Levels Of Activation-induced Cytidine Deaminase Expression Research Articles

C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency.

In activated B cells, activation‐induced cytidine deaminase (AID) generates programmed DNA lesions required for antibody class switch recombination (CSR), which may also threaten genome integrity. AID dynamically shuttles between cytoplasm and nucleus, and the majority stays in the cytoplasm due to active nuclear export mediated by its C‐terminal peptide. In immunodeficient‐patient cells expressing mutant AID lacking its C‐terminus, a catalytically active AID‐delC protein accumulates in the nucleus but nevertheless fails to support CSR. To resolve this apparent paradox, we dissected the function of AID‐delC proteins in the CSR process and found that they cannot efficiently target antibody genes. We demonstrate that AID‐delC proteins form condensates both in vivo and in vitro, dependent on its N‐terminus and on a surface arginine‐rich patch. Co‐expression of AID‐delC and wild‐type AID leads to an unbalanced nuclear AID‐delC/AID ratio, with AID‐delC proteins able to trap wild‐type AID in condensates, resulting in a dominant‐negative phenotype that could contribute to immunodeficiency. The co‐condensation model of mutant and wild‐type proteins could be an alternative explanation for the dominant‐negative effect in genetic disorders.

Abstract C14: A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer

Abstract Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We leverage a “gain-of-function” synthetic lethality approach to selectively target cancers that ectopically express Activation Induced Cytidine Deaminase (AID). AID is a DNA-directed cytidine deaminase normally transiently expressed in maturing B-lymphocytes where it plays a critical role in somatic hypermutation and immunoglobulin class switching. However, AID is dysregulated and inordinately expressed in many cancers, where it acts as a DNA-damaging enzyme mutating widespread locations throughout the genome. This activity leads to the accumulation of point mutations and DNA breaks and promotes DNA replication stress. In the latter context, AID-expressing cells become critically dependent on the homologous recombination factor RAD51 to survive DNA damage-induced replication stress. We have developed a novel small-molecule inhibitor of RAD51, CYT-0851, that is preferentially cytotoxic to AID expressing cells. We have shown in vitro and in vivo that CYT-0851 is efficacious in AID-expressing lymphoma and leukemia mouse models. Genome profiling and gene expression studies have shown many solid tumors, including pancreatic cancer, also aberrantly express AID. To determine whether targeting RAD51 may be an effective approach to the treatment of AID-expressing pancreatic cancer, we evaluated the antitumor activity of CYT-0851 in multiple patient-derived pancreatic cancer xenografts (PDX) models with a range of AID expression levels. All models tested showed tumor growth inhibition (TGI), ranging from 63 to 104% with CYT-0851 treatment. A crossover experiment was performed with one model to determine the activity of CYT-0851 in large, established tumors. We observed tumor regression (137% TGI) in large tumors upon treatment with CYT-0851, resulting in one partial responder and one tumor-free responder. The data presented here demonstrate that CYT-0851 exerts anticancer activity in multiple preclinical models of pancreatic cancer. Considering that recent published studies have shown that these cancers can ectopically overexpress AID, we conclude that CYT-0851 may be an effective treatment for pancreatic cancer. Overall, this provides a rationale for the continued development of CYT-0851 as a novel therapeutic for pancreatic cancer patients. Citation Format: Melinda Day, Jean-Marc Lapierre, Tom O'Shea, Kevin Mills. A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C14.

Exposure to 1,2-Dichloropropane Upregulates the Expression of Activation-Induced Cytidine Deaminase (AID) in Human Cholangiocytes Co-Cultured With Macrophages.

