- Open Access
Combined deletion of Xrcc4 and Trp53 in mouse germinal center B cells leads to novel B cell lymphomas with clonal heterogeneity
© Chen et al. 2016
- Received: 6 November 2015
- Accepted: 28 December 2015
- Published: 7 January 2016
Activated B lymphocytes harbor programmed DNA double-strand breaks (DSBs) initiated by activation-induced deaminase (AID) and repaired by non-homologous end-joining (NHEJ). While it has been proposed that these DSBs during secondary antibody gene diversification are the primary source of chromosomal translocations in germinal center (GC)-derived B cell lymphomas, this point has not been directly addressed due to the lack of proper mouse models.
In the current study, we establish a unique mouse model by specifically deleting a NHEJ gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in GC B cells, which results in the spontaneous development of B cell lymphomas that possess features of GC B cells.
We show that these NHEJ deficient lymphomas harbor translocations frequently targeting immunoglobulin (Ig) loci. Furthermore, we found that Ig translocations were associated with distinct mechanisms, probably caused by AID- or RAG-induced DSBs. Intriguingly, the AID-associated Ig loci translocations target either c-myc or Pvt-1 locus whereas the partners of RAG-associated Ig translocations scattered randomly in the genome. Lastly, these NHEJ deficient lymphomas harbor complicated genomes including segmental translocations and exhibit a high level of ongoing DNA damage and clonal heterogeneity.
We propose that combined NHEJ and p53 defects may serve as an underlying mechanism for a high level of genomic complexity and clonal heterogeneity in cancers.
- Non-homologous end-joining
- Genomic instability
- Chromosomal translocations
- B cell lymphoma
- Clonal heterogeneity
The occurrence of human B cell lymphomas is much more frequent than that of T cell lymphomas . This phenomenon might be attributed to the multiple mechanisms functioning in B lymphocytes that intrinsically generate DNA double-stranded breaks (DSBs) or mutations [2, 3]. Developing B cells in the bone marrow (BM) undergo V(D)J recombination to assemble the variable (V) region exons of Ig genes [4, 5]. V(D)J recombination involves a cut-and-join mechanism initiated by the lymphocyte-specific RAG1/2 endonucleases that recognize and introduce DSBs at recombination signal sequences (RSS) flanking germline V, D, and J segments . Subsequently, broken V, D, and J segments are joined by ubiquitous non-homologous end-joining (NHEJ) . Ongoing RAG-expression in newly generated B cells allows secondary V(D)J recombination, termed “receptor editing”, a process in which additional Ig gene rearrangements may occur in BM immature B cells [8–12]. Ultimately, RAG down-regulation in mature B cells prohibits further V(D)J rearrangement [13, 14]. However, our previous studies suggest that mature B cells may also undergo secondary V(D)J recombination at low frequency in an in vitro culture system . While RAG contributes to the genomic instability of developing B cells [16–18], its role in mature B cell lymphomagenesis is still under debate.
Upon antigen activation, mature B cells undergo further genetic diversification processes, namely, class switch recombination (CSR) and somatic hypermutation (SHM), in specialized secondary lymphoid structures termed germinal centers (GCs) [19–22]. Activation-induced deaminase (AID) initiates CSR and SHM [23, 24], which deaminates cytosines in transcribed DNA and ultimately causes DSBs or point mutations [25–28]. CSR is a region-specific deletional recombination process required for producing isotype-switched antibody such as IgG . AID-initiated DSBs occur at the switch (S) regions within the Igh locus, which are eventually resolved as deletions in cis on the same chromosomes, thereby causing the switch of constant regions of Igh . SHM introduces predominantly point mutations into IgH and IgL V region exons, allowing the selection of B cell clones with increased affinity for antigen . Besides Ig loci, AID can target non-Ig loci to induce genetic lesions, thereby posing a threat to genome stability . Consistently, the dysregulated AID activity contributes to tumorigenesis [31, 32]. We and others have shown that AID is required for generating chromosomal breaks at the Igh locus  and the Igh-c-myc translocations .
