for making the Mll-Af9 translo- chromosomal translocations, however, it will be cator mice is summarized in Figure 6. Homologous necessary to express the Cre recombinase in a tissue recombination was used to introduce loxP recombina- and cell-speci®c manner during haematopoiesis, to tion sites, which are the substrates for Cre recombi- harness the ability of chromosomal translocations to nase, into Mll (mouse chromosome 9) and Af9 (mouse occur in di€erent cell types. In this way, we hope to chromosome 4) genes. These mice were crossed with a determine whether tumours arise in a tissue-speci®c transgenic line expressing Cre recombinase to facilitate manner and we are currently assessing its e€ects on development of inter-chromosomal translocations. The cellular di€erentiation and tumorigenesis. Finally, reciprocal translocation junctions were detectable in other Cre-loxP mediated strategies are also under mice carrying Mll-loxP and Af9-loxP and expressing evaluation in an attempt to ®nd a generic approach Cre recombinase. We conclude that Cre-dependent, to mimicry of tumour-associated chromosomal trans- loxP mediated inter-chromosomal recombination has locations in mice. occurred in the translocator mice creating the derivative der4 and der9 chromosomes during their development (Figure 6). Experimental therapeutics applied to chromosomal Our initial objective was to determine if it were translocation products possible to obtain and detect inter-chromosomal translocation in mice using Cre recombinase expressed The presence of chromosomal translocations in human from a transgene. The ®rst demonstration of this using tumours is both a diagnostic and prognostic feature. In Mll and Af9 genes (Collins et al., 2000); means that the addition, these well characterized mutations occur

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5770

Figure 5 Fusion of Mll and AF9 genes by homologous recombination knock-in. The MLL gene is involved with more than 30 distinct chromosomal translocations in human leukaemias. In order to assess the potential role of the fusion proteins in the tropism of these leukaemias, we established an homologous recombination knock-in strategy for creating the Mll-Af9 gene fusion in mice (Corral et al., 1996). (a) A targeting vector of humanAF9 cDNA sequences fused to exon 8 of mouse Mll was made to facilitate recombination with the Mll gene in ES cells. Homologous recombination would create a fusion of AF9 in one Mll allele, allowing transcription of an Mll-AF9 fusion mRNA and in turn production of Mll-AF9 fusion protein. (b) Targeted ES cells were identi®ed and injected into blastocysts for production of chimaeric mice and breeding to produce heterozygous carriers of the Mll-AF9 fusion gene. (c) Leukaemias arise in both chimaeric and heterozygous Mll-AF9 knock-in mice. The latency was around 6 months and most mice succumb by 14 months. The majority of tumours were Mac-1+, Gr-1+ myeloid tumours (Dobson et al., 1999), similar in phenotype to the majority of tumours found human leukaemias with t(9;11)

somatically and thus the translocated chromosomes, disulphide bonds can be deleterious for intracellular the a€ected genes, their transcripts or translation Folding. As an approach to overcome these problems, product are possible targets for therapy. Although, we have developed a method to isolate antibodies, in highly speci®c to tumour cells, straightforward anti- the form of single chain Fv fragments (scFv), which are body therapy is not an option since chromosomal functional in vivo as intracellular antibodies (ICAbs) translocation-genes and their products are intracellular. and can be expressed, or otherwise introduced into This is a problem for design of therapies. We have cells, to bind their target antigen. As scFv are not well begun to develop experimental therapeutic strategies suited to intracellular folding and stability, we initially for these intracellular targets, with speci®city of found that very few scFv bind to antigen in vivo. This interaction as the main goal. We have described two de®ciency indicated the need for an in vivo screening approaches, one using the recruitment of an endogen- approach and an antibody-antigen interaction screen ous cellular pathway (i.e. the apoptosis pathway) via was developed (Intracellular Antibody Capture Tech- recognition of a chromosomal translocation-antigen nology, IACT) which allows direct selection of (Tse and Rabbitts, 2000) and another using a novel intracellular antibodies (scFv) (Tse et al., 2001; Visintin structured or masked antisense RNA approach (Stocks et al., 1999). The technology uses a three-step approach and Rabbitts, 2000). (Figure 7). First soluble antigen is displayed on a surface and a phage antibody library (Winter et al., 1994) is panned on the antigen. All phage binding to Selection of intracellular antibodies for binding the surface are recovered (Figure 7a), the DNA chromosomal translocations products isolated and the scFv cloned in a yeast expression Chromosomal translocations often result in tumour- vector as a fusion with the VP16 activation domain. speci®c fusion proteins (tumour antigens) which are This yeast `sub-library' comprises antigen-speci®c intracellular targets. While antibodies have evolved to ICAbs together with contaminating ICAbs which bind to antigen with high speci®city, they usually derive from the non-speci®c background. The yeast function outside cells and further the presence of scFv-VP16 fusion clones are screened with an antigen

