non-Ig Loci Research Articles

RAG1/2 induces double-stranded DNA breaks at non-Ig loci in the proximity of single sequence repeats in developing B cells.

In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.

Somatic mutation patterns at Ig and Non-Ig Loci

The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non-Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non-Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non-Ig loci.

Genomic Panorama and Clonal Evolutionary Trajectory of Classical Hairy Cell Leukemia

Classical Hairy Cell Leukemia (HCLc) is a rare B-cell malignancy characterized by hairy-like cell membrane projections. The cell of origin and pathway of malignant transformation in HCLc remain unknown. Early work using whole exome sequencing identified BRAFV600E as a genetic hallmark in HCLc (Tiacci et al 2011 and Weston-Bell et al 2016). However, no other recurrent coding mutations appeared, marking it an uncommon monogenic cancer. As BRAFV600E appears insufficient to drive tumorigenesis in other cancers (Dankort et al 2009; Rad et al 2013), it suggested that other driver alterations may be implicated in HCLc pathogenesis, perhaps in the non-coding genome, overlooked in exome studies alone. To probe this, we carried out whole genome sequencing (WGS) in HCLc. We established the genomic panorama, and importantly leveraged data to derive tumor phylogenies to determine the relative order of early driver mutational events that associate with the most recent common ancestor (MRCA) of all tumor cells. WGS was performed in 10 typical HCLc cases each paired with germline in purified cell sorted populations of disaggregated splenocytes. Sequencing involved 150 bp paired ends on Illumina HiSeq4000. Data analyses included identification of somatic single-nucleotide variants (SNVs) and small indels using CaVEMan and Pindel, respectively; copy-number aberrations (CNA) with Battenberg; and structural variants (SVs) with BRASS. The fitting of COSMIC mutational signatures was performed using mmsig. Mutational SNVs and CNA drivers were used for the analysis of tumor phylogenesis of individual tumors. We mapped the whole genome landscape in HCLc. Somatic coding variants emerged as previously reported. BRAFV600E was present as a clonal mutation in all HCLc tumors. Missense mutations in KLF2 (p.S275N and p.S275I) were identified in 2 tumors. Two somatic SNVs were detected in PRSS3 (p.D95G and p.R94S) and ANKRD30B (p.K388E and p.N392K) in 2 different cases respectively. In the noncoding genome, BCL6 and ZFP36L1 promoters were recurrently mutated in 5/10 cases. Driver mutations in BCL6 promoters have been reported in other B-cell malignancies to disrupt negative autoregulatory circuits (Pasqualucci et al 2003). Analysis of CNA showed a relatively stable genome (ploidy ~2). A large CNA chr5 gain was observed in a single tumor. Two additional focal CNA were spotlighted at chr17q25 (Gain) and chr20q11 (LOH) in two individual tumors. A recurrent interchromosomal translocation at chr12q14.3 locus was identified in 5 individual tumors, and an identical translocation at 14q21.3 with 15q11.2 observed in 2 tumors. Interestingly, novel kataegis-like mutations were observed at TRB loci across all HCLc cohort samples. We further identified somatic hypermutation (SHM) imprints in non-Ig loci, notably in BCL6. The mutational rate of AID targets at TRB and non-Ig loci were comparable to SHM occurring in Ig IGV genes. Deciphering the mutational signatures in HCLc genomes highlighted SBS9, a mutational signature associated with SHM in B-cell leukemia as one of the most relevant mechanisms, allied to the clock-like signatures of SBS1 and SBS5. Next, we utilized estimates of the cancer cell fraction to define which mutations are clonal and associate with the MRCA, and which mutations occured subclonally to derive individual tumor phylogenetic trees using methodology we have reported previously (Rabbie et al 2020; Ansari-Pour 2021). Here, analyses of clonal trajectories revealed that BRAFV600E occurred prior to subclonal diversification in MRCA in all cases. Significantly however, 1 or 2 additional driver mutations were conjointly detected with BRAFV600E in the MRCA in 6/10 HCLc cases prior to any subclonal evolution. Our data provide a comprehensive map of the whole genome in HCLc for the first time. Clonal evolutionary trajectories reveal heterogeneous genetic origins in HCLc, in which BRAFV600E is the sole driver mutation in one subset of disease, but requires 1 or 2 additional mutational drivers in the other subset to promote malignant transformation. Thus, unlike other cancers, BRAFV600E alone appears to suffice to induce malignant transformation in some HCLc. Additionally, SHM in non-Ig loci including BCL6 may implicate early traffic of the tumor cell of origin through the germinal center and early exit prior to acquisition of CD27, or mutational events at ectopic sites.

