Oncogenes and Chromosome Translocation

The success achieved in identifying cellular oncogenes as targets for proviral integration suggested the possibility that other sorts of specific genetic damage in neoplasms might similarly relate to oncogene activation. The potential importance of karyotypic abnormalities in neoplastic cells, including translocations, duplications, deletions, and loss of chromosomes, had been recognized since 1914. Did such abnormalities play a causal role in neoplastic transformation or were they secondary events due merely to the genetic instability of transformed cells? The first insight into the functional importance of chromosomal aberrations came from understanding their relation to oncogene activation. Chromosome Abnormalities in Neoplasms In many neoplasms, chromosomal abnormalities appeared erratic: there was no consistency in the aberrations observed in different tumors of the same type or even between different cells of the same individual neoplasm. In these cases, the observed karyotypic abnormalities do not appear likely to be primary contributors to the genesis of transformation.

In other instances, however, reproducible tumor-specific chromosome abnormalities have been identified. The initial finding was the work of Peter Nowell, reported in 1960. Nowell observed that human chronic myelogenous leukemia (CML) was consistently characterized by the presence of an abnormal small chromosome 22, which was called the Philadelphia chromosome after the city in which it was discovered. The same chromosomal abnormality was found in the leukemic cells of more than 90% of patients with chronic myelogenous leukemia; so the Philadelphia chromosome clearly represented a reproducible aberration that was closely associated with development of this neoplasm. Consistent chromosome abnormalities have since been identified in a number of other types of neoplastic disease. The reproducibility of such lesions suggested the possibility that they represent genetic alterations that impose a selective growth advantage to the neoplastic cells and thus play a CAUSAL ROLE in tumor development-a hypothesis that was validated by a conjunction of the analysis of chromosome translocations with studies of cellular oncogenes. Translocation of c-myc in Burkitt's Lymphomas and Plasmacytomas One hypothesis for the role of chromosome translocations in neoplasia was that they might lead to DNA rearrangements that would result in activation of a cellular oncogene.

The first experimental evidence was derived from studies of the c-myc oncogene, which had previously played a central role in elucidating cellular oncogene activation by non-acutely transforming retroviruses. At the end of 1982, studies from several different groups of investigators implicated translocation of c-myc in the genesis of Burkitt's lymphomas in humans and plasmacytomas in mice. Burkitt's lymphomas and plasmacytomas are B-lymphocyte neoplasms . Moreover, in both diseases, the characteristic translocations occurred at chromosomal loci at which the immunoglobulin genes were located. In Burkitt's lymphomas, a region of chromosome 8 is translocated to either chromosome 14, chromosome 2, or chromosome 22. The sites of these translocations on chromosomes 14, 2, and 22 correspond to the immunoglobulin heavy-chain, k light-chain, and light-chain genes, respectively. In mouse plasmacytomas, translocations occur between a distal region of chromosome 15 and either the immunoglobulin heavy-chain locus on chromosome 12 or the light-chain locus on chromosome 6. Because both Burkitt's lymphomas and plasmacytomas are neoplasms of B lymphocytes, which express immunoglobulin genes, translocation of a proto-oncogene into the immunoglobulin locus could readily be envisaged to result in protooncogene activation. This consideration led to the suggestion that the other partner in these translocations (the locus at the translocation breakpoint on human chromosome 8 and mouse chromosome 15) might be a proto-oncogene.

Based on the activation of c-myc by retroviral insertion in another B-cell neoplasm, chicken bursal lymphomas, several research groups investigated the possibility that c-myc was also activated in Burkitt's lymphomas and mouse plasmacytomas. The question was approached by determining the chromosomal location of c-myc, by probing tumor DNAs directly for c-myc rearrangements, and by investigating the possible presence of c-myc sequences in molecular clones that had been derived from rearranged immunoglobulin genes. These related lines of investigation led to similar conclusions. The c-myc gene was located at the translocation breakpoints on human chromosome 8 and mouse chromosome 15 (Fig. 7.1). In both Burkitt's lymphomas and plasmacytomas, c-myc genes were rearranged by recombination with the immunoglobulin loci. Molecular characterization of the structure and expression of the rearranged c-myc genes has led to the consensus that the translocations result in a loss of normal gene regulation, leading to constitutive cmyc expression. In plasmacytomas, the translocations usually occur within the first intron of c-myc (the first exon is noncoding). In Burkitt's lymphomas, the translocations are more variable, sometimes occurring in either 5' or 3' flanking sequences rather than within the c-myc gene itself.

The mechanisms by which these different rearrangements affect cmyc expression are not entirely understood: possibilities include the linkage of c-myc to immunoglobulin enhancer sequences, deletion of normal c-myc transcriptional regulatory sequences, and alterations in the stability of c-myc mRNA. In any event, the consequence of these rearrangements is abnormal constitutive expression of a c-myc coding sequence that is generally unaltered compared with its normal allele.