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5771 the presence of antigen in the cell and not on its function per se. In our ®rst approach, a technique for speci®c cell killing was developed (the Antibody- antigen Interaction Dependent Apoptosis, Aida, meth- od) in which tumour cells are induced to commit suicide following binding of ICAb scFv to an intracellular antigen. In a model system, we have demonstrated that scFv fused to caspase 3 can cause auto-activation of the caspase 3 after binding to antigen, subsequently causing cell suicide (Tse and Rabbitts, 2000). The rationale is that caspase 3 (the so- called executioner of the programmed cell death or apoptosis pathway) exists as an inactive pro-caspase and on dimerisation can be induced to auto-cleavage (MacCorkle et al., 1998) and activation of downstream caspases of the endogenous programmed cell death or apoptosis pathway (Figure 8b). Therefore, if dimerisa- tion of exogenous caspase 3 could be induced by antigen, we can recruit endogenous downstream caspases and kill the cell. We have achieved this in our Aida method by expressing in tumour cells ICAb as scFv fused to pro-caspase 3 (Figure 8c). Binding to antigen inside cells with appropriate localisation of two Figure 6 The Mll-Af9 translocator mouse. Mouse Mll and Af9 independent scFv-caspase 3 molecules induces apopto- genes are located on chromosomes 9 and 4 respectively (Collins et al., 2000; Ma et al., 1993). The mice which carry both Mll and sis (Figure 8d). Af9 chromosomes with loxP sites introduced by homologous Our model Aida experiment employed b-galactosi- recombination can be induced to undergo inter-chromosomal dase as antigen which is known to be a natural recombination by expressing Cre recombinase, to generate the tetramer (Jacobson et al., 1994) and an anti-b gal ICAb derivative 9 and derivative 4 chromosomes. This translocator mouse line can be maintained with the genotype (Mll-loxP/loxP; scFv (scFv-R4; Martineau et al., 1998) which binds to Af9 loxP/loxP) and therefore tissue-speci®c chromosomal translo- antigen in vivo but does not neutralize its activity. The cations are inducible by inter-breeding with mice expressing Cre rationale was that two or more scFv-caspase 3 in a speci®ed way. For instance, we have found T cell-speci®c molecules should bind per antigen tetramer and initiate inter-chromosomal recombination of Mll and Af9 in mice apoptosis through the regional association and activa- expressing Cre under the control of the Lck T cell promoter (unpublished data) tion of the caspase molecules (Tse and Rabbitts, 2000). Apoptosis was detected in CHO cells transfected with antigen and R4-scFv-caspase 3 as determined by appearance of both the caspase 3 cleavage product bait expressed in yeast as a fusion with the Lex A and of the chromosomal DNA cleavage ladder typical DNA-binding domain to separate speci®c ICAbs from of cells undergoing apoptosis (Tse and Rabbitts, 2000). non-speci®c ones (Figure 7b) (Visintin et al., 1999). This e€ect is antigen-dependent, antibody-speci®c and Finally the selected scFv are veri®ed for binding to required active caspase 3 fused to the ICAb (Figure 9). antigen in a mammalian cell assay. Thus while most Thus cell suicide or apoptosis is caused if antibody, in randomly selected scFv do not bind antigen in vivo the form of scFv physically linked to caspase 3, binds with sucient anity to be useful, those isolated by to antigen in vivo in two or more close positions. the IACT can be used as intracellular reagents. This method adds e€ector functions to the ICAbs and has several important potential uses in cancer therapy targeting chromosomal translocation gene Effector functions on ICAbs: recruitment of cellular products. If two antibodies could be isolated which pathways for therapy and the Aida strategy can bind to sites on a fusion protein (Figure 8d), on An ideal method of cancer treatment would be to either side of the fusion junction, then tumour speci®c selectively destroy tumour cells whilst sparing the cell killing should result. Furthermore, cells expressing normal counterparts. One approach is to recruit oncogenic proteins with mutations could be targeted if endogenous cellular pathways to elicit a response on a mutant-speci®c scFv could be expressed or intro- a target cell, mediated through a target antigen duced in the tumour cells along with a second scFv- speci®cally in that cell (e.g. a chromosomal transloca- caspase molecule binding to a site close enough to the tion fusion protein). There are several endogenous anti-mutant scFv-caspase to allow auto-activation of pathways which could be invoked including apoptosis, the caspase. In those mutant proteins which form proteolysis, signal transduction and transcription path- natural tetramers, such as , it may only be ways (Figure 8a). We have developed a recruitment necessary to introduce one scFv-caspase as the strategy using binding of ICAbs to target antigens to apoptosis induction should occur in a manner activate an endogenous pathway which relies only on analogous to that described for b-galactosidase. The