Mantle cell lymphomas with concomitant MYC and CCND1 breakpoints are recurrently TdT positive and frequently show high-grade pathological and genetic features.

Chromosomal breakpoints involving the MYC gene locus, frequently referred to as MYC rearrangements (MYC - R+), are a diagnostic hallmark of Burkitt lymphoma and recurrent in many other subtypes of B-cell lymphomas including follicular lymphoma, diffuse large B-cell lymphoma and other high-grade B-cell lymphomas and are associated with an aggressive clinical course. In remarkable contrast, in MCL, only few MYC - R+ cases have yet been described. In the current study, we have retrospectively analysed 16 samples (MYC - R+, n = 15, MYC - R-, n = 1) from 13 patients and describe their morphological, immunophenotypic and (molecular) genetic features and clonal evolution patterns. Thirteen out of fifteen MYC - R+ samples showed a non-classical cytology including pleomorphic (centroblastic, immunoblastic), anaplastic or blastoid. MYC translocation partners were IG-loci in 4/11 and non-IG loci in 7/11 analysed cases. The involved IG-loci included IGH in 3 cases and IGL in one case. PAX5 was the non-IG partner in 2/7 patients. The MYC - R+ MCL reported herein frequently displayed characteristics associated with an aggressive clinical course including high genomic-complexity (6/7 samples), frequent deletions involving the CDKN2A locus (7/10 samples), high Ki-67 proliferation index (12/13 samples) and frequent P53 expression (13/13 samples). Of note, in 4/14 samples, SOX11 was not or only focally expressed and 3/13 samples showed focal or diffuse TdT-positivity presenting a diagnostic challenge as these features could point to a differential diagnosis of diffuse large B-cell lymphoma and/or lymphoblastic lymphoma/leukaemia.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting invivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

Templated mutagenesis in B cell non-Ig genes

Abstract The immunoglobulin genes of germinal center B cells (GCB) undergo somatic hypermutation in order to generate antibody diversity. Additionally, several non-immunoglobulin (non-Ig) genes are known to accumulate mutations in the germinal center. There are two methods by which mutations are introduced at the Ig and non-Ig loci. The first is through the accumulation of pseudorandom point mutations as a result of activation-induced cytidine deaminase (AID)-dependent double stranded breaks (DSB). The second is through the process of gene conversion, which involves the use of a template sequence to repair a DSB resulting in the template being copied at the site of repair. Findings from our lab suggest that some mutations in the non-Ig genes of human GCB arise through a gene conversion-like mechanism, referred to as templated mutagenesis. We sorted 4 B cell populations, including light zone and dark zone GCB from a young human tonsil and sequenced ~500bp sections from non-Ig genes including FAS, RHOH, and BTG1 via Illumina MiSeq. Previously, our lab has developed a computational pipeline called TRACE, that is able to match mutation clusters within an input sequence to partially homologous donor templates elsewhere in the genome. TRACE-identified donor templates are statistically likely to have been used in a templated mutagenesis event which led to the mutation of the gene. Through this analysis, we found that unlike gene conversion observed in chickens, human templated mutagenesis occurs between sequences on different chromosomes. Furthermore, donor template sequences tend to originate in introns and intergenic regions, not exons. These findings have opened an avenue into the study of non-Ig templated mutagenesis in human B cells.

Chromoplexy and Chromothripsis Are Important Prognostically in Myeloma and Deregulate Gene Function By a Range of Mechanisms

