ALBERT

Description: Somatic hypermutation (SHM) is a natural process that introduces point mutations into immunoglobulin (Ig) genes during antibody affinity maturation. In addition to this fundamental role in immune diversification, aberrant targeting of SHM contributes to translocations and point mutations of proto-oncogenes associated with B cell malignancy. During the first phase of SHM, the enzyme activation induced cytidine deaminase (AID) converts cytosine (C) to uracil (U) to result in a U-G mismatch. Spontaneous U-G mismatches are normally corrected by high fidelity DNA repair pathways. However, during the second phase of SHM, U-G mismatches are processed by low fidelity base excision and mismatch repair pathways to yield mutations. These second phase pathways are initiated by recognition of the uracil by uracil DNA glycosylase (UNG) and MSH2/MSH6. As a DNA mutator, AID poses a direct threat to genomic integrity but the mechanisms responsible for guiding AID to its genetic target in the first phase and determining whether a mutation will be repaired in a high fidelity or low fidelity manner during the second phase are not understood. Numerous cellular processes are regulated by small inhibitory RNA (siRNA) and microRNA (miRNA) molecules through a mechanism known as RNA interference (RNAi). To test the hypothesis that small RNA molecules are involved in SHM, we introduced two natural inhibitors of RNAi into the DT40 B cell line. DT40 cells express high levels of AID and mutate their Ig loci with high frequency. The protein 3′hExo is a siRNase that has been found to be a general inhibitor of RNAi. VA1 is a non-coding adenoviral RNA that disrupts RNAi by inhibiting nuclear export of pre-miRNA and by direct inhibition of Dicer. Following retroviral infection of 3′hExo or VA1 and empty vector controls into DT40 cells, 24 clones from each group were expanded from single cells over 28 days. DT40 cells are IgM− due to a mutation in the Ig heavy chain gene. When this mutation is corrected by AID activity, they become IgM+, allowing mutation frequency to be followed by flow cytometric analysis of IgM reversion. Compared with mock infected cells and cells infected with empty vector, cells that over-expressed 3′hExo or VA1 demonstrated a 3 and 2-fold reduction in IgM reversion, respectively. Similarly, sequencing of the Ig light chain gene from a minimum of three clones from each group revealed mutation frequencies of 6.2×10−4 mutations/nucleotide (nt) in mock infected cells, 6.8×10−4 mutations/nt in empty vector controls, 1.8×10−4 mutations/nt in cells over-expressing 3′hExo, and 2.7×10−4 mutations/nt in cells over-expressing VA1. No difference in the type of mutations or nucleotide bias was observed. Comparable reductions in RNAi activity with over-expression of 3′hExo or VA1 in DT40 cells were observed in siRNA reporter assays. Thus, general inhibition of RNAi in DT40 cells caused a 2 to 3-fold reduction in AID-mediated mutation events (P

Description: MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLLPTD/WT genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLLPTD/WT blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.

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: Following reports of childhood acute myeloid leukemia (AML) showing that patients with t(9; 11)(p22; q23) have a better prognosis than those with translocations between 11q23 and other chromosomes, we compared response to therapy and survival of 24 adult de novo AML patients with t(9; 11) with those of 23 patients with other 11q23 translocations [t(11q23)]. Apart from a higher proportion of French-American-British (FAB) M5 subtype in the t(9; 11) group (83% v 43%, P = .006), the patients with t(9; 11) did not differ significantly from patients with t(11q23) in terms of their presenting clinical or hematologic features. Patients with t(9; 11) more frequently had an extra chromosome(s) 8 or 8q as secondary abnormalities (46% v 9%, P = .008). All patients received standard cytarabine and daunorubicin induction therapy, and most of them also received cytarabine-based intensification treatment. Two patients, both with t(9; 11), underwent bone marrow transplantation (BMT) in first complete remission (CR). Nineteen patients (79%) with t(9; 11) and 13 (57%) with t(11q23) achieved a CR (P = .13). The clinical outcome of patients with t(9; 11) was significantly better: the median CR duration was 10.7 versus 8.9 months (P = .02), median event-free survival was 6.2 versus 2.2 months (P = .009), and median survival was 13.2 versus 7.7 months (P = .009). All patients with t(11q23) have died, whereas seven (29%) patients with t(9; 11) remain alive in first CR. Seven of eight patients with t(9; 11) who received postremission regimens with cytarabine at a dose of 100 (four patients) or 400 mg/m2 (2 patients) or who did not receive postremission therapy (2 patients) have relapsed. In contrast, 7 (64%) of 11 patients who received intensive postremission chemotherapy with high-dose cytarabine (at a dose 3 g/m2) (5 patients), or underwent BMT (2 patients) remain in continuous CR. We conclude that the outcome of adults with de novo AML and t(9; 11) is more favorable than that of adults with other 11q23 translocations; this is especially true for t(9; 11) patients who receive intensive postremission therapy.