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5772

Figure 7 Strategy for the selection of antigen-speci®c intracellular antibodies. (a) A phage library displaying a repertoire of scFv is screened in vitro with an antigen coated onto a surface. This enriches for those phages displaying the speci®c scFv by binding to the antigen. (b) The phagemid DNA from the bound phage in (a) is prepared and the scFv DNA fragments are sub-cloned into the yeast VP16 expression vector (Visintin et al., 1999) to give a yeast scFv-VP16 library. The library is screened in yeast with the antigen `bait' in which antigen (shown in blue) is expressed as a fusion with the LexA DNA-binding domain (shown in orange). Only those scFv that retain the speci®c binding ability in vivo can activate LexA DNA binding site reporters controlling His3 and LacZ genes. These are isolated and tested in mammalian cell lines with appropriate assays for intracellular binding and function

latter might also apply to situations like AML ± ETO/ short sequence of eight nucleotides (the nucleation MTG8 and PML ± RAR which oligomerize in tumour targeting region) which serves to speci®cally hybridize cells (Minucci et al., 2000). to the ABL side of the BCR ± ABL mRNA junction. Stem/loop II comprises BCR antisense sequences masked in the form of a stable stem loop. This stretch Masked antisense of antisense can only begin to hybridize to the BCR Fusion genes made by chromosomal translocations are mRNA immediately adjacent to the BCR ± ABL transcribed into the corresponding mRNA and the junction when the masked antisense hybridizes to junction between sequences, originating from the target BCR ± ABL mRNA via the nucleation site. exons of the fused genes, represents a unique There is then progressive hybridization of antisense sequence, only found in the mRNA of cells carrying and sense by the thermodynamically favourable process the chromosomal translocation. Short anti-sense of branch-migration. As this proceeds, a longer and oligonucleotides have been designed to target this longer hybrid is formed until a fully stable duplex is sequence and cause mRNA degradation or prevent created. The masked antisense cannot hybridize to protein synthesis from the fusion mRNA. The use of normal BCR mRNA as the BCR antisense is stably short oligonucleotides in vivo has some problems. They masked in stem/loop II and also cannot stably have a limited longevity and form rather unstable hybridize to normal ABL mRNA. hybrids in contact with the target mRNA but, in A masked antisense molecule was assessed for its particular, the hybridization speci®city of short ability to bind to various target RNAs in vitro (Figure oligonucleotides is a potential problem (Wagner and 10b, c). Our data show that only the cognate target Flanagan, 1997). We have developed a procedure RNA was recognized in such a way that a stable which both retains the speci®city of the short hybrid formed between it and the masked antisense. In oligonucleotide binding to the fusion mRNA junction addition, preliminary experiments using expressed and incorporates a ®nal stable hybrid between masked antisense molecules further indicated that oligonucleotide and fusion mRNA (Stocks and these species are expressed, fold adequately and can Rabbitts, 2000). ®nd their target mRNA for hybrid formation in vivo The procedure uses a masked antisense molecule (Stocks and Rabbitts, 2000). The ecacy and designed to target the BCR ± ABL fusion mRNA as a speci®city of masked antisense makes these novel model. The structure of this antisense molecule is RNAs uniquely suited to therapy involving targeting shown in Figure 10a. There are two hair-pin structures to fusion mRNAs emanating from chromosomal (designated stem/loops I and II) between which is a translocations.