1,2-dichloropropane (1,2-DCP) was reclassified recently by IARC as a Group 1 carcinogen based on epidemiological studies on an outbreak of cholangiocarcinoma in offset-printing workers exposed to 1,2-DCP in Japan. However, the underlying mechanism of 1,2-DCP-induced cholangiocarcinoma remains obscure. A previous whole-genome mutation analysis of cholangiocarcinoma of 4 cases exposed to 1,2-DCP suggested the involvement of activation-induced cytidine deaminase (AID), based on specific signatures of mutation patterns. The objective of the present study is to determine whether exposure to 1,2-DCP induces expression of AID in human cholangiocytes. Human MMNK-1 cholangiocytes, differentiated THP-1 macrophages, and co-cultures of MMNK-1/THP-1 cells were exposed to 1,2-DCP at different concentrations and time intervals. The mRNA expression levels of AID and related genes were quantified by real-time PCR. Protein expression was measured by immunostaining. Alkaline Comet assay was performed to examine DNA damage. The results showed that 1,2-DCP alone did not change AID expression in MMNK-1 cholangiocytes. 1,2-DCP significantly increased pro-inflammatory cytokine TNF-α expression in THP-1 macrophages. TNF-α treatment upregulated expression of AID, NF-κB, and IκB in MMNK-1 cholangiocytes. SN50, a NF-κB inhibitor, significantly downregulated TNF-α-induced AID expression, suggesting the involvement of NF-κB pathway in TNF-α-induced AID expression. Exposure to 1,2-DCP significantly increased AID expression in MMNK-1 cholangiocytes co-cultured with THP-1 macrophages. Comet assay showed that 1,2-DCP-induced DNA damage in MMNK-1 cholangiocytes, as indicated by increased tail DNA% and tail moment, was enhanced when co-cultured with macrophages. The results suggest that inflammatory response of macrophages and consequent aberrant AID expression or DNA damage in the cholangiocytes underlie the mechanism of 1,2-DCP-induced cholangiocarcinoma in humans.

Expression and subcellular localisation of AID and APOBEC3 in adenoid and palatine tonsils

Activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA-editing catalytic polypeptide 3 (A3) family are cytidine deaminases that play critical roles in B-cell maturation, antiviral immunity and carcinogenesis. Adenoids and palatine tonsils are secondary lymphoid immune organs, in which AID and A3s are thought to have several physiological or pathological roles. However, the expression of AID or A3s in these organs has not been investigated. Therefore, we investigated the expression profiles of AID and A3s, using 67 samples of adenoids and palatine tonsils from patients, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical analyses. AID and A3s expression levels in the adenoids and the palatine tonsils of the same individual significantly correlated with each other. Of note, AID expression level in the adenoids negatively correlated with the age (r = −0.373, P = 0.003). The younger group with adenoid vegetation and tonsillar hypertrophy showed more abundant AID expression than the older group with recurrent tonsillitis and peritonsillar abscesses (P = 0.026). Moreover, immunohistochemical analysis revealed the distribution of AID and A3s in the epithelial cells as well as germinal centres. The localisation of AID expression and its relation to age may contribute to adenoid vegetation and inflammation.

BCR and Endosomal TLR Signals Synergize to Increase AID Expression and Establish Central B Cell Tolerance

Activation-induced cytidine deaminase (AID) is required to purge autoreactive immature and transitional-1 (immature/T1) B cells at the first tolerance checkpoint, but how AID selectively removes self-reactive B cells is unclear. We now show that B cell antigen receptor (BCR) and endosomal Toll-like receptor (TLR) signals synergize to elicit high levels of AID expression in immature/T1 B cells. This synergy is restricted to ligands for endocytic TLR and requires phospholipase-D activation, endosomal acidification, and MyD88. The first checkpoint is significantly impaired in AID- or MyD88-deficient mice and in mice doubly heterozygous for AID and MyD88, suggesting interaction of thesefactors in central B cell tolerance. Moreover, administration of chloroquine, an inhibitor of endosomal acidification, results in a failure to remove autoreactive immature/T1 B cells in mice. We propose that a BCR/TLR pathway coordinately establishes central tolerance by hyper-activating AID in immature/T1 B cells that bind ligands for endosomal TLRs.