Background: Structural variants are key recurrent molecular features of myeloma (MM) with two types of complex rearrangement, chromoplexy and chromothripsis, having been described recently. The contribution of these to MM prognosis, rapid changes in clinical behavior and punctuated evolution is currently unknown as is the mechanism by which they deregulate gene function. Methods: We analyzed two sets of newly diagnosed MM data: 85 cases with phased whole genome sequencing; and 812 cases from CoMMpass where long-insert whole-genome sequencing was available. Patient derived xenografts from five MM cases were used to generate epigenetic maps for the histone marks, BRD4, MED1, H3K27Ac, H3K4me1, H3K4me3, H3K9me3, H3K36me3 and H3K27me3. Results: In the 10X data the median number of structural events per case was 25 (range 1 - 182); with a median of 14 intra-chromosomal events (range 1 - 179; P<0.001) and 7 inter-chromosomal events (range 0 - 29). Structural events were seen most frequently on chromosomes 14 (64%), 8 (53%), 1 (44%) and 6 (42%). Complex chromosomal rearrangements involving 3 or more chromosomal sites were seen in 46%, 4 or more sites in 20%, 5 or more in 10% and 6 or more in 5% of samples. There were significantly more structural events in the t(4;14) subgroup compared to the t(11;14) subgroup. Significantly more events were also seen in the bi-allelically inactivated TP53 cases. Using an elbow test defined cutoff, we identified cases with high structural variant load in 10% of cases. Chromoplexy called by "Chainfinder" was seen in 18% of cases. Chromothripsis called by "Shatterseek" was seen in 9% of cases. Cases with a high structural load alone were not associated with an adverse outcome whereas cases with chromoplexy or chromothripsis were associated with adverse PFS and OS, p=0.001. A new high-risk subgroup comprising approximately 5% of cases was identified with chromoplexy, chromothripsis and a high structural load. Gene set enrichment analysis of cases with chromoplexy and chromothripsis showed an excess of MYC, E2F and G2M targets, and a reduction in RAS signaling. Interferon a and g responses, an excess of TP53 and reduction in TRAF3 mutations was associated predominantly with chromothripsis. How chromoplexy and chromothripsis are tolerated by the cell is unknown and the association with the cGAS/STING response is further being explored. To determine how chromoplexy may deregulate multiple genes we identified the full spectrum of structural variants to the immunoglobulin (Ig) and non-Ig loci. A range of genes are deregulated by Ig loci including MAP3K14 at a frequency of 2% confirming the importance of non-canonical NFkB signaling. A novel intra-chromosomal rearrangement to ZFP36L1 was upregulated in 10% of cases but was not prognostic. Gene upregulation by non-Ig super enhancers is frequent and targets include PAX5, GLI3, CD40, NFKB1, MAP3K14, LRRC37A, LIPG, PHLDA3, ZNF267, CENPF, SLC44A2, MIER1, SOX30, TMEM258, PPIL1, and BUB3. The topologically associating domain (TADs) containing super enhancers bringing about gene deregulation include TXNDC5, FOXO3, FCHSD2, SP2, FAM46C, CACNA1C, TLCD2 and PIK3C2G. These super enhancers frequently contain important MM genes, the coding sequence of which are disrupted by the rearrangement and could contribute to the clinical phenotype. Accurately reconstructing the structure of the complex rearrangements will allow us to identify the mechanism of gene deregulation and to distinguish between either gene stacking, receptor stacking or both. Conclusions: Upregulation of gene expression by super enhancer rearrangement is a major mechanism of gene deregulation in MM and complex structural events contribute significantly to adverse prognosis by a range of mechanisms as well as simple gene overexpression. Disclosures Boyle: Amgen, Abbvie, Janssen, Takeda, Celgene Corporation: Honoraria; Amgen, Janssen, Takeda, Celgene Corporation: Other: Travel expenses. Walker:Celgene: Research Funding. Thakurta:Celgene: Employment, Equity Ownership. Flynt:Celgene Corporation: Employment, Equity Ownership. Davies:Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor; Janssen, Celgene: Other: Research Grant, Research Funding. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding.

Comprehensive detection of chromosomal translocations in lymphoproliferative disorders by massively parallel sequencing

Balanced translocations have diagnostic and prognostic value in B-cell lymphoproliferative disorders (LPDs). Most of these translocations involve the juxtaposition of a strong immunoglobulin (Ig) enhancer to proto-oncogenes, such as BCL2, BCL6, and MYC, leading to their overexpression. These rearrangements generally do not result in mRNA fusions, and fluorescent in situ hybridization (FISH) remains the gold standard for assessing of recurrent translocations in LPDs. With the growing use of massively parallel sequencing for the detection of both point mutations and large structural rearrangements, we aimed at evaluating the utility of this method for the molecular work-up of B-cell LPDs side by side with FISH. We describe a method using solution capture for enrichment of known translocation breakpoints and massively parallel sequencing for the detection of balanced translocation in formalin-fixed tissues with a B-cell LPD. We detected a total of 57 rearrangements with a high concordance of 94.2% when compared to FISH. We detected translocations between BCL2, BCL6, and MYC and the three Ig loci and non-Ig loci, including novel partners for MYC and BCL6. In addition, massively parallel sequencing allowed a detailed analysis of the structure of the resulting chromosomal fusions. Our comparison shows the feasibility of using massively parallel sequencing for detecting balanced translocations in B-cell LPDs and advantages and disadvantages to both methods, and how they can complement each other.

