Insertion of excised IgH switch sequences causes overexpression of cyclin D1 in a myeloma tumor cell

Oncogenes are often dysregulated in B cell tumors as a result of a reciprocal translocation involving an immunoglobulin locus. The translocations are caused by errors in two developmentally regulated DNA recombination processes: V(D)J and IgH switch recombination. Both processes share the property of joining discontinuous sequences from one chromosome and releasing intervening sequences as circles that are lost from progeny cells. Here we show that these intervening sequences may instead insert in the genome and that during productive IgH mu-epsilon switch recombination in U266 myeloma tumor cells, a portion of the excised IgH switch intervening sequences containing the 3' alpha-1 enhancer has inserted on chromosome 11q13, resulting in overexpression of the adjacent cyclin D1 oncogene.     

Minimal peripheral blood cells carrying clonal markers of B cell disorders: evidence for monoclonality of circulating lymphocytes in patients with multiple myeloma

Peripheral blood lymphocytes (PBL) of 20 patients with multiple myeloma (MM) were assayed for clonality by Southern blot and cell surface marker analysis. Eight samples showed monoclonal origin of circulating lymphocytes by demonstrating rearrangements of the heavy chain immunoglobulin gene (IgH). In selected experiments, comparison of IgH rearrangements of bone marrow plasma cells and peripheral blood-derived mononuclear cells, highly enriched for B lymphocytes, proved to be identical. However, monoclonal circulating cells could not be detected in samples with rearranged IgH genes by surface marker phenotyping using one-color immunofluorescence analysis and a panel of monoclonal and polyclonal antibodies to various B lineage-associated antigens. These results indicate that in a substantial proportion of MM, monoclonal growth involves circulating B lymphocytes and underscores the clinical usefulness of Southern analysis of IgH gene rearrangements for monitoring this disease.     

AID is required for c-myc/IgH chromosome translocations in vivo

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.     

AID and Igh switch region-Myc chromosomal translocations

Chromosomal translocations involving Ig heavy chain switch regions and an oncogene, like Myc, represent early initiating events in the development of many B cell malignancies. These translocations are widely believed to result from aberrant class switch recombination (CSR). Recent reports have produced conflicting models for the role of activation-induced cytidine deaminase (AID) in this process. Here, we discuss possible roles of AID, CSR, and somatic hypermutation in generating chromosomal translocations and in tumor progression.     

Assessing the pathogenic potential of the V(D)J recombinase by interlocus immunoglobulin light-chain gene rearrangement.

Chromosomal translocations involving antigen receptor genes and oncogenes have been observed in several forms of lymphoid malignancy. Observations of their lymphocyte-restricted occurrence and a molecular analysis of some translocation breakpoints have suggested that some of these rearrangements are generated by V(D)J recombinase activity. However, a direct correlation between this activity and the generation of such rearrangements has never been established. In addition, because these aberrant rearrangements are usually detected only after a tumor has been formed, the frequency with which the recombinase machinery generates translocations has never been assessed directly. To approach these issues, immunoglobulin light-chain gene rearrangements were induced in pre-B cells transformed by temperature-sensitive mutants of Abelson murine leukemia virus and PCR was used to identify interlocus recombinants. Vlambda Jkappa and Vkappa Jlambda rearrangements as well as signal joints resulting from the recombination of Vlambda and Jkappa coding elements were recovered and were found to be similar in structure to conventional intrachromosomal joints. Because these products were detected only when the cells were undergoing active intralocus rearrangement, they provide direct evidence that translocations can be generated by the V(D)J recombinase machinery. Dilution analyses revealed that interlocus rearrangements occur about 1,000 times less frequently than conventional intralocus rearrangements. Considering the large numbers of lymphocytes generated throughout life, aberrant rearrangements generated by the V(D)J recombinase may be relatively common.     

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.     

Amino-terminal phosphorylation of activation-induced cytidine deaminase suppresses c-myc/IgH translocation

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

H2AX prevents DNA breaks from progressing to chromosome breaks and translocations

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.     

Site-specific recombination of the tal-1 gene is a common occurrence in human T cell leukemia.

The tal-1 gene is altered as a consequence of the t(1;14) (p32;q11) chromosome translocation observed in 3% of patients with T cell acute lymphoblastic leukemia (T-ALL). tal-1 encodes a helix-loop-helix (HLH) domain, a DNA binding and dimerization motif found in a number of proteins involved in cell growth and differentiation. We now report that an additional 25% of T-ALL patients bear tal-1 gene rearrangements that are not detected by karyotype analysis. These rearrangements result from a precise 90 kb deletion (designated tald) that arises independently in different patients by site-specific DNA recombination. Since the deletion junctions resemble the coding joints of assembled immunoglobulin genes, tald rearrangements are likely to be mediated by aberrant activity of the immunoglobulin recombinase. Moreover, t(1;14)(p32;q11) translocations and tald rearrangements disrupt the coding potential of tal-1 in an equivalent manner, and thereby generate a common genetic lesion shared by a significant proportion of T-ALL patients.     