The Correlation of APOBEC Gene Family Member Expression with Worse CLL Patient Outcome Suggests a Role in CLL Mutational Evolution

A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL.CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System.CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P <0.05) with shorter time to first treatment (TFT), which was anticipated based on previous reports. Interestingly APOBEC3B and APOBEC3F expression differences showed possible trends correlating with worse patient outcome. Therefore, we tested select APOBEC gene family members by qPCR. For qPCR, we utilized the CLL patient cohort (N= 83) previously found to indicate that AID expression was a risk factor for worse patient outcome in a multivariate analysis (Patten et al. 2012 Blood 120:4802). RNA expression by qPCR followed the same pattern as the microarray data: AID and APOBEC3B had very low levels, APOBEC3H had modest levels, and APOBEC3F and 3G had high levels. Similar to AID, patients could be grouped based on the presence or absence of detectable APOBEC3B, with its presence showing a significant correlation (P <0.05) with worse TFT and overall survival. Higher levels of APOBEC3F and 3H showed a trend towards a correlation with shorter TFT, while differences in APOBEC3G expression had no significant correlation with patient outcome. Thus, not only did we confirm the correlation of AID expression with worse patient outcome, but we also found APOBEC3B and potentially APOBEC3F and 3H correlate with worse patient outcome.To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented.In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. DisclosuresBarrientos:Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

Increased frequencies of memory and activated B cells and follicular helper T cells are positively associated with high levels of activation‑induced cytidine deaminase in patients with immunoglobulin A nephropathy.

The aim of the present study was to examine the frequencies of different subsets of B and follicular helper T (Tfh) lymphocytes in patients with immunoglobulin A nephropathy (IgAN), and investigate the potential underlying mechanism. A total of 27 patients with IgAN and 10 healthy controls (HC) were recruited for analysis of the frequencies of different subsets of B and Tfh cells. ELISA was used to analyze the concentration of serum interleukin (IL)‑21. The transcriptional levels of activation‑induced cytidine deaminase (AID) in the B cells were determined using reverse transcription‑quantitative polymerase chain reaction, while the translational levels of AID were analyzed using western blotting. The frequencies of circulating memory and activated B cells and Tfh cells were found to be significantly increased in the IgAN groups, compared with those of the HC group, although the number of plasma cells were not significantly different between the two IgAN groups. In addition, the serum levels of IL‑21 were found to be higher in the patients with IgAN, and correlated with 24‑h proteinuria. IL‑21 also enhanced the expression levels of AID in the B cells. The data of the present study revealed that the high levels of memory and activated B cells and Tfh cells were positively associated with the progression of IgAN, and that this may be mediated by the overexpression of AID, which is potentially regulated by IL‑21.

Expression of activation-induced cytidine deaminase is associated with a poor prognosis of diffuse large B cell lymphoma patients treated with CHOP-based chemotherapy

Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation and class switch recombination processes in the antibody formation. The AID activity induces gene mutations and could be associated with transformation processes of B cells. Nevertheless, the relation between AID expression and the prognosis of B cell lymphoma patients remains uncharacterized. We examined expression levels of the AID gene in 89 lymph node specimens from lymphoma and non-lymphoma patients with Northern blot analysis and investigated an association with their survival. The AID gene was preferentially expressed in B cell lymphoma in particular in diffuse large B cell lymphoma and follicular lymphoma. We confirmed AID protein expression in the mRNA-positive but not in the negative specimens with Western blot analysis and immunohistochemical staining. Survival of the patients treated with cyclophosphamide-/doxorubicin-/vincristine-/prednisone-based chemotherapy demonstrated that the prognosis of diffuse large B cell patients was unfavorable in the mRNA-positive group compared with the negative group, and that AID expression levels were correlated with the poor prognosis. In contrast, AID expression was not linked with the prognosis of follicular lymphoma patients. AID expression is a predictive marker for an unfavorable outcome in DLBCL patients treated with the chemotherapy.

AID expression in peripheral blood of children living in a malaria holoendemic region is associated with changes in B cell subsets and Epstein-Barr virus.