Elucidation of distinct mutational patterns between diffuse large B cell lymphoma subtypes utilizing circulating tumor DNA.

7538 Background: Patients with diffuse large B cell lymphoma (DLBCL) exhibit significant differences in clinical outcome based on cell-of-origin (COO). Patients are categorized as having germinal-center-like (GCB) or activated-B-cell-like (ABC) disease based on RNA microarray and histopathological analyses of tumor biopsies. We recently described an accurate sequencing-based method for determination of COO in DLBCL utilizing stereotyped differences in mutations (Scherer et al., 2016). Here, we further explore the mutational patterns in patients with differing molecular subtypes of DLBCL based on sequencing of circulating tumor DNA. Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) to pretreatment plasma samples and matched germline from a cohort of 115 patients with DLBCL. We then identified somatic alterations, which were used to determine COO molecular subtypes as previously described. Finally, we compared mutational patterns in patients with GCB and non-GCB DLBCL. Results: We detected a significantly greater number of total mutations (GCB: 1766 ± 160 mutations per Mb of targeted sequencing; non-GCB: 1364 ± 150 mutations per Mb of targeted sequencing; p < 0.05) and coding mutations (GCB: 145 ± 21 mutations per Mb of targeted sequencing; non-GCB: 28 ± 8.5 mutations per Mb of targeted sequencing; p < 0.001), particularly in immunoglobulin (Ig) regions (p < 0.05). In addition, GCB and non-GCB samples exhibited distinct mutational patterns within Ig regions. GCB samples were enriched for mutations in regions of switch mu (Sμ) (p < 0.01) and IGHV2-70 (p < 0.01), while non-GCB samples were enriched for mutations in regions of IGHG3 (p < 0.03), IGHV4-34 (p < 0.03), and IGLL5 (p < 0.05). GCB samples were also significantly enriched for coding mutations in SOCS1 (p < 0.01), a gene not included in our original COO classifier. Conclusions: Patients with GCB and non-GCB DLBCL exhibit distinct mutational patterns across both Ig and non-Ig loci of the genome. These differences in mutational patterns can be used to classify molecular subtypes noninvasively, potentially providing further utility to noninvasive genotyping and liquid biopsies.

Novel IGH and MYC Translocation Partners in Diffuse Large B-Cell Lymphomas.

Chromosomal translocations involving an immunoglobulin (IG) locus and a proto-oncogene play a major role in diffuse large B-cell lymphoma (DLBCL) pathogenesis. Recurrent IG translocation partners in DLBCL are the BCL6, BCL2, and MYC genes, but other rare translocation partners are also known. We studied 20 DLBCL with fluorescence in situ hybridization-based evidence for IG heavy chain (IGH) locus-associated translocations not involving BCL6, BCL2, MALT1, or MYC by long distance inverse PCR to identify the translocation partners. Moreover, we studied eight DLBCL with MYC translocations not involving IG or known non-IG loci as translocation partner to search for novel MYC translocations. We identified three novel IGH-associated translocations. Chromosomal breakpoints involved the IMMP2L gene in 7q31, the BCAS2 gene in 1p13, and the PVRL2 gene in 19q13. The latter gene, which is recurrently translocated in T-cell lymphomas, is significantly higher expressed in the biopsy with the translocation compared to cases without this genetic aberration, indicating a pathogenetic role of PVRL2 also in DLBCL. In one case with a MYC break we obtained a novel MYC-SOCS1 translocation representing an unusual translocation of a proto-oncogene with a tumor suppressor gene. Indeed, we demonstrate that the oncogene was deregulated and the tumor suppressor gene inactivated. As both genes undergo aberrant somatic hypermutation in the region of the chromosomal breakpoints, this translocation likely happened as a byproduct of the hypermutation process. Overall, our study suggests that chromosomal translocations in DLBCL are more heterogeneous than previously known. © 2016 Wiley Periodicals, Inc.

