Activation-induced Cytosine Deaminase Research Articles

In This Issue

In This Issue

The role of microRNA-142 in B cell activation and effector functions

Abstract Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immune responses despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function and terminal differentiation. To more precisely dissect the role of miR 142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDcre). To label activated B cells in miR-142fl/fl AIDcre mice, we bred them to mice carrying a loxP-flanked STOP cassette, allowing for the expression of fluorescent Zsgreen1 reporter protein in the presence of Cre recombinase. As expected, B cell development was largely unperturbed in miR-142fl/fl AIDcreZsgreen+/− mice. In contrast, FACS analysis of naive and antigen-challenged miR-142fl/fl AIDcre Zsgreen+/− mice revealed a significant decrease in germinal center (GC) B cells and plasma cells (PCs). RNA sequencing of GC B cells suggests dysregulation of transcription factor networks such as miR-142-3p targets BACH2 and IRF4 may contribute to the phenotypes observed in miR 142fl/fl AIDcre Zsgreen+/− mice. Future experiments will investigate molecular pathways governing terminal B cell differentiation and effector function in miR-142fl/fl AIDcre Zsgreen+/− mice, thereby elucidating the role of miR-142 in promoting a healthy response to antigen.

Tandem Substitutions in Somatic Hypermutation.

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.

Detection of R-Loops by In Vivo and In Vitro Cytosine Deamination in Saccharomyces cerevisiae.

R-loops are transcriptional by-products formed by a hybrid of the nascent RNA molecule with its DNA template and the displaced nontemplate DNA strand. The single stranded nature of the displaced nontemplate strand makes it vulnerable to attack. This property is used in nature to cause directed mutagenesis and breaks by the action of the activation-induced cytosine deaminase (AID) enzyme and can thus be exploited to detect the presence of R-loops even when they form at low frequencies by overexpressing this enzyme in vivo or by in vitro treatment with the bisulfite anion, which further allows nucleotide resolution. This is of particular relevance given the fact that R-loops have the potential to hamper DNA replication and repair, threatening genome integrity. Here, we describe the protocols used in the yeast Saccharomyces cerevisiae to infer the presence ofR-loops through increased AID-induced DNA damage, measured as increased recombination or Rad52 foci formation as well as to detect single R-loop molecules and determine their length at particular genomic sites via bisulfite treatment and amplification.

Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown.We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum.In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism.Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions.Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5‘ and 3‘ base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Mutation Landscape of Follicular Lymphoma and Chronic Lymphocytic Leukemia Reveals a Distinctive Frequency and Composition of Aid-Related Mutation Signatures