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5773

Figure 8 Recruitment of endogenous cellular pathways for therapeutics: cell death mediated by the Aida procedure. (a) Multi- protein complexes are involved in many cellular activities such as signal transduction, transcription, proteolysis and apoptosis. These pathways can be recruited and activated by introduction into cells of one or more exogenous components linked to antigen-speci®c moieties, such as intracellular antibodies. The pathways can thereby be controlled by interaction of intracellular antibodies with a target antigen. (b) An example of this strategy is the Aida method (Antibody-antigen Interaction Dependent Apoptosis). Apoptosis or programmed cell death is carried out by activated caspases which form an irreversible cascade leading to cell destruction. A central enzyme in this process is caspase 3 (the `executioner' of the pathway) which is present as a pro-caspase and which can be activated by dimerization, resulting in auto-cleavage and cell death. (c) The Aida strategy comprises linking of pro-caspase 3 to antigen-speci®c scFv as shown and expressing pairs of scFv-CP3 fusions in cells with target antigen. (d) If there are two close binding sites on a single target antigen molecule, auto-activation of caspase 3 results from dimerisation and in initiation of apoptosis. This process is antigen dependent, antibody-speci®c and caspase dependent

Conclusions . Translation of experimental therapeutics into clinical management of cancer. Our work has been aimed at de®ning the types of gene activated by chromosomal translocations and using one example particularly, viz. the LMO2 gene, to illustrate Definition of the role of chromosomal translocations in some key features of master regulators controlling cell tumour cell type fate in normal and tumour settings. We have begun to evaluate the potential for the chromosomal transloca- An understanding of the molecular basis of chromo- tion gene products as therapeutic targets. In addition, somal translocation tropism is an important goal. Why we are developing mouse models of chromosomal do particular chromosomal translocations occur in translocations to study basic biological cell-speci®c speci®c cell types, and how do these chromosomal consequences of chromosomal translocations and as translocations work at the molecular level? In acute experimental therapeutic systems for pre-clinical assess- cancers, chromosomal translocation-genes are up- ment of therapeutic strategies and reagents. stream master genes encoding transcription factors I would like to highlight four areas where future and enforced expression of oncogenes/fusion oncogenes research and development will be valuable. These are cause cell fate/developmental abnormalities. Therefore, inter-connected and should yield important molecular a major part of the tropism of these chromosomal information about chromosomal translocations to help translocations is due to the speci®city of transcription devise new therapies: factor interactions which ensue after the chromosomal . De®nition of the role of chromosomal translocations translocation and the consequent e€ect on transcrip- in tumour cell type. tion pathways. The identity of target genes, which are . Studies of chromosomal translocations in epithelial activated or repressed in downstream pathways, is a tumours. key issue. The downstream genes will not only be . Development of new experimental therapeutic tech- crucial in our understanding of chromosomal translo- nologies for application to cancer treatment using cations in cancer development but will also become chromosomal translocations and primary or down- part of our repertoire of target molecules for stream products of genes a€ected by them. therapeutics.