Apart from programmed DSBs, B lymphocytes harbor general DSBs arising from genotoxic agents such as oxidative damage or DNA replication errors. To preserve genome integrity, two major DSB repair pathways operate in mammalian cells: homologous recombination (HR) and NHEJ. While HR-directed repair requires homologous templates, NHEJ can repair DSBs with little or no sequence homology . The NHEJ pathway joins programmed DSBs in lymphocytes including RAG- or AID-initiated DSBs  and repairs general DSBs in all types of cells . The NHEJ pathway includes Ku70, Ku80, DNA-PKcs, XLF, Artemis, XRCC4, and DNA Ligase 4 (Lig4) . XRCC4, Lig4, and possibly XLF form a complex to catalyze the end-ligation step of NHEJ [34, 36]. Germline deletion of NHEJ results in severe combined immune deficiency due to inability to complete V(D)J recombination [4, 7]. Conditional deletion of Xrcc4 or Lig4 in peripheral B cells reduces the CSR level and causes a high level of chromosomal breaks and translocations at the Igh locus due to inability to repair AID-initiated DSBs [15, 37]. While defective DSB repair leads to genomic instability, cell cycle checkpoints can protect organisms from adverse downstream effects, such as transformation, by eliminating damaged cells. As DSB repair and checkpoint mechanisms complement each other, loss of both can cause dramatic predisposition to transformation in mouse lymphocytes, often leading to lymphomas due to the inappropriate repair of programmed or general DSBs . For instance, deficiency of Xrcc4, Lig4, and Xrcc6 (Ku70) in conjunction with Trp53 deficiency causes pro-B cell lymphomas carrying co-amplified Igh-c-myc loci [39–43]. TP53 is a well-known tumor suppressor gene, which encodes p53 protein capable of responding to diverse cellular stresses by regulating the expression of its target genes, thereby inducing cell cycle arrest, apoptosis, or senescence, modulating DNA repair or metabolism and serving as the guardian of the genome [44–46].
We previously showed that conditionally deleting Xrcc4 in Trp53-deficient peripheral B cells resulted in the development of surface Ig negative lymphomas from editing and switching B cells (termed CXP lymphomas) . Although CXP tumors have mature B cell characteristics, they appear to be very different from human mature B cell lymphomas. For instance, CXP lymphomas do not express IgH or IgL chain protein on the surface or intracellularly and show no SHM in the rearranged VDJ exon . In contrast, most of human mature B cell lymphomas are surface Ig positive except classical Hodgkin’s lymphoma and a few others . These differences suggest that the mechanism of lymphomagenesis and the developmental stage of tumor progenitors are very different between CXP and human mature B cell lymphomas. Such difference may be due to the relatively early deletion of Xrcc4 via CD21cre. CD21 begins to be expressed between the immature and the mature B cell stages, specifically in transitional B cells . Thus, in mice performing CD21cre-mediated Xrcc4 deletion, it is likely that some DSBs are generated before the cells are recruited into the GC reaction. In the current study, we delete Xrcc4 and Trp53 at a later stage of mature B cell development during the GC reaction, which leads to B cell lymphomas that possess GC B cell features and harbor frequent Ig loci translocations, ongoing DNA damage and a high level of clonal heterogeneity.
Deletion of Xrcc4 but not Trp53 via Cγ1cre leads to a high level of genomic instability at the Igh locus
Combined deletion of Xrcc4 and Trp53 via Cγ1cre predisposes B cells to lymphomagenesis
Phenotypic characterization of G1XP lymphomas
We performed Southern blotting to assay G1XP tumor DNA for Igh rearrangements and found that 7 out of 11 analyzable primary G1XP tumors from the first cohort exhibited clonal Igh rearrangements, along with varying degrees of a germline JH band (Additional file 1: Figure S1C). However, the germline band usually occurred in low levels indicating derivation from non-B lineage cells within the tumor. Consistently, the tumor samples analyzed from the second cohort also displayed clonal rearrangements of the JH allele (Fig. 2b). Histological analysis of the tumor samples showed that the lymphoma cells possessed diffusely enlarged cytoplasm, salient nuclei, and nucleoli (Fig. 2c), consistent with immunoblastic B cells. Phenotypic characterization of G1XP lymphomas was performed by flow cytometry (Additional file 1: Table S1). We found that all of the lymphoma samples consistently expressed B220, CD24, CD38, CD43, CD93, and CD138. The majority of lymphomas also expressed PNA, a marker for GC B cells (see below), and about 40 % of them were surface IgG positive (Additional file 1: Table S1).