Introduction: Somatic mutations associated with distinctive types of cancer can be globally classified by the trinucleotide context of single nucleotide variants into mutational signatures. Distinct signatures can be attributed to underlying causative mechanisms such as UV irradiation or smoking. B-cell lymphomas have only been analyzed globally and carry a signature characterized by APOBEC activity (Alexandrov, 2014).The two most common indolent B-cell neoplasms, chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), are characterized by distinctive pathogenic processes, but mutagenesis caused by the APOBEC enzyme activation-induced cytosine deaminase (AID) may be shared by both malignancies. AID deaminates deoxycytidine to deoxyuridine, which is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double-strand breaks. AID activity and subsequent DNA repair create two different mutation signatures: the canonical signature characterized by C>T/G transitions at RCY motifs and the non-canonical defined by A>C transversions at WAN motifs. More recently a third AID signature characterized by C>T transitions at RCG motifs has been associated with AID-mediated CpG-methylation dependent mutagenesis.We here investigate the hypothesis that AID mutational signatures contribute to the pathogenesis of FL, a lymphoma characterized by constitutional expression of AID, but also of CLL since high AID expression levels may account for a more aggressive disease, and a subset of CLL B-cells with AID expression display dissociation between class switch recombination and somatic hypermutation.Methods: We analyzed the mutational landscape in whole exome sequencing data from 31 FL and 24 CLL cases. Somatic variants were called by comparison of tumor and germline DNA with an in-house developed pipeline. The relative contribution of mutations at canonical and non-canonical AID hotspot motifs was computed by direct quantification of variants occurring in known AID motifs. Student's t test was used for comparison between CLL and FL.Mutational signatures were defined according to the 96-base substitution model (Alexandrov, 2013) by an unsupervised machine learning approach, with a local implementation of the SomaticSignatures R package.Results: The frequency of C>T/G transitions at canonical AID hotspot was more prevalent in CLL (9.0%; 95% CI: 8.1-10.0%) than in FL (6.6%, 95% CI: 5.9-7.3%)(p<0.001). Likewise, A>C transversions at non-canonical AID sites were significantly more frequent in CLL (1.9%; 95% CI: 1.6-2.2%) as compared to FL (0.5%; 95% CI: 0.4-0.6%)(p<0.0001).In the unsupervised analysis of FL and CLL cases, 97.7% of the mutation spectrum variance was explained by only 5 signatures (Figure 1). In FL, the mutational landscape was dominated by a signature (S2) characterized by C>A transversions in the trinucleotide KCH context (40%, 95% CI: 33-36%). The second most frequent signature (S3; 22%, 95% CI: 15-30%) was characterized by C>T transitions in the context of a hybrid between the canonical AID and the CpG hotspot motifs (RCY/RCG).In contrast, the mutational landscape of CLL was dominated by a signature (S5; 42%, 95% CI: 34-49%) characterized by C>T transitions in the RCY motif and A>C transversions in the WAN context, consistent with an overlapping between the canonical and the non-canonical AID hotspots (Kasar, 2016).Conclusions: The mutational landscape of both CLL and FL seems to be strongly shaped by AID activity. In FL, AID-induced mutations are mainly restricted to canonical AID hotspots and CpG-methylation dependent mutagenesis sites. In strong contrast, both canonical and non-canonical AID motifs contributed to the mutational landscape of CLL. In addition, FL carries a high prevalence of C>A substitutions that are associated with exposure to polycyclic aromatic hydrocarbons that produce bulky DNA adducts followed by the nucleotide excision repair process. These compounds are present in several industrial processes (food products, beverages, tobacco) that have been suggested as occupational risk factors for follicular lymphomas (Mester, 2009).Overall, our data suggest a much stronger role of AID in CLL pathogenesis than hitherto appreciated. The differential contribution of canonical versus non-canonical AID activity points to different regulation of AID in CLL compared to FL and warrants further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ATR kinase activity promotes antibody class switch recombination in B cells through cell cycle regulation without suppressing DSB resection and microhomology usage.

Class switch recombination (CSR) changes the effector functions of antibodies and is carried out by classical and alternative nonhomologous end joining (c-NHEJ and A-EJ) of repetitive switch (S) region double-strand breaks (DSBs). The master DNA damage response (DDR) kinase ataxia-telangiectasia mutated (ATM) is critical for CSR in part by suppressing S region DSB resection. However, whether another related DDR kinase ATM- and Rad3-related (ATR) plays similar role in CSR remains elusive. In this study, we investigated the requirement for ATR kinase activity on CSR in both c-NHEJ competent and deficient B cell lines with high-throughput sequencing of S-S junctions. We found that ATR kinase inhibition efficiently blocked both c-NHEJ- and A-EJ-mediated CSR without affecting germline transcription and activation-induced cytosine deaminase expression. In contrast to ATM, ATR does not suppress S region DSB resection and microhomology usage. In addition, ATR kinase inhibition did not affect Cas9-generated DSB end joining by either c-NHEJ and A-EJ. ATR kinase-inhibited stimulated B cells proliferate much slower than controls and exhibited altered cell cycle profile with increased G1 and G2/M phase cells. In summary, our data revealed a role for ATR in promoting both c-NHEJ- and A-EJ-mediated CSR through regulating cell proliferation upon damage without negatively influencing DSB end-joining features.