PI3Kdelta Inhibitors Increase Genomic Instability By Upregulating Aid Expression

▪Activation-induced cytidine deaminase (AID) is a B cell-specific enzyme that initiates class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes, essential mechanisms to generate different classes of antibody and antibody diversity for the antigens. At lower frequency, AID also promiscuously introduces DNA structural lesions at non-Ig loci and it is involved in the pathogenesis of B cell lymphoma. Thus, its expression is tightly controlled in B cells to limit its genotoxic effects. Phosphatidylinositol 3-kinase (PI3K) p110δ isoform acts downstream of the B-Cell Receptor (BCR) to suppress AID expression, whereas blockade of PI3K signaling enhances the expression of AID. Potent oral PI3K inhibitors such as the p110δ inhibitor idelalisib (GS-1101, CAL-101) have been recently approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenstrom macroglobulinemia (WM), whereas dual PI3K p110δ and γ inhibitors such as duvelisib (IPI-145) showed promising results for the treatment of CLL and other hematologic malignancies.Here we investigated whether the increased of AID expression caused by idelalisib or duvelisib induced genomic instability in normal and malignant B cells.We observed that in splenic purified mouse B cells activated with IL4 and αCD40 to induce AID expression and CSR, treatment with idelalisib and duvelisib significantly increased both AID protein and mRNA levels, compared to controls. As a result, both drugs strongly promoted CSR. The regulation of AID expression was strictly dependent on PI3K p110δ activity because a selective p110γ inhibitor (AS-604850) did not have any effect. In contrast, B cells expressing a constitutively active PI3Kδ showed suppression of CSR and reduced AID expression compared to inactive PI3Kδ.To gain insights into the degree of genomic instability induced by increased AID expression upon PI3Kδ inhibition, we applied a genome-wide translocation technique we previously developed (High-Throughput Genomic Translocation Sequencing approach, HTGTS) to identify translocation partners from DNA double strand breaks (DSBs) introduced into the c-myc locus (Chiarle et al, Cell 2011). By HTGTS, we isolated thousands of translocations from activated primary mouse B cells distributed widely across the genome. Remarkably, in B cells treated with idelalisib or duvelisib, we identified a significantly higher number of translocations in known AID off-target genes as well as novel hotspots of translocations (48 for idelalisib, 50 for duvelisib). Unbiased genome-wide analysis of translocation formation revealed a consistency in the translocation patterns, with AID target hotspots localized in the TSS region and predominantly grouped within super-enhancers and regulatory clusters. HTGTS analysis performed on activated AID knock-out (AID KO) B cells showed that the vast majority of these translocations were dependent on AID. Hence, our data demonstrate that in normal B cells PI3Kδ inhibitors increase genomic instability by an AID-dependent mechanism.Finally, we observed that both idelalisib and duvelisib increased AID expression in human EBV-immortalized and lymphoma cell lines (MCL and CLL). We adapted the HTGTS technique to human cells by introducing DSBs in the human myc locus by CRISPR/Cas9 technology and sequencing genome-wide the formation of chromosomal translocations. By this approach, we demonstrated that also in human neoplastic B cells the treatment with idelalisib or duvelisib increased the formation of translocations to known AID off targets.In conclusion, we showed that idelalisib or duvelisib increase genomic instability in normal and neoplastic B cells by enhancing AID expression. Since several B cell malignancies imply treatment with these drugs for years, these effects of PI3Kδ inhibitors on the genomic stability of B cells should be carefully taken into account for therapeutic outcomes and protocol design. DisclosuresNo relevant conflicts of interest to declare.

AID-initiated DNA lesions are differentially processed in distinct B cell populations (IRM8P.701)

Abstract Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at immunoglobulin (Ig) loci. How AID-initiated lesions are prevented from inducing genome-wide damage is not completely understood. Prior studies proposed a differential DNA repair mechanism that protects certain non-Ig loci such as c-myc from AID attack. However, determinants that regulate such protective mechanisms remain largely unknown. To test whether target DNA sequences might modulate protective mechanisms via modifying the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance genomic instability of c-myc, due to error-free repair of AID-initiated lesions, in antigen-stimulated germinal center B cells. In contrast, cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Our findings reveal a complex interplay between target sequence, DNA repair mechanism, and cellular context associated with differential transcription, which coordinately regulate genome stability. Our studies could provide mechanistic insight into differential routes to AID-mediated genetic alterations in distinct subtypes of common B cell lymphomas.

Generation and repair of AID-initiated DNA lesions in B lymphocytes.

Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions.