The development of endemic Burkitt's lymphoma (eBL) is closely associated with Epstein-Barr virus (EBV) infection and holoendemic malaria infections. The role of EBV in the development of malignancy has been studied in depth, but there is still little known about the mechanisms by which malaria affects Burkitt's lymphomagenesis. Activation induced cytidine deaminase (AID) expression is necessary for the introduction of c-myc translocations that are characteristic of BL, but a link between AID and EBV or malaria is unclear. To determine whether frequency of malaria exposure leads to increased AID expression in peripheral blood mononuclear cells (PBMC) we examined two cohorts of children in western Kenya with endemic and sporadic malaria transmission dynamics. High frequency of malaria exposure led to increased expression of AID, which coincided with decreases in the IgM(+) memory B cells. In the children from the malaria endemic region, the presence of a detectible EBV viral load was associated with higher AID expression compared to children with undetectable EBV, but this effect was not seen in children with sporadic exposure to malaria. This study demonstrates that intensity of malaria transmission correlates with AID expression levels in the presence of EBV suggesting that malaria and EBV infection have a synergistic effect on the development of c-myc translocations and BL.

A new pathway for central B cell tolerance: interaction of AID, MyD88, and BCR (IRC3P.467)

Abstract Expression of activation-induced cytidine deaminase (AID) in immature and transitional B cells in mice and humans is genetically linked to the first tolerance checkpoint. In the absence of AID, autoreactive immature/transitional-1 (T1) B cells are inefficiently purged and exhibit increased resistant to receptor-induced apoptosis. These substantial effects are surprising given that AID expression in the immature/T1 B cells is only 3% of that in germinal center B cells. We now show that in immature/T1 B cells, but not mature B cells, B-cell antigen receptor and Toll-like receptor (TLR) signaling synergize to elicit high levels of AID expression. This synergy is restricted to intracellular TLR ligands, requires the phospholipase D-dependent compartmentalization of intracellular TLR and activation of the TLR through acidification, and acts to ensure self-tolerance through a process that requires Myd88. In a 3H9 mouse model, we show that suppression of intracellular acidification by administration of chloroquine increased the generation of immature/T1 B cells in proportion to small pre-B cells, suggesting that chloroquine treatment support the survival of autoreactive 3H9 immature/T1 B cells in bone marrow. These findings identify a novel mechanism for central B-cell tolerance and resolve several weaknesses of current models for the first tolerance checkpoint. We suggest that this AID mediated pathway may be the primary mechanism for central B-cell tolerance by deletion.

Can Activation Induced Cytidine Deaminase Lead to Genetic Instability in Bcr-Abl Negative Myeloproliferative Neoplasms?

AbstractAbstract 5056 Objective:Most patients with myeloproliferative neoplasms (MPN) have mutations that lead to constitutive activation of the tyrosine kinase signal transduction pathways. JAK2V617F mutation is present in about 97% of polycythemia vera (PV), and in 60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. JAK2-exon-12 mutations, MPL exon-10-mutations are also seen in MPNs. In addition to this mutations, some patients with MPN may acquire mutations of other genes, such as in genes involved in negative regulations of signaling pathways (LNK, CBL, SOCS1, SOCS2, SOCS3) and epigenetic regulations (TET2, ASXL1, DNMT3A, or EZH2). IKZF1 deletions and TP53 mutations are mainly found post-MPN acute myeloid leukemia (AML). Loss of function mutations in TET2, ASXL1 and EZH2 genes, may be early events preceding JAK2V617F but may also secondary genetic event. These mutations are more frequently observed in PMF than in PV and ET. They are also present in other myeloid malignancies such as myelodysplasia and AML. Moreover several cases have been described that are positive for BCR-ABL and JAK2V617F mutation. It is thought that the mutagenic phenotype existing inherently in patients with MPN can generate new mutations. With the contribution of these mutations, PV, ET and PMF might evolve into secondary myelofibrosis or leukemia at different ratios. Activation induced cytidine deaminase (AID) is required for a B cell terminal differentiation mechanism shared by somatic hypermutation and class-switch recombination. Aberrant expression of AID can induce imperfect mutations in non-immunoglobulin genes and was detected in human lymphomas, lung cancer and gastric or intestinal metaplasia. High AID expression is associated with poor prognosis in follicular lymphomas, chronic lymphocytic leukemia and promotes B lymphoid blast crisis in chronic myeloid leukemia. Current study aims to investigate AID expression levels in myeloproliferative neoplasms and their relationship to genetic instability in MPN patients. Material and methods:The patient group consisted of 120 patients with bcr-abl negative myeloproliferative neoplasms (PV: 32 patients, ET: 62 patients and PMF: 18 patients, post-ET or post-PV myelofibrosis: 5 patients, unclassified MPN: 1 patient, MPN-AML: 2 patients). The patients were admitted to Istanbul University, Istanbul Medical Faculty, Division of Hematology Outpatient Clinics. The control group consisted of 69 healthy persons. We evaluated AID expression levels of the patients and of the control group. RNA was extracted from peripheral blood samples of patients and controls, RNA was converted to complementary DNA (cDNA). Using the primers and probes of the target gene (AID) and reference gene HPRT (hypoxanthine phosphoribosyltransferase), cDNA was amplified by realtime polymerase chain reaction and expression levels of the genes were evaluated. Results:We found that AID expression levels in the patient group were significantly higher than in the control group (Target-AID/Reference-HPRT, mean value: 0. 1747 vs 0. 0417, respectively, p<0. 001). We found that the patients age, blood hemoglobin levels, history of thrombosis, bone marrow reticulin levels, serum LDH levels, organomegaly and having JAK2V617F mutation were not correlated with AID expression levels. Conclusion:We showed that AID expression levels in patient with myeloproliferative neoplasms were significantly higher than in healthy control group. The role and the biological significance of high AID expression levels in genetic instability in MPNs need to be elucidated. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Quantitative Analysis of AID Expression and Somatic Hypermutation Identifies Isotype-Switched and Non-Switched Follicular Lymphomas As Distinct Biological Subgroups,

