Molecular Biology Of Burkitt Lymphoma

Burkitt lymphoma (BL) is an aggressive malignancy consisting of highly proliferative B-cells which infiltrate lymph nodes and other organs. The tumor cells are distinguished as B-cells by their expression of IgM and k or X light chains and by specific surface markers such as CD19 and CD20. The high proliferative rate is obvious from numerous mitotic figures. Staining for markers such as Ki67 or PCNA, which are characteristic of cycling cells, shows that almost all tumor cells are participating in proliferation. The rapid proliferation in this cancer is only partly compensated by a high apoptotic rate, evident most straightforwardly from interspersed macrophages phagocytosing the apoptosed tumor cells. (Figure 10.4)

BL was first described as a tumor in young people in equatorial Africa where it is endemic in some regions. It is much rarer in Europe and North America, although more frequent in the context of AIDS and in other immunocompromised patients. In tropical areas, most BLs harbor Epstein-Barr virus (EBV), which is not as consistently associated with the disease in countries of the temperate zones. Treatment by chemotherapy and stem cell transplantation12 can be successful in some cases.

The three characteristic translocations in BL all involve the MYC locus at 8q24.1. The most frequent, 'major' translocation joins the gene to the IGH locus at 14q32 which encodes the immunoglobulin heavy chain. The 'minor' translocations involve the IGL (IgX) locus at 22q11 or the IGK (Igk) locus at 2p12 (Figure 10.5). These loci encode the two immunoglobulin light chains, either of which can be used in B cells. One of these three translocations is found in each case of BL. The diagnosis of BL therefore is made contingent on their presence. The converse is not true, i.e. these translocations are also found in other lymphomas, all of which are also aggressive.

Figure 10.4 Histology of Burkitt lymphoma Note the macrophage phagocytising an apoptotic cell.

In each translocation, the MYC gene is brought under the influence of an immunoglobulin gene enhancer which is strongly active at this stage of B-cell differentiation. BL is thus an important example of the gene activation mechanism by translocations (Figure 10.3A). The translocations in BL result in deregulation and overexpression of a potent oncogene, which normally ought to become down-regulated in mature B-cells. While the overall result is the same, the mechanisms leading to deregulation differ in detail between the various translocations (Figure 10.6).

12 Stem cells were formerly transferred by bone marrow transplantation; today they can be harvested from peripheral blood following administration of G-CSF. They can be identified by their CD34 marker.

Figure 10.5 Translocations in Burkitt lymphoma See text for further details.

In most major translocations, the translocation breakpoint lies at some distance upstream of the MYC gene, which remains intact. The translocated IGH locus is oriented in inverse orientation, with the enhancer positioned towards the MYC gene. This configuration appears to result in a 'classical' enhancer activation mechanism, in which the strong enhancer activates the unchanged MYC promoters.

In some of the major translocations, the breakpoints are located in the first intron of the MYC gene, so that the first exon with the P1 and P2 promoters is removed. The gene is transcribed from the otherwise 'cryptic' (i.e. unused) P3 promoter located in the first intron. In this configuration, all elements in the MYC upstream region controlling transcription are deleted. Specifically, the translocation inactivates an attenuation mechanism in the first intron which makes transcription of the gene contingent on continuous stimulation. In these translocations, the placement of the IGH enhancer next to the gene does not seem to be absolutely essential for MYC activation, but the presence of an active IGH gene is required, perhaps to guarantee an open chromatin structure.

In the minor translocations, the breakpoints are located downstream of the MYC gene, sometimes at a considerable distance (>100 kb). The regulatory regions of the MYC gene remain largely intact, with the possible exception of a negative regulatory element presumed to lie downstream of the gene. The light chain loci are located in inverse orientation with their downstream enhancers oriented towards MYC. These seem to be mainly responsible for driving MYC over-expression. In the IGK locus, further elements may contribute, including an intronic enhancer.

In all translocations, the translocated MYC gene becomes susceptible to mutations that augment the effects of the enhancers. In translocations retaining the MYC regulatory regions, point mutations are found which inactivate negative regulatory elements in exon 1 and the first intron, thereby exacerbating overexpression. In all translocations, mutations are also found in the N-terminal coding region of MYC. In general, these stabilize the protein. For instance, a common mutation in Thr58 prevents a regulatory phosphorylation that targets the gene for proteosomal degradation. Interestingly, an according mutation is also common in the v-myc oncogenes of retroviruses (^Figure 4.1).

The genetic alterations at the MYC locus found in Burkitt lymphoma can be understood as accidents during the maturation of B-cells. During the differentiation of B-cells, V(D)J recombination of the IGH and either IGL or IGK immunoglobulin genes is employed to generate the antibody repertoire necessary for the immune response to many different antigens. Later, a further recombination event accompanies the switch from expression of IgM to that of IgG. Furthermore, in the germinal centers of the lymphoid organs, somatic hypermutation is directed to the rearranged immunoglobulin genes to generate additional antibody variants with

Figure 10.6 Mechanisms of MYC activation in Burkitt lymphoma A more detailed illustration of the mechanisms leading to MYC deregulation. Note that the figure is not to scale.

higher affinities and improved specifity towards the encountered antigens. These processes are recognizable in BL, although deviant.