Protecting the Genome: Mechanisms Targeting Somatic Hypermutation

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes to support the generation of high affinity antibodies through affinity maturation. SHM is quite dangerous, having been shown to affect many non-Ig genes in germinal center and malignant B cells. Nonetheless, the reaction exhibits a strong preference for Ig loci over non-Ig loci. The targeting mechanism responsible for this preference has remained elusive despite many years of study. We and others have used the chicken Ig light chain (IgL) locus and the chicken Dt40 B cell line as a model system for the study of SHM targeting. This system has several advantages, including efficient homologous recombination and a compact IgL locus, making the search for targeting sequences easier than in the large, complex mammalian Ig loci. We and others have reported that sequences downstream of the chicken IgL constant region are cis-acting SHM targeting sequences, which we refer to as Diversification Activator (DIVAC). Dissection of the chicken DIVAC region reveals that targeting activity is dispersed among numerous sequence elements that function in an additive, synergistic, and/or redundant manner. We find that the E-box motif (CANNTG) is critical for DIVAC function, but has no detectable DIVAC activity unless embedded in the appropriate sequence context. We have recently developed a more sensitive mutation reporter assay in Dt40 cells that allows us to assess the targeting activity of short DNA sequence elements. Two "core" portions of the chicken DIVAC region, both evolutionarily conserved among avian species, have particularly strong SHM targeting activity: the IgL enhancer, and a downstream region rich in transcription factor binding sites characteristic of Ig enhancers. Remarkably, some human and mouse Ig locus enhancers have even stronger DIVAC function in our Dt40 assay than the chicken core sequences. In the mouse Igλ locus, where the canonical enhancers have relatively weak SHM targeting activity, strongly active "shadow enhancers" work synergistically with the canonical enhancers to create powerful DIVAC elements. DIVAC fragments enhance SHM in a position- and orientation-independent manner, and deletion or mutation of certain E-box, NFκB, Mef2, PU.1, or Ets family binding sites – all known to be important for the transcriptional role of Ig enhancers – impair or abolish DIVAC activity. Importantly, the increase in SHM mediated by DIVAC is not mediated by an increase in transcription. We conclude that Ig enhancers and shadow enhancers are the central components of the cis-acting DNA sequences that target SHM to Ig loci, that targeting relies on cooperation between multiple well-known transcription factor binding motifs, and that the targeting mechanism is likely to be conserved from birds to mammals. We are currently investigating whether these insights can be used to understand the widespread mistargeting of SHM that occurs in both normal and malignant B cells. Since multiple myeloma (MM) cells can be induced to express activation-induced cytidine deaminase (AID), we are also studying the targeting of AID in the MM genome and how AID might contribute to the progression of the disease. Disclosures:No relevant conflicts of interest to declare.

Identification of the Novel Chimeric Gene, PVT1-WWOX, in Multiple Myeloma with 8q24 Abnormality

Abstract 1823Genetic abnormalities play a crucial role in the pathogenesis of various malignancies, including multiple myeloma (MM). Secondary cytogenetic abnormalities implicated in MM progression include 8q24 rearrangements, gain of the long arm of chromosome 1 (1q+), and loss of the short arm of chromosome 17 (17p-). The 8q24 rearrangements, including MYC and PVT1, have been identified by conventional cytogenetic analysis in 3.5–5.0% of MM patients and by fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) in 9.5–20%. 8q24 rearrangements are frequently associated with advanced disease in MM patients and MM cell lines. Ig chromosomal translocations, such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% of MMs with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14 and 16q22, in which no candidate genes have been delineated so far, have also been identified as translocation partners. MYC has long been a possible candidate target gene for 8q24 rearrangements; however, many of the breakpoints within 8q24 have been assigned to various regions that encompassed more than 2 Mb centromeric or telomeric to MYC.We have previously found frequent PVT1 rearrangements in MM. PVT1 rearrangements were detected in 7 of 12 patients (58.3%) and in 5 of 8 cell lines (62.5%) with 8q24 abnormalities. A combination of SKY, FISH, and oligonucleotide array identified several partner loci of PVT1 rearrangements, such as 4p16, 4q13, 13q13, 14q32 and 16q23–24, and identified a chimeric gene, PVT1 - NBEA, resulting in high expression of abnormal NBEA in a cell line with t(8;13)(q24;q13), AMU-MM1, suggesting PVT1 rearrangements play significant roles in myelomagenesis.In this study, we analyzed RPMI8226 cell line in detail to identify other partner genes of PVT1 in these partner loci. SKY analysis revealed a complex karyotype including der(16)t(16;22)ins(16;8)(q23;q24) in this cell line. Oligonucleotide array analysis clearly demonstrated that the copy number change at 8q24 occurred within intron 1 of PVT1, and at 16q23, the copy number change occurred within intron 8 of WWOX, indicating that the translocation breakpoints of 8q24 and 16q23 were within intron 1 of PVT1 and intron 8 of WWOX, respectively. Based on these results, RT-PCR analysis was performed to detect chimeric products and direct sequencing of this product revealed the fusion of 5’-PVT1 exon 1 with WWOX exon 9-3’. The expression level of WWOX exon 9 was higher than WWOX exon 8–9 detected by semi-quantitative RT-PCR in RPMI8226, suggesting that high expression of WWOX derived from PVT1 - WWOX chimeric transcript, like PVT1 - NBEA.WWOX is generally considered to be a candidate tumor suppressor gene, and known to have a proapoptotic effect by participating in the tumor necrosis factor (TNF) apoptotic pathway and via direct physical interaction with p53 and its homolog p73. However, immunohistochemistry revealed that WWOX protein level were rather elevated in gastric and breast carcinoma. Therefore, WWOX seemed not to act as tumor suppressor gene simply. Although both NBEA and WWOX are located at common fragile site, usually contributing to gene inactivation, FRA13A and FRA16D respectively, these genes highly express via fusion to PVT1. These findings indicate that PVT1 rearrangements play significant roles in myelomagenesis via translocation and fusion to cancer-related genes. Disclosures:No relevant conflicts of interest to declare.