Abstract 3666 Introduction:Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. Deamination of cytosine residues by AID leads to double-strand breaks and point mutations in immunoglobulin (Ig) genes by subsequent mechanisms. Physiological AID expression is tightly regulated and restricted to germinal center (GC) B cells. AID acts not exclusively on Ig loci but effects aberrant SHM (ASHM) throughout the genome (Liu et al., 2008). High-fidelity mutation repair mechanisms protect most non-Ig AID target genes against mutation accumulation. Some genes including BCL6, the most highly mutated non-Ig gene, also undergo error-prone repair and consequently accumulate aberrant somatic mutations (Liu et al., 2009). In contrast to normal B cells, AID is constitutively expressed in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) (Greeve et al., 2003; Lossos et al., 2004). These lymphoma entities are derived from GC B cells and continue to accumulate somatic mutations in the immunoglobulin heavy variable region (IGHV) through ongoing SHM (Kosmas et al., 1998). In FL, the 5` noncoding region of BCL6 is altered by aberrant somatic mutations. Histological transformation of FL to DLBCL is associated with ASHM of this gene segment (Pasqualucci et al., 2001; Lossos et al., 2000). To further elucidate the role of AID in FL pathogenesis, we performed precise quantitative correlations between AID expression, frequency of ongoing SHM of IGHV and ASHM of BCL6 in 16 isotype-switched (IgG-expressing) and 23 non-switched (IgM-expressing) FL. Methods:AID expression was measured by quantitative real-time PCR. SHM of IGHV and ASHM of the 5` noncoding region of BCL6 were determined by PCR-based cloning and sequencing, followed by alignment with their consensus and germ-line sequences, respectively. Detected mutations were further categorized into consensus mutations, defined by their presence in the majority of clones, and sporadic mutations, defined by their presence in the minority of clones. Consensus and sporadic mutations are taken to represent previous and current AID activity, respectively. Results:All FL had readily detectable AID transcripts, albeit at highly variable levels. Overall, AID expression levels did not differ between isotype-switched and non-switched FL (p=0.99). Sporadic IGHV mutations were slightly more prevalent in switched FL (p=0.026). Consensus IGHV mutations (p=0.99) and both sporadic and consensus mutations in BCL6 did not differ between the FL subgroups (p=0.91 and p=0.77, respectively). In non-switched FL, AID expression levels were positively correlated with sporadic (r=0.44, p=0.041) and consensus IGHV mutations (r=0.42, p=0.049) and sporadic BCL6 mutations (r=0.51, p=0.016). Consensus mutations in BCL6 were independent of AID levels (r=0.23, p=0.30). In switched FL, AID expression was inversely correlated with consensus IGHV mutations (r= -0.71, p=0.004). There was no correlation between AID expression and sporadic IGHV mutations (r= -0.055, p=0.85), nor with sporadic or consensus BCL6 mutations (r=0.056, p=0.84 and r= -0.058, p=0.82, respectively). Conclusions:Only in non-switched FL, AID expression levels determine its enzymatic activity with respect to expected mutations in its physiological and unphysiological target genes IGHV and BCL6 in a manner that appears to be quantitatively independent from AID cofactors or repair mechanisms. In contrast, the rates of ongoing IGHV mutations and of ASHM in isotype-switched FL are not directly dependent on AID expression levels. The unexpected inverse correlation to consensus IGHV mutations points to efficient high-fidelity mutation repair or alternative mechanisms that suppress AID activity in isotype-switched FL. Therefore, our data demonstrate hitherto unrecognized biological differences between isotype-switched and non-switched FL. These results predict that the combination of IgM isotype with high AID expression define an FL subgroup that is genetically less stable over time than their IgG-expressing or AID low-expressing counterparts. As a consequence, we postulate that continuous AID activity and the resulting ASHM of BCL6 and perhaps other proto-oncogenes cause a more aggressive clinical behaviour and an inferior prognosis in this FL subgroup, including a predisposition to histological transformation. Disclosures:No relevant conflicts of interest to declare.