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5774

Figure 9 Aida cell killing of CHO cells expressing target antigen. In model experiments to validate the Aida strategy (Tse and Rabbitts, 2000), we used an anti-b-galactosidase scFv R4 (Martineau et al., 1998) which binds to b-galactosidase in vivo but does not neutralize its function. In the assay, the level of b-galactosidase activity is monitored as a re¯ection of cell viability (Tse and Rabbitts, 2000). CHO cells were transfected with plasmids expressing b-galactosidase and the various scFv fusion proteins as indicated on the histogram. b-galactosidase levels were measured 60 h after transfection and the data shown are the representative of two independent experiments, each carried out in duplicate. b-galactosidase levels are expressed relative to those obtained with the pEF-bgal expression vector co-transfected with vector (pEF-BOS) alone (which was taken as 100%). When the anti-b- galactosidase scFv R4 was expressed as a fusion with caspase 3 as an ICAb, cell viability was reduced to around 10% and cell death by apoptosis was con®rmed by detection of the caspase 3 cleavage product and chromatin fragmentation studies (Tse and Rabbitts, 2000). The scFv-induced apoptosis is antigen speci®c as it only occurred in the presence of b-galactosidase, it is dependent of speci®c antibody binding as non-relevant scFv (F8) (Tavladoraki et al., 1993) fused to caspase 3 showed no e€ect, and it is dependent on the activity of the fused caspase as an inactivating mutation (C163S) abrogated the e€ect. Finally, antigen binding alone was insucient to activate apoptosis as scFv R4 fused to a VP16 fragment was ine€ective

addition, the publication of the and Studies of chromosomal translocations in epithelial cDNA sequences (Nature, 2001; Science, 2001) pro- tumours mises much in the way of expression analysis which The `solid' tumours make up more than 95% of human may indirectly identify chromosomal translocation cancers. We know rather little about the existence of gene products. For instance, if fusion genes are a recurrent chromosomal translocations in this group, feature of epithelial tumours, cDNA cloning and except for the sarcomas, despite a wealth of cytogenetic sequencing should identify these, given that the human information (see http://cgap.nci.nih.gov/Chromosomes/ genome sequence is now available. However, this may Mitelman). Some consistent changes are known, such still prove beyond the capacity of current technology as as the translocation t(3;5) in renal cell carcinoma most cDNA library construction techniques are not (Kovacs et al., 1987), but signi®cantly this is an adequate for the purpose. Most cDNA clones are not unbalanced translocation, implying a di€erent mechan- full length, many do not even contain coding sequence ism compared haematopoietic tumours and sarcomas. and most cDNA libraries are representations rather It may be that di€erences in epithelial cell biology, than normalized in any useful way for a mass compared with for instance haematopoiesis, account sequencing approach. Finally, many generally used for di€erences in the types of chromosomal transloca- cDNA cloning methods may generate pseudo-fusion tions found and the chromosomal translocation master transcripts. Whilst these can easily be sifted by gene concept may not be applicable to epithelial restriction enzyme scanning and by inter-exon RT ± tumour development. PCR testing of cDNA made from the source mRNA, New approaches to the question of chromosomal each fusion identi®ed will need to be evaluated. Despite translocations in epithelial tumours are now possible these caveats, the currently available genome sequence, using multi-colour ¯uorescence in situ hybridization and its future re®nements and completion, will provide (FISH) and analysis of primary tumour material. In the data for epithelial cancers to be fully described in

