ALBERT

Description: Somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation induced cytidine deaminase (AID). Resulting uracil-guanine (U-G) mismatches are processed by UNG-dependent base-excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with B cell lymphomas, the role of downstream AID-associated DNA repair pathways in lymphomagenesis is not defined. Through deregulated expression of BCL6, IµHABcl6 mice develop an AID-dependent GC-derived lymphoma that resembles human diffuse large B cell lymphoma (DLBCL). We have previously demonstrated that IµHABcl6 Ung-/-Msh2-/- mice have a similar incidence (35% vs 27%) but a 2.5-fold shorter median time to development of B220+ IgM+ PNAhi CD138- DLBCL compared with IµHABcl6 mice (6.5 months vs. 16.2 months; P = 0.0003). This suggests that AID-associated DNA repair pathways serve to protect the GC B cell and delay BCL6-driven lymphomagenesis. To investigate the individual contribution of BER and MMR in the pathogenesis of GC-derived lymphoma, we have now generated IµHABcl6 Ung-/- and IµHABcl6 Msh2-/- single-deficient mice. The majority of IµHABcl6 Ung-/- mice remained healthy beyond 20 months with only 3 of 22 (13.6%) mice becoming sick starting at ∼16 months. Sick mice were found to have splenic lymphomas comprised of mature B220+ IgM+ PNAlow CD138- B cells. Histological examination revealed expanded follicles with a population of small lymphocytes, consistent with a follicular B cell lymphoma which has been shown to arise in Ung-/- mice. In contrast, 18 of 22 (81.8%) IµHABcl6 Msh2-/- mice rapidly succumbed to malignancy starting at ∼3 months and had a median survival of 6 months. Of 15 tumors available for analysis, there was 1 histiocytic sarcoma, 1 squamous cell carcinoma, 4 T cell lymphomas, and 9 B220+ IgM- PNA- CD138- pre-B cell lymphomas (determined by histology, immunophenotyping and gene expression profiling). None of the IµHABcl6 Ung-/- or IµHABcl6 Msh2-/- mice developed DLBCL. Since lack of UNG is strongly protective when MSH2 is present, we conclude that in the setting of deregulated BCL6, UNG promotes the development of DLBCL. In contrast, MSH2 is protective against the development of tumors in general and does not facilitate DLBCL in the absence of UNG. Combined with the observation that IµHABcl6 Ung-/-Msh2-/- mice develop DLBCL with a significantly shorter latency than IµHABcl6 mice, this data indicates that a complex interplay between AID-associated BER and MMR produces a net protective effect against lymphomagenesis. In the absence of UNG and MSH2, AID-generated U-G mismatches are not processed into strand lesions and are simply replicated, yielding C/G to T/A transition mutations. Thus, to assess how combined lack of UNG and MSH2 might promote the accelerated development of BCL6-driven lymphoma, we carried out spectral karyotyping and sequence analysis of AID target genes (IgJH4, cMyc, Pim1, RhoH, Cd79a, CD79b, H2afx, Pax5, and Cd83) in lymphomas from the different genotypes. IµHABcl6 DLBCLs (3/3) harbored various complex chromosome abnormalities, consistent with previous findings. Numerous clonal and sub-clonal chromosome abnormalities including translocations, duplications, deletions, and aneuploidies were also detected in IµHABcl6 Ung-/-Msh2-/- (4/4) and IµHABcl6 Ung-/- (2/2) lymphomas. Pre-B cell tumors from IµHABcl6 Msh2-/- mice could not be stimulated to produce metaphase chromosomes. Clonal and non-clonal mutations of the IgJH4 intronic region were identified in lymphomas from IµHABcl6 (2/3), IµHABcl6 Ung-/-Msh2-/- (4/4), and IµHABcl6 Ung-/- (2/3) mice, consistent with ongoing AID activity. No mutations were detected in 3 pre-B cell lymphomas, consistent with their pre-GC origin. Six clonal mutations within AID hotspots (all C/G to T/A) were identified in Pim1, RhoH, and Pax5 in 2 of 4 IµHABcl6 Ung-/-Msh2-/- DLBCLs. None of the other genotypes carried any clonal mutations of non-Ig genes. Thus, chromosome abnormalities in GC B cell lymphomas can arise through mechanisms independent of BER and MMR but may be due to off-target effects of AID on other genes that regulate cell cycle, apoptosis, or genomic stability. Disclosures: No relevant conflicts of interest to declare.