Aberrant activation-induced cytidine deaminase expression is associated with mucosal intestinalization in the early stage of gastric cancer

Although Helicobacter pylori is a risk factor for gastric cancer (GC), its detailed carcinogenesis remains unclear. Recently, aberrant expression of activation-induced cytidine deaminase (AID) was demonstrated in gastric epithelium with H. pylori infection and seems to cause the accumulation of mutation. This investigation aims to elucidate whether or not AID expression plays an important role in the carcinogenesis of early GC. We examined the correlation between immunohistochemical AID expression and histological characteristics, including pre-existing chronic gastritis and cellular mucin phenotype in 138 cases of intramucosal GC. Furthermore, we investigated the relationship between AID, p53 protein, and β-catenin. The low degree of polymorphonuclear neutrophil activity, and the high degree of glandular atrophy and intestinal metaplasia were significantly correlated with the high levels of AID expression in non-neoplastic mucosa (P = 0.007, P ≤ 0.001, and P = 0.003). With regard to mucin phenotype of carcinoma, the intestinal phenotype tended to have the higher AID expression levels (P = 0.052). AID showed close correlations with Cdx2 and nuclear staining of β-catenin (P = 0.003, P = 0.034). As for p53 protein, no correlation was found with AID expression. Our findings suggest that aberrant AID expression is correlated with persistent inflammatory condition induced by H. pylori infection and may contribute to the development of GC through an inflammatory condition and intestinalization.

B cell distribution and activation-induced cytidine deaminase expression in rheumatoid synovitis: clinical and bio-molecular correlates

Background and objectivesDevelopment of functional B cell niches in peripheral tissues has been associated to chronicity and pathologic severity in different human conditions such as kidney allograft rejection and multiple...

Ectopic expression of activation-induced cytidine deaminase caused by epigenetics modification

Recently studies have shown that ectopic expression of activation-induced cytidine deaminase (AID) plays an important role in carcinognesis and cancer progression of inflammatory-associated cancers. Here, we examined the molecular mechanism of ectopic expression of AID in cancer cells, and whether or not nitric oxide (NO) modulates this expression, as NO is known to cause chemical deamination of the cytidine. In several cancer cell lines, treatment with the DNA methyltransferase (Dnmt) inhibitor 5-Aza-dC effected expression of AID by TNF-α, and expression was further induced by additional treatment with histone deacetylase (HDAC) inhibitors with no stimulation. The CpG sites located in the promoter and exon 1 region of the AID gene in cancer cells were found to be hypomethylated in correlation with AID expression levels. Further, administration of HDAC inhibitors also induced expression of inducible nitric oxide synthase (iNOS) in cancer cells treated with 5-Aza-dC. Interestingly, administration of S-nitroso-L-glutathione (GSNO) a nitric oxide (NO) donor, was found to enhance AID and iNOS expression in LoVo cells treated with 5-Aza-dC. Our findings suggest that AID and iNOS expression in cancer cells may be modified by epigenetic mechanisms, and that NO may further enhance AID and iNOS expression. Given recent plans to introduce Dnmt and HDAC inhibitors as novel cancer treatments, these findings regarding the potential for Dnmt and HDAC inhibitors to enhance expression of AID and iNOS, resulting in further cancer progression, might be taken into consideration.