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5775

Figure 10 Masked antisense molecules to target BCR ± ABL fusion mRNA. The structured or masked antisense format was designed to speci®cally hybridize and inactivate fusion mRNA, either by causing RNA degradation or inhibiting protein synthesis. (a) The sequence and putative secondary structure of a masked antisense speci®c for BCR ± ABL p190 (MAS190a) is shown. Antisense residues are shown in bold. The antisense molecule has two stem-loop structures of which stem-loop two contains 31 bases complementary to BCR. The targeting region (nucleation site) is highlighted in yellow and comprises 8 bases complementary to the ABL sequence at the junction with BCR. The targeted region of BCR-ABL mRNA is shown in orange (indicated 3'?5'). The two forms of the MAS190 molecule, designated a and b, di€er only in the descending strand of stem-loop II (the di€erences present in MAS190b form are shown in red brackets). Coloured lines indicate the regions complementary to the oligonucleotides used for blocking experiments which con®rmed that the nucleation site was the primary site of hybridization (red=oligo 1, green=oligo 2 and blue=oligo 3). (b) The time course of interaction of either MAS190 a or MAS190 b masked antisense RNA with the p190 BCR-ABL RNA and related sequences. Radio-labelled p190 mRNA fragment (p190(+)RNA) was mixed with MAS190a or MAS190b masked antisense and incubated at 378C. At the indicated times, aliquots were removed and immediately loaded on a continuously electrophoresing 5% native polyacrylamide gel. Samples were run into the gel for 2 min at 10 V/cm then the voltage was reduced to 1 V/cm until the next sample was loaded. After the last sample was loaded (taken at 120 min), electrophoresis was completed at 10 V/cm followed by auto-radiography. The indicated bands correspond to antisense-sense hybrid (AS/(+) RNA), un- annealed p190 RNA (p190(+)) and unhybridized antisense (either hAS190a or b). (c) Speci®city of masked antisense interaction with target mRNA molecule. 32P-labelled masked antisense to BCR ± ABL p190 (MAS190a) or p210 (MAS210) was mixed with the unlabelled target mRNA species indicated at the top of the gel and allowed to associate at 378C for 30 min. The mixtures were resolved by gel electrophoresis, followed by autoradiography. Target mRNA fragments (top) were: BCR ± ABL fusion RNA=p190(+) or p210(+); normal ABL RNA=ABL(+); normal BCR RNA=BCR(+) and an antisense fragment of BCR- ABL p190=p190(7). tRNA indicates reaction carried out with only carrier tRNA terms chromosomal translocations (and of course other may be biologically relevant to the speci®c tumour but mutations). Technology improvement, as is invariably in any event, techniques such as the recruitment of the case, will be crucial to rapidly answer issues about endogenous pathways described above (e.g. Aida cell epithelial tumours, to broaden the endeavours into the suicide) which rely on the presence of a protein target, biological role of chromosomal translocations, as well but not its function, will be applicable in this situation. as the goals of improving cancer treatment and early detection strategies. Developing new therapeutic technologies for cancer While the existence of speci®c chromosomal translo- treatment cations is uncertain in epithelial tumours, these tumours seem to contain non-recurrent chromosomal A long term strategy for cancer treatment is tailored- translocations which are idiopathic changes (discussed therapy for individual cancers. This might be possible in Rabbitts, 1994). Do these changes have any by exploiting technologies capable of rapid sequencing signi®cance for the biology of the individual tumours of chromosomal translocation-gene products and and are the gene products of any interest as therapeutic application to rapid methods for production of targets? If tumour progression, which is associated with patient-speci®c therapeutic `drugs'. In the future, it genome instability, is accompanied by the accumula- will be possible to pro®le an individual cancer genome tion of idiopathic chromosomal translocations these shortly after `diagnosis', and in addition diagnosis will

Oncogene Chromosomal translocations and therapeutic strategies TH Rabbitts 5776 be possible at a much earlier stage of disease rather need for technological development in this quest, as than at the time of presentation with overt cancer. This cancer cells are notoriously good at evading therapy. individual pro®ling will facilitate these tailor-made The strategy we describe here for recruiting endogen- therapeutics. ous cellular pathways after introducing an exogenous reagent into the cell is one approach. With an increase in the sophistication of techniques for identifying new Translation of experimental therapeutics into clinical target molecules and their biological pathways, we management of cancer need to improve strategic technologies and continue to A goal of molecular biology is to describe cancer develop new ones for treatment. Mouse models of biology at the molecular level and identify those areas chromosomal translocations remain at the forefront of of molecular pathology which are consistent changes. our requirements for a balanced portfolio of methods The molecular biology of chromosomal translocations to translate the molecular biology of chromosomal is an example where signi®cant progress has been translocations into pre-clinical experimental therapeu- achieved. We are now in a position to capitalize on this tics and ®nally into patient management. wealth of information for therapy since we can use the molecular information for drug design and develop new strategies to kill or inhibit cancer cells. Protein ± Acknowledgments protein interactions are likely to provide many speci®c This work was supported by the Medical Research targets. It is important to keep in mind the continuing Council.

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