Aid-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Differentiation Stages in a Locus-Dependent Manner.

Abstract 2376Activation induced deaminase (AID) initiates U:G mismatches that are subsequently converted into point mutations or DNA double-stranded breaks. AID-mediated DNA alterations in switch (S) regions at the Igh locus frequently occur in both antigen-stimulated germinal center (GC) B cells and cytokine-activated B cells. To investigate whether AID-initiated U:G lesions are differentially processed in a differentiation stage-specific manner at non-Ig loci to maintain genome stability, we established a knock-in model by inserting an Sg2b region into the first intron of proto-oncogene c-myc. We found that the inserted Sg2b region mutated at an extremely low level and did not enhance genomic instability of c-myc locus in antigen-stimulated GC B cells. In contrast, the inserted Sg2b region mutated more frequently and increased c-myc locus abnormalities in cytokine-activated B cells. Furthermore, uracil glycosylase deficiency led to increased mutation frequency at the c-myc locus. These results reveal that AID-initiated lesions are differentially processed via error-free or error-prone repair in a differentiation stage-specific and locus-dependent manner. Our data might provide mechanistic explanation for differential frequency of AID-mediated genetic alterations in distinct subtypes of common B cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Frequent Involvement of PVT1 in Multiple Myeloma Carrying 8q24 Rearrangement and Identification of Novel PVT1-NBEA Chimeric Gene,

Abstract 3917The 8q24 rearrangement has been identified in 3.5–5.0 % of multiple myeloma (MM) patients with conventional cytogenetic analyses, such as G-banding, and in 9.5–20% of MM patients with fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) techniques. Of note, 8q24 rearrangements accompany with advanced MM patients and MM cell lines more frequently. Chromosomal translocation of the immunoglobulin gene (Ig), such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% in MM with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14, and 16q22, have been also identified as translocation partners where no candidate genes have been delineated so far. In this study, we precisely investigate molecular features of chromosomal 8q24 rearrangements to access pathophysiology of MM. FISH, SKY, and RT-PCR analyses were performed as described previously. DNA gain and loss assay based on oligonucleotide array (GeneChip Human Mapping 50K, 250K, or 6.0 array, Affymetrix) was performed on the genomic DNA extracted from MM cells. Oligonucleotide array data was analyzed using the CNAG3.0 or 3.3 programs. Various types of 8q24 rearrangements were detected in 12 (22.2 %) of 54 MM patients and 8 (72.7 %) of 11 MM cell lines by means of FISH procedure. A breakpoint cluster of approximately 360 kb region containing myelocytomatosis oncogene (MYC) and plasmacytoma variant translocation 1 (PVT1) genes at 8q24 was divided into three subclusters according to rearranged region, that is, PVT1 region, region which is centromerically adjacent to PVT1 (including MYC), and region which is 120kb centromeric to MYC. Seven of 12 patient-derived cells and 5 of 8 cell lines with 8q24 abnormalities showed PVT1 rearrangements with various partner loci, such as 4p16, 4q13, 13q13, 14q32, or 16q23–24 in SKY combined with FISH analyses (SKY-FISH). Oligonucleotide array combined with SKY analysis delineated the candidate genes within partner loci of 8q24 rearrangements by mapping boundary breakpoints of copy number gains and losses, identifying MMSET, EPHA5, NBEA, and WWOX as candidate genes at 4p16, 4q13, 13q13, and 16q23–24, respectively. We identified the novel chimeric gene PVT1-NBEA in AMU-MM1 cell line with t(8;13)(q24;q13). The PVT1-NBEA fusion transcript in which PVT1 exon 1 was fused to NBEA exon 3 was accompanied with overexpression of abnormal NBEA lacking its part of N-terminus. In conclusion, PVT1 rearrangement may play pivotal roles in pathophysiology of MM harboring 8q24 abnormalities. Disclosures:No relevant conflicts of interest to declare.