The fact that such deregulated expression of a normal c-myc protein is sufficient to activate c-myc as an oncogene has been demonstrated directly by gene transfer experiments. Molecular clones of c-myc that result in constitutive expression of a normal Myc protein (EXPRESSION VECTORS) are able to induce transformation in a number of different cell culture systems. Studies of c-myc rearrangement in these neoplasms thus established chromosome translocation as a mode of cellular oncogene activation. In addition, these results further intensified studies of c-myc, which had now been identified as an oncogene important in the development of a human lymphoma as well as several different animal neoplasms. The Philadelphia Translocation and abl The discovery of c-myc translocation heightened interest in the chromosomal locations of other proto-oncogenes. Perhaps additional proto-oncogenes could be identified at translocation breakpoints that would then prove to be activated by rearrangement. As noted before, the first consistent chromosomal abnormality identified in a neoplasm was the Philadelphia chromosome in chronic myelogenous leukemia. More than ten years after its initial description, the Philadelphia chromosome was found to be the result of a reciprocal translocation between chromosomes 9 and 22 (Fig. 7.3).

In 1982, the abl proto-oncogene was mapped to chromosome 9 and was found to be translocated to the Philadelphia chromosome in chronic myelogenous leukemias. These findings clearly raised the prospect that abl was activated by this translocation. Substantiation of this hypothesis was forthcoming from molecular analysis of the effect of translocation on abl structure and function. Elucidation of the mechanism of abl activation by the Philadelphia translocation came both from analysis of translocation breakpoints and from studies of abl expression. The abl proto-oncogene contains two alternative first exons (designated 1A and IB), each with its own promoter, which are spliced to a common exon 2 to yield two different proto-oncogene transcripts. THE PHILADELPHIA TRANSLOCATION BREAKPOINTS ON CHROMOSOME 9 OCCUR OVER A RANGE OF 50 KB AND CAN FALL EITHER UPSTREAM OR DOWNSTREAM OF ABL EXON 1A (Fig. 7.4). On chromosome 22, most of the breakpoints fall within a region of 6 kb, which has been termed the breakpoint cluster region (bcr). Molecular analysis established that bcr is a functional gene, which is disrupted near its middle by the translocations. A translocation therefore creates a fusion gene, in which the 5' half of bcr is joined upstream of abl in the same transcriptional orientation.

Transcription of the bcrl-abl fusion gene is initiated at the bcr promoter and continues through abl, yielding a long primary transcript containing both bcr and abl sequences. The bcr and abl coding sequences are then joined by RNA splicing, which results in formation of a fused bcr-abl mRNA of -8,5 kb. The splice donor site of the last remaining bcr exon joins the first splice acceptor site in abl, which occurs at the start of abl exon 2, to form the junction of bcr and abl coding sequences. Because abl exon 1A does not contain a splice acceptor site, it is deleted from the spliced bcr/abl mRNA. This fused mRNA is then translated to yield a Bcr/Abl fusion protein in which the normal amino terminus of the abl proto-oncogene protein has been deleted and replaced with bcr amino-terminal coding sequences. A similar translocation activates the abl proto-oncogene in some acute lymphocytic leukemias, except that in these cases the translocation breakpoint falls farther upstream in the bcr gene. THE MECHANISM BY WHICH TRANSLOCATIONS ACTIVATE ABL THUS APPEARS TO BE FUNDAMENTALLY DIFFERENT FROM ACTIVATION OF C-MYC: THE ABL TRANSLOCATIONS LEAD TO FORMATION OF AN ALTERED PROTEIN, WHEREAS C-MYC TRANSLOCATIONS RESULT IN ABNORMAL EXPRESSION OF THE NORMAL GENE PRODUCT.

Interestingly, the BCR/ABL FUSION PROTEIN CLOSELY RESEMBLES THE VIRAL ABL ONCOGENE PRODUCT, WHICH IS A FUSION PROTEIN CONTAINING VIRAL GAG SEQUENCES AT ITS AMINO TERMINUS. In both of these fusion proteins, the normal amino terminus encoded by the abl proto-oncogene is deleted. Furthermore, the Bcr/Abl protein has been found to display ENHANCED TYROSINE KINASE ACTIVITY, similar to that of the viral oncogene protein. Substantiation of the biological significance of this aberrant gene product has also been provided by the ability of the bcr/abl fusion gene to induce cell transformation. It thus appears that the Philadelphia chromosome translocation results in oncogene activation as a consequence of structural and functional changes in the Abl protein, rather than by an alteration in gene expression. More TranslocationsMore Oncogenes With chromosome translocation clearly established as a mode of oncogene activation, it became reasonable to ask whether those translocations that were reproducible features of neoplasm development would regularly be associated with activation of cellular oncogenes, potentially including new genes that had not previously been described. Activation of c-myc by translocation to the immunoglobulin locus has been reported in some human B-cell malignancies in addition to Burkitt's lymphomas. Moreover, the c-myc oncogene is also activated in occasional human T-cell neoplasms by translocation to the locus encoding one of the Tcell receptor subunits.