The role of microRNA-142 in B cell activation and effector functions

Abstract Control of gene expression by microRNA (miRNA) has recently emerged as a critical mechanism that regulates B cell activation and function. However, the role of specific miRNAs in this process is unclear. Using germline knockout (KO) mice, we have previously shown that miR-142 ablation impairs humoral immunity despite significant expansion of the B cell compartment, suggesting that miR-142 is critical for B cell effector function. We found that mice lacking miR-142 cannot generate specific antibody responses upon antigen challenge and display impaired germinal center (GC) formation and plasma cell differentiation. To more precisely dissect the role of miR-142 in B cell effector responses, we developed an activated B cell-specific miR-142 KO mouse by breeding conditional miR-142 KO (miR-142fl/fl) mice with mice expressing Cre recombinase driven by activation-induced cytosine deaminase (AIDCre). AID is required for class switch recombination and somatic hypermutation, and is induced in B cells upon antigen encounter. To label activated B cells in miR-142fl/flAIDCre mice, we bred them to Ai6 mice that carry a loxP-flanked STOP cassette, allowing for the expression of fluorescent ZsGreen1 reporter protein in the presence of Cre recombinase. FACS analysis of Peyer’s patches from naive miR-142fl/flAIDCreZsgreen+/− mice revealed a significant decrease in GC B cells, while the number of ZsGreen1+ activated B cells increased, suggesting an aberrant B cell activation and GC response. We will discuss at the meeting the role of miR-142 in promoting humoral responses to antigen by presenting the findings from our ongoing studies of B cell differentiation and effector function in immunized miR-142fl/flAIDCreZsgreen+/− mice.

I.19 New Insights of EGFR Inhibition: How to Overcome Resistance &amp; New Agents

The main mechanism of resistance to first-generation EGFR TKIs is a single-nucleotide transition mutation in EGFR, cytosine to thymine (C>T) at position 2369 (ACG/ATG) causing threonine to methionine amino acid change at codon 790 (T790M). The T790M mutation occurs in cis with the original driver mutation and leads to steric hindrance and increased binding affinity for ATP. Activation-induced cytosine deaminase (AICDA) is expressed in germinal center B-lymphocytes upon antigen exposure. AICDA translocates to the nucleus, deaminates cytosine in single stranded DNA and converts it to uracil. Gefitinib, afatinib or osimertinib increase AICDA expression. A significant increase in the AICDA expression occurs in the PC9 and other EGFR-mutant cell lines upon treatment with different EGFR TKIs. AICDA is induced through the non-canonical nuclear factor-ĸB (NFĸB) (Figure). NFĸB was noted to be a mechanism of resistance to erlotinib and further studies of the Bivona group have further demonstrated that NFĸB is activated in EGFR-mutant LADCs. AICDA knockdown decreases the frequency of T790M and targeting AICDA activity directly or indirectly with NFĸB inhibitors, as well as DNA damage response inhibitors, could delay or prevent the appearance of T790M. The function of AICDA supports the rationale for an NFĸB-driven generation of T790M. It was demonstrated that, regardless of the use of gefitinib or osimertinib, NFĸB, STAT3 and YAP1 were activated in EGFR-mutant cell lines, including the H1975 (that harbors T790M). The downstream effectors were inhibited when the EGFR TKI was combined with blockers of the STAT3 and Src pathway. Osimertinib showed a PFS of 10.1 months versus 4.4 with platinum-pemetrexed chemotherapy in EGFR-mutant patients with T790M. Screening for EGFR mutations is of great usefulness in customizing therapy in LADCs. However, combinatory therapies with EGFR TKIs is warranted to avoid the mechanisms of drug resistance that occur following single EGFR TKIs. The activation of STAT3 occurred a few hours after treatment with EGFR TKIs. AKT pathway is commonly activated following treatment with first- or third-generation EGFR TKIs (Figure). In addition, co-expression of other receptor tyrosine kinases is commonly present with EGFR mutations. We demonstrated that AXL expression can be driven by the Src- yes-associated protein 1 (YAP1) activation, providing a glimpse for adequate combinatory approaches. Recent articles identify AXL as a mechanism of intrinsic and acquired resistance to osimertinib. YAP1 directly regulates the expression of PD-L1 in EGFR TKI-resistant LADCs. Another downstream central effector is the protein tyrosine phosphatase SHP2 (PTPN11). The urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1) correlates with poor prognosis in EGFR-mutant LADCs (Figure). The suppression of CPS1 potentiates the effects of EGFR inhibition and, therefore, opens an additional angle for optimizing therapy in EGFR-mutant LADCs that could most likely be extrapolated to other, oncogene driven LADCs. Among the transcriptional targets of YAP1 and its effector Forkhead box protein M1 (FOXM1), are the aurora kinases A and B (AURKA and AURKB) (Figure). Both AURKA and AURKB have been associated with resistance to EGFR TKIs. EGFR-mutant LADCs do not respond to immune checkpoint inhibitors. NFĸB, T790M, STAT3, CPS1, LADCs

Progesterone attenuates hypertension and autoantibody levels to the angiotensin II type 1 receptor in response to elevated cadmium during pregnancy