To test whether G1XP lymphomas derived from B cells that had undergone SHM/CSR, we cloned the VDJH exons from three G1XP lymphomas using a PCR approach . We found that G1XP tumors contained structurally normal in-frame or out-of-frame V(D)J rearrangements; more importantly, we sequenced these VDJH exons and downstream JH introns and found that these three tumors harbored 13, 1, and 3 mutations, respectively, in the JH regions (Additional file 1: Figure S2). These results further confirm that inactivation of Xrcc4 and Trp53 via Cγ1cre leads to B cell lymphomas capable of undergoing SHM. Thus, we conclude that the deletion of both Xrcc4 and Trp53 genes via Cγ1Cre results in the development of novel B cell lymphomas.
Analysis of Ig loci translocation junctions identifies known and novel translocation partners
Notably, an interesting pattern emerged among these CTXs that apparently separated into two categories. First, all of the 10 CTXs occurring around S or C regions of the Igh locus were translocated to the c-myc or plasmacytoma variant translocation 1 (Pvt1) locus on chromosome 15 (8 vs 2, respectively) (Fig. 3a). The translocation breakpoints at the c-myc locus clustered in the 5′ non-coding regions on chromosome 15qD1 (Fig. 3b, Additional file 1: Table S2). Second, all of the 10 CTXs occurring close to V gene segments of Igh, Igκ, and Igλ were translocated to various chromosomes involving random genetic or intergenic regions (Fig. 3a) (Additional file 1: Table S2). This intriguing pattern of translocation partners suggests that distinct mechanisms operate to mediate these two categories of translocations, probably involving AID vs RAGs, respectively. NGS data revealed a novel translocation partner of Igh locus, which targeted the Pvt1 locus (Fig. 3c), located about 30 kb telomeric of the c-myc locus. The Pvt1 locus contains a long non-coding RNA gene, which is a frequent translocation partner in mouse plasmacytomas and variants of human Burkitt’s lymphomas (BL) [50, 51]. NGS data identified Igh breakpoints located within Sμ or Sα regions, strongly implicating the involvement of AID (Fig. 3d). The junctional sequences harbored micro-homology (MH), an indicator of alternative end-joining (A-EJ) [35, 52]. Taken together, our data suggest that Ig loci translocations are likely caused by AID or RAG activity.
Apart from CTX junctions, we also identified V(D)J recombination junctions from our NGS data. Since Xrcc4 was deleted in the GC B cells, these lymphoma cells harbored normal D-J or V-D-J recombination junctions at the Igh locus (Additional file 1: Figure S3, junction 1 and 2, respectively). In contrast, we detected aberrant Igλ locus V-J rearrangements, which harbored large deletions of Vλ1 or Jλ3 exon including 41 bp of Vλ1 exon and the entire Jλ3 exon (Additional file 1: Figure S3, junction 3). These data are consistent with our previous results showing that CD21cre-mediated Xrcc4 deletion led to aberrant Igλ rearrangements [15, 47]. We identified MH at the V-J junction (Additional file 1: Figure S3, junction 3), consistent with the involvement of A-EJ. Taken together, our data suggest that these aberrant Igλ rearrangements occurred in the absence of Xrcc4, probably in the context of secondary V(D)J recombination.
Validation of clonal translocations involving Ig loci in G1XP lymphomas
G1XP lymphomas harbor ongoing DNA damage and a high level of clonal heterogeneity
A high level of genomic complexity and clonal heterogeneity may contribute to relapse or therapy resistance [54, 55]; however, key determinants regulating their generation have not been clearly addressed. In the current study, we establish a unique lymphoma model by specifically deleting Xrcc4 and Trp53 in the subset of B cells proposed to be prone to lymphomagenesis, namely, GC B cells . Our mutant mouse B cells spontaneously develop B cell lymphomas, and we employed multiple approaches to characterize their genomic instability. Our studies reveal several important discoveries: (1) Ig loci translocations can be attributed to distinct mechanisms including AID- or RAG-associated DSBs in mature B cells; (2) AID-associated Igh translocations target oncogenes such as c-myc whereas RAG-associated translocations appear to involve random genomic loci; and (3) G1XP lymphomas harbor complicated genomes including segmental translocations, and exhibit a high level of ongoing DNA damage and clonal heterogeneity. Taken together, we propose that combined NHEJ and p53 defects may serve as an underlying mechanism for a high level of genomic complexity and clonal heterogeneity in cancers.