Promiscuity of translocation partners in multiple myeloma

Multiple myeloma (MM) is characterized by karyotypic instability, including chromosomal translocations involving the IGH locus. MM cells display a promiscuity of translocation partners, only some of which are recurrent. We propose that several factors, including temporal and spatial nuclear positioning of potential partner loci, “off‐target” IGH diversification mechanisms, and aberrant repair pathways contribute to the promiscuity of translocation partners in MM. We speculate that in MM, IGH diversification processes [V(D)J recombination, somatic hypermutation, and class switch recombination] in B cells may not be restricted to specific stages of B‐cell development or within specific immune tissues, but may occur in different temporal “windows.” Before or during MM evolution, off‐target activities of the enzymes involved in IGH modification processes may contribute to the generation of double‐strand breaks (DSB) in translocation partner loci. In the parent B cells from which MM originates, spatial proximity within the nucleus of IGH and potential translocation partners contributes to the selection of a translocation partner and the clinical frequency at which a specific translocation occurs. The spatial proximity of IGH and specific translocation partners may be temporal and contribute not only to partner selection but also to the promiscuity of partners seen in MM. Lastly, aberrant repair mechanisms in MM progenitors (including the possibility that a Ku 86 variant allows for positional instability at DSBs) may also contribute to the promiscuity of chromosome translocation partners in MM. J. Cell. Biochem. 109: 1085–1094, 2010. © 2010 Wiley‐Liss, Inc.     

Monoclonal gammopathy of undetermined significance and Russell body formation in Helicobacter pylori gastritis

BACKGROUND: Infection by Helicobacter pylori has been linked to monoclonal gammopathy of undetermined significance (MGUS). MGUS is thought to develop due to chronic antigenic stimulation in people with a specific genetic predisposition. METHODS AND RESULTS: We describe a patient presenting with dyspepsia associated with H. pylori-related erosive gastritis. Histopathologic findings revealed infiltration with plasma cells containing accumulated condensed intercisternal immunoglobulins, the so-called 'Russell bodies'. In addition, MGUS was present with total immunoglobulins within the normal range but a significantly decreased serum concentration of IgG subtype 3. Molecular analyses demonstrated IgH formation, T-cell receptor gamma rearrangement, and alterations within the IgHG3 gene sequence. Following H. pylori eradication, gastritis and dyspepsia gradually resolved but MGUS persisted for at least 22 months. CONCLUSIONS: This is the first report to demonstrate that upon infection with H. pylori, an impaired secretory capacity of plasma cells due to specific molecular changes can present as Russell body gastritis. The molecular findings question a pathogenetic link between Russell bodies and H. pylori, but suggest genetic alterations in the immunoglobulin locus as the possible cause for both MGUS and Russell body gastritis.     

Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors

We employed gene targeting to study H2AX, a histone variant phosphorylated in chromatin surrounding DNA double-strand breaks. Mice deficient for both H2AX and p53 (H(delta/delta)P(-/-)) rapidly developed immature T and B lymphomas and solid tumors. Moreover, H2AX haploinsufficiency caused genomic instability in normal cells and, on a p53-deficient background, early onset of various tumors including more mature B lymphomas. Most H2AX(delta/delta)p53(-/-) or H2AX(+/delta)p53(-/-) B lineage lymphomas harbored chromosome 12 (IgH)/15 (c-myc) translocations with hallmarks of either aberrant V(D)J or class switch recombination. In contrast, H2AX(delta/delta)p53(-/-) thymic lymphomas had clonal translocations that did not involve antigen receptor loci and which likely occurred during cellular expansion. Thus, H2AX helps prevent aberrant repair of both programmed and general DNA breakage and, thereby, functions as a dosage-dependent suppressor of genomic instability and tumors in mice. Notably, H2AX maps to a cytogenetic region frequently altered in human cancers, possibly implicating similar functions in man.     

AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.     

Localization of human variable and constant region immunoglobulin heavy chain genes on subtelomeric band q32 of chromosome 14

Analysis of a group of human/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human variable region (VH), junctional (JH), and constant region (C epsilon) heavy chain immunoglobulin genes indicates that all of these IgH genes are localized on the subtelomeric (q32) band of chromosome 14. Somatic cell hybrids were isolated in selective medium after fusing human fibroblasts with hprt- Chinese hamster cells. The human parental cells contained two translocation chromosomes representing a reciprocal translocation between chromosomes X and 14. Only those hybrid cell lines retaining a complete human autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32) retained the human IgH genes. Retention of these genes did not correlate with the presence of the other translocation chromosome, 14/X. These results indicate that all human IgH genes (VH, JH, and CH) map to the same chromosomal band (14q32) which is commonly involved in reciprocal translocations with human chromosome 8 (8q24) in B-cell neoplasms.