The RAG recombinases mediating V(D)J rearrangements recognize particular sequence motifs in the immunoglobulin genes which are found in the vicinity of the breakpoints in the translocated immunoglobulin genes, although not usually in the MYC gene. It is therefore possible that the translocations are initiated by the physiological introduction of double-strand breaks into the immunoglobulin genes by B-cell specific recombinases. By accident, these breaks are then wrongly connected to the MYC gene. Likewise, placement of the MYC gene into an immunoglobulin gene cluster may set it up as a target for somatic hypermutation. The accidents that initiate BL are likely to be very rare, even if favored by the unknown agents that cause endemic BL. However, activation of MYC provides a strong proliferation stimulus that may provide a large pool of cells from which the lymphoma arises, likely by further alterations that are less conspicuous than the translocations. Since BL is more frequent in immunocompromised individuals, an immune defense may exist which normally eliminates B cells with aberrantly rearranged immunoglobulin genes.

Activation of MYC to an oncogene accounts for many of the properties of BL cells. MYC stimulates many aspects of cell proliferation and cell growth (Table 10.3). In particular, in many cell types overexpression of MYC is sufficient to maintain them active in the cell cycle, independent of growth factors. It may therefore account for the high proliferative fraction and rate in BL. MYC, through its action on telomerase, may also contribute to immortalization. Moreover, some proteins regulated by MYC are involved in cell adhesion, e.g. LFA-1 in lymphocytes. This may lead to altered interactions of BL cells with other immune cells.

Table 10.3. Effects of MYC on various cellular functions

Function

Some proteins regulated by MYC*

Metabolism

Adhesion

Apoptosis

Cell growth

Cell proliferation

Cell differentiation

T RNA polymerase, nucleolar proteins, ribosomal proteins, splice factors, eIF proteins, CAD, polyamine biosynthesis (ODC, spermidine synthase), chaperones, T Cyclin D2, Cyclin B1, CDK4, CDC25A, E2F1, CUL1, hTERT 4 p21CIP1, p27KIP1, MYC

4 cell -type specific bHLH transcriptional activators, ID proteins

T LDH , PFK, enolase 4 LFA1, PAI1 T E2F1 (p14ARF), BAX

* t up-regulation or activation i- down-regulation or direct interference

The oncogenic potential of MYC is normally restrained by two factors. (1) The gene and the protein are tightly regulated by a variety of mechanisms which are subverted by the translocations and mutations in BL (see above). (2) MYC is a strong inducer of apoptosis. This induction is, at least partly, mediated by induction of p14ARF by E2F1 which is a transcriptional target of MYC. p14ARF then blocks HDM2/MDM2 activating TP53 to elicit apoptosis (^5.3). The high apoptotic rate found in BL suggests that a mechanism of this sort may indeed be active, but not sufficiently so as to compensate for the increase in proliferation.

It is therefore thought that the development of BL requires at least one further genetic alteration which limits apoptosis. Different recurrent genetic alterations, each observed in a fraction of BLs, may account for this requirement. About one third of all cases have mutations in TP53 and LOH at 17p, others have lost TP73, a related protein and a potential mediator of TP53-induced apoptosis. The BCL6 gene at 3q27 is affected in BL, as well as in other B-cell lymphomas, by translocations and mutations, which may also stem from somatic hypermutation. The function of the transcriptional repressor BCL6 is probably specific to B-cells at a certain stage of differentiation in germinal centers. It is down-regulated during terminal differentiation. The various translocations, which are typically promoter substitutions, appear to keep the gene active beyond its time. Importantly, BCL6 does not stimulate cell proliferation, but influences the expression of specific B cell proteins and decreases apoptosis.

Since EBV is found in so many BL, it is tempting to speculate that this herpes virus may provide a complementary function to that of MYC in the development of this tumor. The relationship has, however, emerged as more roundabout. EBV occurs in >90% of all adults, but only in a fraction of B-cells. It is capable of immortalizing cells of this type. This ability is therefore used in the laboratory to generate permanent lymphoblastoid lines. The virus exists as an episome, whose maintenance and replication require minimally the expression of the EBNA-1 protein (Figure 10.7). In lymphoblastoid lines and in long-term infected cells in vivo, further genes are expressed which down-regulate apoptosis and help to avoid immune detection. These genes are obvious candidates for synergizing with MYC in BL, but most of them are more weakly expressed in the actual disease than normally. The EBER RNAs of the virus are also good candidates for cooperating with MYC. Conversely, there is evidence that strong MYC expression may support the maintenance of the virus.

Several alternative plausible explanations are therefore considered for the evident association of the virus with the disease. For instance, EBV-infected cells may be more susceptible to accidents during attempted V(D)J recombination or the BL cells may be initially protected from apoptosis and elimination by the immune system by EBV proteins, while later on the same functions would be provided by other alterations such as translocations or mutations of BCL6.

EBFR-RNAs

EBFR-RNAs

Figure 10.7 Structure of the EBV genome The 172 kb circular DNA genome of the virus with selected important features. Specifically, only the transcription units most important in stably infected cells are shown. TR: terminal repeat; LMP: latent membrane protein; OriP: viral origin of replication.
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