Description: DNA methylation and demethylation at cytosine residues are epigenetic modifications that regulate gene expression associated with early cell development, somatic cell differentiation, cellular reprogramming and malignant transformation. While the process of DNA methylation and maintenance by DNA methyltransferases is well described, the nature of DNA demethylation remains poorly understood. The current model of DNA demethylation proposes modification of 5-methylcytosine followed by DNA repair-dependent cytosine substitution. Although there is debate on the extent of its involvement in DNA demethylation, activation-induced cytidine deaminase (AID) has recently emerged as an enzyme that is capable of deaminating 5-methylcytosine to thymine, creating a T:G mismatch which can be repaired back to cytosine through DNA repair pathways. AID is expressed at low levels in many tissue types but is most highly expressed in germinal center B cells where it deaminates cytidine to uracil during somatic hypermutation and class switch recombination of the immunoglobulin genes. In addition to this critical role in immune diversification, aberrant targeting of AID contributes to oncogenic point mutations and chromosome translocations associated with B cell malignancies. Thus, to explore a role for AID in DNA demethylation in B cell lymphoma, we performed genome-wide methylation profiling in BL2 and AID-deficient (AID-/-) BL2 cell lines (Burkitt lymphoma that can be induced to express high levels of AID). Using Illumina’s Infinium II DNA Methylation assay combined with the Infinium Human Methylation 450 Bead Chip, we analyzed over 450,000 methylation (CpG) sites at single nucleotide resolution in each line. BL2 AID-/- cells had a median average beta value (ratio of the methylated probe intensity to overall intensity) of 0.76 compared with 0.73 in AID-expressing BL2 cells (P 〈 0.00001), indicating a significant reduction in global methylation in the presence of AID. Using a delta average beta value of ≥ 0.3 (high stringency cut-off whereby a difference of 0.3 or more defines a CpG site as hypomethylated), we identified 5883 CpG sites in 3347 genes that were hypomethylated in BL2 versus BL2 AID-/- cells. Using the Illumina HumanHT-12 v4 Expression BeadChip and Genome Studio software, we then integrated gene expression and methylation profiles from both lines to generate a list of genes that met the following criteria: 1) contained at least 4 methylation sites within the first 1500 bases downstream of the primary transcriptional start site (TSS 1500; AID is most active in this region during somatic hypermutation); 2) average beta value increased by 〉0.1 in the TSS 1500 region in BL2 compared with BL2 AID-/- cells; and 3) down-regulated by 〉50% in BL2 compared with BL2 AID-/- cells. This analysis identified 31 candidate genes targeted for AID-dependent demethylation with consequent changes in gene expression. Interestingly, 15 of these genes have been reported to be bound by AID in association with stalled RNA polymerase II in activated mouse B cells. After validating methylation status in a subset of genes (APOBEC3B, BIN1, DEM1, GRN, GNPDA1) through bisulfite sequencing, we selected DEM1 for further analysis. DEM1 encodes an exonuclease involved in DNA repair and contains 16 CpG sites within its TSS1500, with only one site 〉50% methylated in BL2 cells compared with 8 of 16 in BL2 AID-/- cells. To assess a direct role for AID in DEM1 methylation status, a retroviral construct (AIDΔL189-L198ER) driving tamoxifen-inducible expression of a C-terminal deletion mutant of AID (increases time spent in the nucleus) was introduced into BL2 AID-/- cells. BL2, BL2 AID-/-, and BL2 AIDΔL189-L198ER cells were cultured continuously for 21 days in the presence of tamoxifen, 100 nM. Bisulfite sequencing of DEM1 TSS 1500 did not demonstrate any significant changes in methylation at day 7. However, at day 21, 13 of the 16 DEM1 TSS 1500 methylation sites in BL2 AIDΔL189-L198ER cells were found to have an increase in the ratio of unmethylated to methylated clones ~10-25% above that of BL2 AID-/- cells. By qPCR, this correlated with a 1.75-fold increase in DEM1 gene expression to levels that were equivalent to that seen in BL2 cells (P = 0.003). Although further investigations are needed, this data supports the notion that AID is able to regulate target gene expression in B cell malignancy through active DNA demethylation. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 397 Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER (Ung−/−) and MMR (Msh2−/−). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung−/−Msh2−/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung−/−Msh2−/−mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p

Description: Abstract 3244 Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. AID converts cytosine to uracil and resulting U-G mismatches are subsequently processed by low-fidelity base excision and mismatch repair pathways to yield point mutations (for SHM) and DNA strand breaks (for CSR). Under normal conditions, genotoxic stressors activate DNA damage response pathways that result in DNA repair or cell cycle arrest and apoptosis. However, in normal germinal center B cells, key DNA damage checkpoint factors such as ATR, p53 and p21 are repressed by the germinal center transcriptional repressor BCL6. BCL6 thus creates a permissive environment to allow the accumulation of mutations within the Ig loci. However, in addition to this fundamental role in immune diversification, aberrant targeting of AID contributes to point mutations and translocations of proto-oncogenes associated with B cell malignancy. Indeed, the combined effect of BCL6 and AID poses a direct threat to genomic integrity but the mechanism responsible for regulating the mutation threshold in germinal center B cells is not understood. To determine if B cells have a mechanism for down-regulating AID activity in response to genotoxic stress, we subjected the Ramos-A23 cell line to continuous low-dose exposure to several genotoxic agents. Ramos-A23 is a Burkitt lymphoma line that constitutively expresses AID and mutates the Ig heavy chain variable region with high frequency. Mutation frequency can be monitored by flow cytometry through loss of surface expression of IgM (due to AID-dependent nonsense and missense mutations) and by direct sequencing of the variable region. IgM+ Ramos-A23 cells were maintained in continuous culture in the presence or absence of etoposide (100 nM), cytarabine (5 nM), 5-azacytidine (10 nM), or trichostatin A (15 nM). Drug concentrations were titrated to the highest dose that would minimally affect proliferation or survival when compared with untreated cells. After 4 weeks in culture, untreated clones were 20–22% IgM-, consistent with ongoing SHM. In contrast, clones treated with the DNA damaging agents etoposide or cytarabine were only 4–6% IgM- (p

Description: Tumor microenvironment (TME) is commonly implicated in regulating the growth of tumors, but whether it can directly alter the genetics of tumors is not known. Genomic instability and dendritic cell (DC) infiltration are common features of several cancers, including multiple myeloma (MM). Mechanisms underlying genomic instability in MM are largely unknown. Here, we show that interaction between myeloma and DCs, but not monocytes, leads to rapid induction of the genomic mutator activation-induced cytidine deaminase (AID) and AID-dependent DNA double-strand breaks (DSBs) in myeloma cell lines as well as primary MM cells. Both myeloid as well as plasmacytoid DCs have the capacity to induce AID in tumor cells. The induction of AID and DSBs in tumor cells by DCs requires DC-tumor contact and is inhibited by blockade of receptor activator of NF-κB/receptor activator of NF-κB ligand (RANKL) interactions. AID-mediated genomic damage led to altered tumorigenicity and indolent behavior of tumor cells in vivo. These data show a novel pathway for the capacity of DCs in the TME to regulate genomic integrity. DC-mediated induction of AID and resultant genomic damage may therefore serve as a double-edged sword and be targeted by approaches such as RANKL inhibition already in the clinic.