Description: Abstract 94 Somatic hypermutation (SHM) is a natural process that introduces point mutations into immunoglobulin (Ig) genes during antibody affinity maturation. During the first phase of SHM, the enzyme activation-induced cytidine deaminase (AID) converts cytosine (C) to uracil (U) to result in a U-G mismatch. Spontaneous U-G mismatches are normally corrected by high-fidelity DNA repair pathways. However, during the second phase of SHM, U-G mismatches are processed by low-fidelity DNA repair pathways to yield mutations. These second phase pathways are initiated by recognition of the uracil by uracil DNA glycosylase (UNG) and MSH2/MSH6. As a DNA mutator, AID poses a direct threat to genomic integrity, a notion supported by reports demonstrating that aberrant targeting of AID contributes to translocations and point mutations of proto-oncogenes associated with B cell malignancy. Transgenic mice with constitutive and ubiquitous expression of Aid (Aid-Tg) provide a unique system for investigating the poorly understood dynamics of Aid targeting and DNA repair. In these animals, development of B and T lymphocytes is indistinguishable from that of wild-type littermates. However, within 5 to 7 months, a subset of Aid-Tg mice develop monoclonal T cell lymphomas with high levels of SHM of the T cell receptor locus (Tcr), cMyc, Pim1, Cd4 and Cd5 genes. Interestingly, these animals do not develop B cell malignancies, suggesting that B cells have a specific mechanism for regulating Aid activity and/or high-fidelity versus low-fidelity DNA repair of non-Ig genes. To further investigate this mechanism, we crossed Aid-Tg mice with Ung−/−Msh2−/− double knockout mice. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not repaired and are simply replicated, leaving a footprint of Aid targeting in the form of C/G to T/A transition mutations. Our plan was to compare Aid targeting (Aid-Tg Ung−/−Msh2−/−) and DNA repair (Aid-Tg) patterns between ‘normal' splenic B and T cells, prior to the development of genomic instability and malignant transformation. Splenic B and T cells from 4-month-old Aid-Tg and Aid-Tg Ung−/−Msh2−/− mice were obtained by fluorescence activated cell sorting. Genomic DNA was prepared and a ∼1-kilobase (kb) region spanning the first 1.5-kb downstream of the major promoter of cMyc, Pim1 and H2afx was PCR-amplified and sequenced (∼40 to 60-kb/gene). Surprisingly, mutation frequencies of all genes were found to be extremely low in all cell types (range, 8.4 × 10−6 to 7.8 × 10−5 mutations/bp) and well below the mutation frequencies of 4 × 10−4 to 9 × 10−4 mutations/bp we have previously seen for these genes in germinal center B cells from Ung−/−Msh2−/− mice. In fact, these mutation frequencies approach the background mutation frequency attributable to the sequencing procedure of 1.6 × 10−5 mutations/bp (previously determined from Aid−/− mice). Additional sequencing in the T cells also did not demonstrate any mutations in Cd4 or Tcrb, contrasting the high mutation frequencies found in tumors from Aid-Tg mice. To explore the possible cause of these unexpected results, we proceeded with further analysis of Aid expression in our system. Resting splenic B and T cells were obtained from wild-type and Aid-Tg mice and divided into one group that was used to make resting total cellular RNA and protein lysates and a second group that was activated ex vivo with lipopolysaccharide and IL4 (for B cells) or anti-CD3/CD28 beads and IL2 (for T cells). Real-time RT-PCR demonstrated high and equal expression of Aid transcript in resting and activated Aid-Tg B and T cells that was ∼3 to 4-fold higher than activated wild-type B cells. Analysis of Aid expression by Western blotting demonstrated that Aid protein is expressed at equal levels in activated wild-type and Aid-Tg B cells but is undetectable in resting wild-type B and T cells, resting Aid-Tg B and T cells, and activated Aid-Tg T cells. Thus, the Aid transgene is highly transcribed but likely fails to initiate SHM of known target genes in splenic B and T cells because Aid protein is absent. Our findings suggest that, in this model, B and T cells have an internal mechanism for negatively regulating Aid protein, preventing aberrant SHM and malignant transformation. Further investigation is needed to determine whether Aid protein is regulated at the level of mRNA translation or protein stability and how this mechanism breaks down during the pathogenesis of lymphoid malignancy. Disclosures: No relevant conflicts of interest to declare.