The NHEJ and p53 deficiency models have made significant contributions to our understanding of translocation and lymphomagenesis, more importantly, the molecular mechanism of DNA repair [15, 37, 39–43, 47]. Emerging evidence suggests that defects in DSB repair can lead to oncogenic genomic instability and, in support of this notion, mutations in DNA break repair factors are implicated in a number of human tumors, including breast, colon, and lung cancers . In addition, somatic mutations in NHEJ factors have been identified in different types of human tumors including hypomorphic mutations of Artemis in EBV-associated lymphomas , mutations of Lig4 or XLF associated with non-Hodgkin’s diffuse large B cell lymphoma [58–60], and mutations of DNA-PKcs in glioblastoma and lung cancer . TP53 mutations were associated with human BL, its leukemic counterpart L3-type B cell acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia (CLL), and, in particular, its stage of progression known as Richter’s transformation . Richter syndrome (RS) is characterized by the transformation of CLL to high-grade non-Hodgkin’s lymphoma. Consistently, a recent study by performing a comprehensive molecular characterization of 86 pathologically proven RS reveals that TP53 disruption (47.1 %) and c-myc abnormalities (26.2 %) were the most frequent alterations in RS , both of which are present in our models. Therefore, it is likely that defects in both NHEJ and p53 or in the modulators of these pathways may contribute to the development of human lymphomas, at least, a subset of them.
Our NGS data identified Igh translocation partners, c-myc and Pvt-1, which are often observed in BL and a subset of diffuse large B cell lymphomas [51, 63–67]. Thus, our model might provide a unique platform to better elucidate the molecular mechanisms of translocations in B cell lymphomagenesis. Prior studies demonstrate an important role of AID in promoting translocations in B cells . We and others also prove that the NHEJ deficiency-induced Igh locus instability  or the generation of Igh-c-myc translocation is completely dependent on AID . Consistently, we found that the majority of Igh translocations in G1XP lymphomas probably originated from AID-initiated DSBs, further solidifying its role in inducing Igh locus genomic instability. Furthermore, we find that half of Ig translocations occur in close proximity to V gene segments in the Igh, Igκ, or Igλ locus, strongly implicating these translocations catalyzed by RAGs. Notably, the partners of these Ig V gene translocations are random genetic loci or intergenic regions scattered all over the genome. We suggest that the generation of such translocations probably is largely influenced by mechanistic factors , such as the increased frequency of RAG-mediated DSBs at the Igh or Igl locus in the context of secondary V(D)J recombination. In this regard, these results are consistent with our previous findings that a small percentage of peripheral B cells harbor RAG-dependent Igλ breaks/translocations in the absence of Xrcc4 . Thus, our conclusion is further corroborated that mature B lymphocytes can undergo secondary V(D)J recombination, which may contribute to mature B cell lymphomagenesis.
Our data reveal that Trp53 deficiency is essential to cause B cell lymphomas; however, Trp53 deficiency per se does not increase the level of DSBs markedly. Thus, we propose that Trp53 deficiency enhances the tolerance threshold of B cells for genomic instability induced by DNA repair deficiency in our model, thereby predisposing to lymphomagenesis. Consistent with our hypothesis, it has been shown that, in response to DSBs, p53 is phosphorylated and activated by ATM , then monitors DSBs in the context of G1 checkpoints, and signals arrest and/or apoptosis . Trp53 deficient mice usually succumb to thymic lymphomas that are aneuploid but lack translocations [72–75]. Of note, CD21cre-mediated deletion of Trp53 in peripheral B cells results in the development of mature B cell lymphomas (IgM+) that lack recurrent clonal translocations involving Ig or c-myc loci . Overall, these findings support our hypothesis that Trp53 deficiency enables B cells to tolerate genomic instability. Furthermore, we propose that the regulation of genomic instability tolerance is more p53-dependent in B cells than in other cell lineages. This notion is supported by the findings that NHEJ/p53 germline deficient mice developed only pro-B cell lymphomas [39, 43]. Thus, our unique mouse model may facilitate the discovery of critical components of p53-mediated effector cascades that regulate genomic instability tolerance. Furthermore, we were able to establish cell lines from our lymphoma model (data not shown), which would facilitate subsequent studies. Addressing these fundamental questions potentially identifies targets that specifically attack cancer cells with unstable genomes, while leaving genetically stable normal cells unaffected.