IntroductionPreeclampsia is associated with the presence of pathogenic angiotensin-receptor-activating autoantibodies. Cadmium is an increasingly prevalent environmental pollutant that can mimic oestrogens, which may enhance immunoglobulin production. Progesterone exerts opposite effects to oestrogen. MethodsWe measured the levels of cadmium and progesterone in preeclamptic patients and controls. Pregnant rats exposed to cadmium (0.125 mg/kg body weight) from gestational day 9–12 were treated with/without progesterone (3 mg/kg) beginning from gestational day 9 to delivery. We analysed the main features of preeclampsia and circulating level of the angiotensin II type 1 receptor agonistic autoantibody. We also measured the expression of activation-induced cytosine deaminase in B cells. ResultsThere were higher cadmium levels and lower progesterone levels in the blood of preeclamptic women than in the blood of those with a healthy pregnancy. Based on this finding, a rat model of preeclampsia was established by intraperitoneally administrating low-dose cadmium on gestational days 9–12. Rats were then treated with/without progesterone. Key features of preeclampsia, including hypertension, proteinuria and placental abnormalities, appeared in pregnant rats after cadmium injection and improved after treatment with progesterone. Cadmium increased immunoglobulin production, mainly angiotensin II type 1-receptor-agonistic autoantibodies, by increasing the expression of activation-induced cytosine deaminase in B cells; progesterone exerted an opposite effect. ConclusionCadmium induced immune abnormalities that may be a key pathogenic contributor to preeclampsia. Progesterone supplementation to correct hormonal imbalance may be a viable strategy for preeclampsia management.

R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching

R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from μ–γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G+C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process.

Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system.

Germinal centres (GCs) promote humoral immunity and vaccine efficacy. In GCs, antigen-activated B cells proliferate, express high-affinity antibodies, promote antibody class switching, and yield B cell memory. Whereas the cytokine milieu has long been known to regulate effector functions that include the choice of immunoglobulin class, both cell-autonomous and extrinsic metabolic programming have emerged as modulators of T-cell-mediated immunity. Here we show in mice that GC light zones are hypoxic, and that low oxygen tension () alters B cell physiology and function. In addition to reduced proliferation and increased B cell death, low impairs antibody class switching to the pro-inflammatory IgG2c antibody isotype by limiting the expression of activation-induced cytosine deaminase (AID). Hypoxia induces HIF transcription factors by restricting the activity of prolyl hydroxyl dioxygenase enzymes, which hydroxylate HIF-1α and HIF-2α to destabilize HIF by binding the von Hippel-Landau tumour suppressor protein (pVHL). B-cell-specific depletion of pVHL leads to constitutive HIF stabilization, decreases antigen-specific GC B cells and undermines the generation of high-affinity IgG, switching to IgG2c, early memory B cells, and recall antibody responses. HIF induction can reprogram metabolic and growth factor gene expression. Sustained hypoxia or HIF induction by pVHL deficiency inhibits mTOR complex 1 (mTORC1) activity in B lymphoblasts, and mTORC1-haploinsufficient B cells have reduced clonal expansion, AID expression, and capacities to yield IgG2c and high-affinity antibodies. Thus, the normal physiology of GCs involves regional variegation of hypoxia, and HIF-dependent oxygen sensing regulates vital functions of B cells. We propose that the restriction of oxygen in lymphoid organs, which can be altered in pathophysiological states, modulates humoral immunity.

Cadmium-induced immune abnormality is a key pathogenic event in human and rat models of preeclampsia

With increased industrial development, cadmium is an increasingly important environmental pollutant. Studies have identified various adverse effects of cadmium on human beings. However, the relationships between cadmium pollution and the pathogenesis of preeclampsia remain elusive. The objective of this study is to explore the effects of cadmium on immune system among preeclamptic patients and rats. The results showed that the cadmium levels in the peripheral blood of preeclamptic patients were significantly higher than those observed in normal pregnancy. Based on it, a novel rat model of preeclampsia was established by the intraperitoneal administration of cadmium chloride (CdCl2) (0.125 mg of Cd/kg body weight) on gestational days 9–14. Key features of preeclampsia, including hypertension, proteinuria, placental abnormalities and small foetal size, appeared in pregnant rats after the administration of low-dose of CdCl2. Cadmium increased immunoglobulin production, mainly angiotensin II type 1-receptor-agonistic autoantibodies (AT1-AA), by increasing the expression of activation-induced cytosine deaminase (AID) in B cells. AID is critical for the maturation of antibody and autoantibody responses. In addition, angiotensin II type 1-receptor-agonistic autoantibody, which emerged recently as a potential pathogenic contributor to PE, was responsible for the deposition of complement component 5 (C5) in kidneys of pregnant rats via angiotensin II type 1 receptor (AT1R) activation. C5a is a fragment of C5 that is released during C5 activation. Selectively interfering with C5a signalling by a complement C5a receptor-specific antagonist significantly attenuated hypertension and proteinuria in Cd-injected pregnant rats. Our results suggest that cadmium induces immune abnormalities that may be a key pathogenic contributor to preeclampsia and provide new insights into treatment strategies of preeclampsia.