Activation of c-myc in these lymphoid neoplasms thus appears to be a consequence of translocation of the oncogene into a new locus that is expressed in the target cell type. Such translocations may readily occur because the immunoglobulin and T-cell receptor loci normally undergo genetic rearrangements during maturation of B and T lymphocytes. The recombination systems responsible for these normal rearrangements may also increase the frequency of aberrant recombination events: if such events were to result in oncogene activation, the resultant cell would acquire enhanced growth potential. On the basis of these considerations, one approach to identifying additional oncogenes that take part in the development of human neoplasms is molecular cloning and characterization of genes that are located near the breakpoints of characterized chromosome translocations. The rationale for this approach is that such studies would allow direct isolation of the candidate oncogene, whether or not it was a gene that had been previously described. THE PROTOTYPE NEOPLASM that has been analyzed in this way is follicular B-cell lymphoma, which is characterized by a translocation between chromosomes 14 and 18. The translocation site on chromosome 14 is the immunoglobulin heavy-chain locus, suggesting the hypothesis that the translocation might activate an oncogene from chromosome 18 (Fig. 7.5). MOLECULAR CLONING INDICATED THAT THE BREAKPOINTS CLUSTERED IN A SMALL REGION OF CHROMOSOME 18 DNA.

Probes from this region were then used to establish the existence of an active transcriptional unit in this locus, thereby identifying a new candidate oncogene that was designated bcl-2. Cloning and further analysis of both normal and translocated bcl-2 transcripts have determined the sequence of the Bcl-2 protein, which has been identified in human cells. In addition, these studies have indicated that bcl-2 (like cmyc) is abnormally expressed as a result of translocation into the immunoglobulin heavy-chain locus. Such deregulated expression as a consequence of a consistent chromosome translocation clearly supports the suggestion that bcl-2 is a functional oncogene, as has been directly demonstrated by functional analysis. Continuing molecular analysis of translocation breakpoints has identified a number of additional oncogenes that are activated in human neoplasms, particularly LEUKEMIAS AND LYMPHOMAS (TABLE 7.1). One example is the gene encoding the growth factor interleukin-3, which is translocated and abnormally expressed in some leukemias. Two other genes activated by translocations (erg and fli-1) are related to the ets oncogenes, first identified in avian retroviruses. In addition, translocations activate both the human homolog of the retroviral rel oncogene and On the basis of these considerations, one approach to identifying additional oncogenes that take part in the development of human neoplasms is molecular cloning and characterization of genes that are located near the breakpoints of characterized chromosome translocations.

 The

transcriptional enhancers of immunoglobulin (Ig) chains are highly active in B lymphocytes Translocation of the bcl-2 gene on chromosome 18 near the enhancers of the immunoglobulin heavy chain gene on chromosome 14 (t14;18) leads to inappropriately high transcription levels of bcl-2

D bcl2

En

Cm bcl2 J En Cm

Ig enhancer makes bcl-2 promoter more active

THE FIRST CLINICAL APPLICATION OF MOLECULAR ONCOGENETICS

Treatment of leukemia may be successful by conventional chemotherapy. The major limitation to this approach is extremely rapid development of drug resistance in malignant cells. For this reason additional treatments with chemotherapeutic drugs are not effective. Drug resistance develops in malignant cells surviving the first cycle of treatment. So, after the first treatment it is essential to know if there are surviving malignant cell(s) capable of clonal expansion. Traditional detection of Ph+ cells involved cytogenetic chromosome examination. This is highly impractical since 1 ml of bone marrow contains 10exp9 cells an formidable task for cytogenetic approach. This is quite feasible with PCR based analysis that is capable of analyzing of 10exp7 cells in a single analysis. This approach has to be specific for Ph+ cells only. That means that PCR has to be targeted to fusion brc-abl mRNA with primers derived from sequences specific for both genes that generate the fusion mRNA.

 RNA

clean-up using DNase

28S rRNA 18S rRNA DNA

Before

After

BCR BCR F

ABL ABL F ABL R

Semi-nested RT-PCR followed by direct sequencing

Nested PCR uses two sets of amplification primers. One set is targeted within the amplification product of the other set, and two rounds of amplification in order to amplify the specific target DNA. The amplicon (amplification product) of the second reaction is shorter than that of the first (Figure 2). This procedure is designed to increase the sensitivity of PCR by directly reamplifying the product from a primary PCR with a second PCR.

The Philadelphia chromosome translocation fuses the bcr and abl genes

healthy individual bcr Chr. 22 abl Chr. 9

Groffen et al. Cell 36, 93 (1984)

leukemic patient

bcr-abl

9; 22 Translocation fuses bcr and abl bcr= (breakpoint cluster region)

Fluorescence In Situ Hybridization (FISH) a tool for diagnosing CML

bcr

abl

Fluorescence In Situ Hybridization (FISH)

fusion 9 abl/bcr

fusion 22 bcr/abl

abl

bcr

RNA DNA

Normal proto-oncogene

RNA
DNA

Translocation of strong promoter/ enhancer

RNA DNA

Translocation of coding region

Imatinib (Gleevec)