With regard to the potential of our model in clinical applications, such as biomarkers for diagnosis and therapy , we suggest that our unique model might potentially provide novel insights into the biomarker development in predicting the onset of the B cell lymphomas, given that this lymphoma model has a relatively long latency and low penetrance. In addition, novel therapies have been developed rapidly to treat B cell lymphomas or CLL, for example, Ibrutinib and new agents are effective for TP53 mutant lymphoma cells; thus, there is the potential of clinical applications of our lymphoma model for testing new agents [78–80]. Mechanistically, it would be of interest to elucidate which signaling pathway is required for the survival of these lymphoma cells.
Deletion of Xrcc4 and Trp53 via Cγ1Cre leads to novel B cell lymphomas that appear to derive from GC B cells. These B cell lymphomas harbor ongoing DNA damage and exhibit a high level of clonal heterogeneity for characteristic c-myc translocations. We propose that combined NHEJ and p53 defects may serve as an underlying mechanism for a high level of genomic complexity and clonal heterogeneity in cancers.
Generation of mouse models
Cγ1Cre knock-in (KI) mice , Xrcc4 , or Trp53  conditional knock-out (KO) mice were generated previously. These mice were in mixed genetic background of C57BL/6, 129/Ola, and FVB/N [37, 49, 81]. Animal work was approved by the Institutional Animal Care and Use Committee of University of Colorado Anschutz Medical Campus (Aurora, CO), National Jewish Health (Denver, CO), and Children’s Hospital in Boston (Boston, MA).
B cell culture, FISH, and Southern Blot analysis
Splenic B cells were isolated from naïve mice, purified by negative selection kit (Stem Cell Technologies, Canada), activated with anti-CD40 and IL4 as described previously , and collected 4 days after culture for metaphase preparation and FISH analysis. FISH analysis was performed with specific BAC probes as previously described  (see details in Additional file 1). Genomic DNA was isolated from tumor masses or normal tissues from control mice, and Southern blotting was performed as previously described .
H&E staining, flow cytometry, and H2AX foci staining
Tumor masses usually presented in the abdomen and were adjacent to gut-associated lymphoid tissues such as mesenteric lymph nodes or Peyer’s patch. Tumors were dissected, fixed in 10 % formalin, and processed for H&E histology staining. Tumor single-cell suspensions were prepared and subjected to flow cytometry for phenotypic characterization. Samples were analyzed using a FACSCalibur (BD Bioscience), and FACS analysis was performed with FlowJo software. Single-cell suspensions prepared from tumors or wt naïve or GC B cells were stained for γ-H2AX foci according to the manufacturer’s instructions (BD Bioscience). The generation of splenic GC B cells was induced by in vivo immunization as described previously .
NGS library preparation, sequencing platform, and data analysis
Tumor DNA samples were employed to generate the NGS paired-end library using the standard TruSeq DNA library preparation kit (Illumina, San Diego, CA). The libraries were subjected to whole genome sequencing on the Illumina Hi-Seq 2000 platform (pair-ended, 2 × 100 bp per read). Details of NGS analysis are provided in the Additional file 1 including usage of CREST software  and generation of Circos plots .
We thank Drs. Frederick W. Alt for his generous support of this study and Ryan T. Phan for his contribution to the model. We thank Drs. Klaus Rajewsky for the Cγ1Cre KI mice and Xiayuan Liang and Zenggang Pan for the histology analysis. We thank Dr. Roberta Pelanda for the critical reading of the manuscript. NGS experiments were performed by the Genomics and Microarray Core Facility at University of Colorado Anschutz Medical Campus. This work was supported by the University of Colorado School of Medicine and Cancer Center startup funds, a Boettcher Foundation Webb-Waring Biomedical Research Award, an American Society of Hematology Scholar Award, a fund from Cancer League of Colorado, NIH-R21CA184707, and NIH-R01CA166325 to JHW and MTE is supported by NIH-3R01CA166325-02S1.
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