Translocation-Capture Sequencing Reveals the Extent and Nature of Chromosomal Rearrangements in B Lymphocytes

Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma.

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.

Clinical and Pathological Features of Non-Hodgkin Lymphomas Harboring Concurrent t(14;18) and 8q24 Anomalies

AbstractAbstract 3134 Background:Concurrent t(14;18) and 8q24 translocations involving BCL2 and MYC in non-Hodgkin lymphomas (NHL) are rare, but are associated with a inferior overall survival (OS) regardless of the presenting or antecedent histological features (Johnson N, et al. Blood 2009). We sought to confirm these observations in an independent cohort of patients with NHL. Methods:Metaphase karyotypes and/or fluorescence in-situ hybridization (FISH) were used to identify cases of NHL with cytogenetic abnormalities involving 18q21 and 8q24 (BCL2 and MYC). Clinical and cytogenetic characteristics of these patients were assessed for correlations with pathological and clinical variables including outcome. Histological diagnoses were determined according to the 2008 World Health Organization Classification. Overall survival (OS) was calculated from the date a biopsy demonstrated an abnormality involving MYC to the last follow up date or death, as some cases acquired these lesions during their clonal evolution. Result:Among ∼1700 NHL patients diagnosed and/or treated at Stanford University Medical Center on whom cytogenetic studies were routinely performed, we identified 26 patients with evidence for concurrent cytogenetic abnormalities involving BCL2 and MYC. The histological diagnoses at the time of BCL2 and MYC rearrangements were available for 25 of the patients and included follicular lymphoma (FL1-2, n=3; FL3A, n=4), diffuse large B-cell lymphoma (DLBCL, n=2), and B cell lymphoma, unclassifiable, with features intermediate between Burkitt Lymphoma and DLBCL (BCLU, n=16).Cytogenetic analysis revealed that 13/26 cases with both BCL2 and MYC rearrangements had MYC translocations involving the immunoglobulin (Ig) loci. However, in striking contrast to the <10% prevalence of IgL or IgK partners in NHL harboring Ig-MYC, without Ig-BCL2 rearrangements, 9 of these13 cases (69%) involved IgL t(8;22), and only 4 of these 13 cases (31%) involved IgH t(8;14). 9/26 MYC translocations involved various non-Ig loci (35%). One case (1/26) demonstrated amplification of ?both the BCL2 and the MYC loci by FISH, while one case showed trisomy 8 (1/26). Three cases had FISH analysis only, precluding assessment of the MYC rearrangement partner. No correlation between MYC partner and histology was observed, although this analysis was limited by small sample sizes. Non-FL histology correlated with significantly poorer than expected outcome, with a median OS of 4 months compared to 2 y for FL (p=0.003).Interestingly, we found cases with low-grade follicular lymphoma (FL1-2) histology which harbored both BCL2 and MYC rearrangements. To better define the frequency of 8q24 anomalies in an unselected cohort of follicular lymphoma cases, FISH for MYC and BCL2 rearrangements was performed on 192 independent low-grade follicular lymphoma (FL1-2) cases. While a subset of these cases (71) showed the expected frequency of BCL2 rearrangements (72%) by FISH, none showed concomitant 8q24 anomalies by FISH, suggesting that routine FISH analysis of low-grade follicular lymphoma for MYC rearrangements would have a low diagnostic yield. Conclusion:NHL with rearrangements of both BCL2 and MYC are under-recognized due to lack of routine karyotyping or FISH analysis. Comprehensive analysis of BCL2 and MYC status should be performed on all HGBCL in the new WHO (2008) classification, though the low frequency of these lesions among FL tumors precludes a similar conclusion for low grade NHL. Disclosures:No relevant conflicts of interest to declare.