High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease

AbstractInteraction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

Somatic Hyperrepair: A Novel Tumor Suppression Mechanism for Germinal Center B Cells.

Abstract 92 BACKGROUND:All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS:We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS:Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS:Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures:No relevant conflicts of interest to declare.

Increased Expression of Activation‐Induced Cytidine Deaminase is Associated with Anti‐CCP and Rheumatoid Factor in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID(+) germinal centres were observed in six of the 12 RA synovial samples, and AID(+) cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID(+) cells. Significantly higher levels of AID mRNA (Aicda) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10(+)IgM(+)CD20(+) B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P </= 0.0001) and anti-cyclic citrullinated peptide (CCP) (P = 0.0005). Serum levels of IFN-gamma (P = 0.0005) and IL-17 (P = 0.007), but not IL-4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN-gamma and IL-17, leading to the development of anti-CCP and RF.

Activation-Induced Cytidine Deaminase Expression and Activity in the Absence of Germinal Centers: Insights into Hyper-IgM Syndrome

Somatic hypermutation normally occurs as a consequence of the expression of activation-induced cytidine deaminase (AID) by Ag-activated, mature B cells during T cell-dependent germinal center responses. Nonetheless, despite their inability to express CD154 and initiate GC responses, patients with type 1 hyper-IgM syndrome (HIGM1) support populations of IgM(+)IgD(+)CD27(+) B cells that express mutated Ig genes. The origin of these mutated B cells is unknown; the IgM(+)IgD(+)CD27(+) cells do not express AID and appear to acquire mutations independent of stringent selection by Ag. Here, we demonstrate that immature/transitional 1 B cells from the bone marrow of CD154-deficient mice express AID and acquire Ig mutations that lack the hallmarks of antigenic selection via BCR signaling. Comparable levels of AID expression was found in developmentally immature B cells recovered from murine fetal liver and from human immature/transitional 1 B cells recovered from umbilical cord blood. AID expression in human fetal liver was also robust, approaching that of human tonsil tissue and the human germinal center B cell line, Ramos. These observations led us to conclude that AID expression in developing human B cells is the origin of the mutated IgM(+)IgD(+)CD27(+) B cells present in HIGM1 patients, and we propose that both mice and humans share a latent, AID-dependent pathway for the preimmune diversification of B lymphocytes that is more prominent in chicken, sheep, and rabbits.

CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

Class switch recombination and somatic hypermutation occur in mature B-cells in response to antigen stimulation. These processes are crucial for the generation of functional antibodies. During HIV-1 infection, loss of memory B-cells, together with an altered differentiation of naïve B-cells result in production of low quality antibodies, which may be due to impaired immunoglobulin affinity maturation. In the current study, we evaluated the effect of HIV-1 infection on class switch recombination and somatic hypermutation by studying the expression of activation-induced cytidine deaminase (AID) in peripheral B-cells from a cohort of chronically HIV-1 infected patients as compared to a group of healthy controls. In parallel, we also characterized the phenotype of B-cells and their ability to produce immunoglobulins in vitro. Cells from HIV-1 infected patients showed higher baseline levels of AID expression and increased IgA production measured ex-vivo and upon CD40 and TLR9 stimulation in vitro. Moreover, the percentage of CD27−IgA+ and CD27−IgG+ B-cells in blood was significantly increased in HIV-1 infected patients as compared to controls. Interestingly, our results showed a significantly increased number of somatic hypermutations in the VH genes in CD27− cells from patients. Taken together, these results show that during HIV-1 infection, CD27− B-cells can also produce class switched and somatically hypermutated antibodies. Our data add important information for the understanding of the mechanisms underlying the loss of specific antibody production observed during HIV-1 infection.