Ayurvedic herbal medicine and lead poisoning

Although the majority of published cases of lead poisoning come from occupational exposures, some traditional remedies may also contain toxic amounts of lead. Ayurveda is a system of traditional medicine that is native to India and is used in many parts of world as an alternative to standard treatment regimens. Here, we report the case of a 58-year-old woman who presented with abdominal pain, anemia, liver function abnormalities, and an elevated blood lead level. The patient was found to have been taking the Ayurvedic medicine Jambrulin prior to presentation. Chemical analysis of the medication showed high levels of lead. Following treatment with an oral chelating agent, the patient's symptoms resolved and laboratory abnormalities normalized. This case highlights the need for increased awareness that some Ayurvedic medicines may contain potentially harmful levels of heavy metals and people who use them are at risk of developing associated toxicities

Negative Regulation of Activation-Induced Cytidine Deaminase Protein Prevents Aberrant Somatic Hypermutation and Lymphomagenesis.

Abstract 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. </jats:sec

Imatinib Resistance and Progression of CML to Blast Crisis: Somatic Hypermutation AIDing the Way

Very little is known about how acquired oncogenic mutations arise. In this issue of Cancer Cell, Klemm and colleagues present evidence supporting a role for the antibody diversification enzyme activation-induced deaminase (AID) in the generation of mutations associated with disease progression and drug resistance in chronic myeloid leukemia

A Role for Small RNA Molecules during the DNA Repair Phase of Somatic Hypermutation

Abstract 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&lt;0.001). Neither 3′hExo nor VA1 had a significant effect on the rate of cell proliferation, and Ig gene expression and AID expression were equivalent in all experimental groups. To distinguish a role for small RNA molecules in AID targeting versus repair of the resulting U-G mismatch, we performed the same experiments in UNG-deficient DT40 cells. In the absence of UNG, U-G mismatches are not recognized or repaired but are simply replicated, revealing the footprint of AID activity in the form of C to T transition mutations. In contrast to wild type DT40 cells, over-expression of 3′hExo or VA1 in UNG-deficient DT40 cells did not result in a reduction in mutation frequency and, as expected, all observed mutations were C to T transitions. Thus, in UNG-deficient cells, AID targeting was intact despite abrogation of the RNAi pathway by 3′hExo and VA1 (confirmed by siRNA reporter assay). This suggests that the reduced mutation frequency when 3′hExo or VA1 is over-expressed in wild type DT40 cells may be due to augmented high fidelity DNA repair rather than reduced AID targeting. Based on these results, we propose that small RNA molecules may play a role in mediating the balance of high fidelity and low fidelity DNA repair during SHM. This balance must be carefully maintained in order to preserve genomic integrity without compromising immune diversification. Further characterization of this mechanism is critical to our understanding of how these processes break down during malignant transformation of B cells.</jats:p

Regulation of the Mutation Threshold During Immune Diversification by Activation Induced Cytidine Deaminase,

Abstract 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&lt;0.0001). Cells treated with the epigenetic modifiers 5-azacytidine or trichostatin A were 15–19% IgM-. Sequence analysis of the Ig variable region (3 clones from each group) demonstrated 6.2 and 3.8-fold reductions in mutation frequency in cells treated with etoposide and cytarabine, respectively, when compared with untreated cells. The mutation frequency in the cells treated with epigenetic modifiers did not differ significantly from untreated controls. This suggests that while epigenetic stress has little effect on SHM, genotoxic stress leads to a significant reduction in AID activity. It has previously been demonstrated that genotoxic stress leads to an ATM-dependent down-regulation of BCL6, a finding that we also observed in our model. In addition, we found that genotoxic stress causes a dose and time-dependent decrease in AID protein and transcript levels, thus explaining the decrease in SHM in our system. Like BCL6, repression of AID by genotoxic stress was inhibited by pre-treating the cells with the ATM inhibitor KU-55933. Further analysis of markers of B cell differentiation revealed up-regulation of IRF4 and Blimp1 with complete down-regulation of PAX5 (a transcriptional regulator of AID) in response to genotoxic stress. Lentiviral expression of BCL6 shRNA in Ramos cells also lead to down-regulation of AID and SHM, consistent with the observation that BCL6-deficient B cells do not undergo SHM. It has also recently been demonstrated that DNA damage in germinal center B cells leads to ATM-dependent inactivation of the CREB transcriptional co-activator CRTC2 with subsequent down-regulation of CRTC2 target genes, including AID. Altogether, these data suggest that, during the germinal center response, a mutation threshold recognized by ATM leads to repression of the SHM machinery and reprogramming to facilitate B cell maturation. Additional investigation is needed to further define the critical steps in this regulatory pathway and how it might breakdown during the pathogenesis of B cell malignancy. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Activation Induced Cytidine Deaminase-Associated DNA Repair Pathways Influence Germinal Center B Cell Lymphomagenesis

Abstract 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. </jats:sec

The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Abstract 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&lt;0.0001). All 5 of the Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung−/−Msh2−/−mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung−/−Msh2−/−mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung−/−Msh2−/−tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung−/−Msh2−/−mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung−/−Msh2−/−tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes (cMyc, Pim1, RhoH, Pax5, Cd79a, Fas, H2ax and OcaB) in tumors from 3 Iμ-Bcl6 Ung−/−Msh2−/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10−4, suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung−/− and Iμ-Bcl6 Msh2−/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures: No relevant conflicts of interest to declare. </jats:sec

DNA Demethylation By Activation-Induced Cytidine Deaminase in B Cell Lymphoma

Abstract 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 &lt; 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 &gt;0.1 in the TSS 1500 region in BL2 compared with BL2 AID-/- cells; and 3) down-regulated by &gt;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 &gt;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. </jats:sec