The many layers of epigenetic dysfunction in B-cell lymphomas.

Perturbation of the epigenome is emerging as a central driving force in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and follicular lymphoma. The purpose of this review is to explain how alteration of different layers of the epigenome contributes to the biology and clinical features of these tumors. Key new findings implicate DNA methylation heterogeneity as a core feature of DLBCL. Epigenetic diversity is linked to unfavorable clinical outcomes, clonal selection at relapse, and is driven at least in part because of the actions of activation-induced cytosine deaminase, which is a unique feature of B-cell lymphomas. Somatic mutations in histone modifier genes drive lymphomagenesis through the establishment of aberrant gene-specific histone modification signatures. For example, EZH2 somatic mutations drive silencing of bivalent gene promoters through histone 3 lysine 27 trimethylation, whereas KMT2D (MLL2) mutations disrupt specific sets of enhancers through depletion of histone 3 lysine 4 mono and dimethylation (H3K4me1/me2). Appreciation of the epigenome in determining lymphoma clonal heterogeneity and in driving lymphoma phenotypes through altered promoter and enhancer histone modification profiles is leading to a paradigm shift in how we understand and design therapies for DLBCL and follicular lymphoma.

Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells.

The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.

Effects of prostaglandin E2on p53 mRNA transcription and p53 mutagenesis during T‐cell‐independent human B‐cell clonal expansion

Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5 × 10(-4)) greater than the baseline error rate (<0.8 × 10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.

Abstract IA20: Epigenetic deregulation of gene expression

Abstract Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss of function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCPG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using alpha-ketoglutarate (aKG) as a substrate. 2HG can disrupt the activity of enzymes that use aKG as a cofactor, including TET2 and the Jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibit TET2 hydroxymethylation and Jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards reduced histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is abundance in hematopoietic stem cells, and displays specific distribution patterns, and yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B-cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Citation Format: Ari Melnick. Epigenetic deregulation of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA20.

BCL6 breaks occur at different AID sequence motifs in Ig–BCL6 and non-Ig–BCL6 rearrangements

AbstractBCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig–BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig–BCL6 and non-Ig–BCL6 rearrangements.

Genome-Wide Detection of Genes Targeted by Non-Ig Somatic Hypermutation in Lymphoma

The processes of somatic hypermutation (SHM) and class switch recombination introduced by activation-induced cytosine deaminase (AICDA) at the Immunoglobulin (Ig) loci are key steps for creating a pool of diversified antibodies in germinal center B cells (GCBs). Unfortunately, AICDA can also accidentally introduce mutations at bystander loci, particularly within the 5′ regulatory regions of proto-oncogenes relevant to diffuse large B cell lymphomas (DLBCL). Since current methods for genomewide sequencing such as Exon Capture and RNAseq only target mutations in coding regions, to date non-Ig promoter SHMs have been studied only in a handful genes. We designed a novel approach integrating bioinformatics tools with next generation sequencing technology to identify regulatory loci targeted by SHM genome-wide. We observed increased numbers of SHM associated sequence variant hotspots in lymphoma cells as compared to primary normal germinal center B cells. Many of these SHM hotspots map to genes that have not been reported before as mutated, including BACH2, BTG2, CXCR4, CIITA, EBF1, PIM2, and TCL1A, etc., all of which have potential roles in B cell survival, differentiation, and malignant transformation. In addition, using BCL6 and BACH2 as examples, we demonstrated that SHM sites identified in these 5′ regulatory regions greatly altered their transcription activities in a reporter assay. Our approach provides a first cost-efficient, genome-wide method to identify regulatory mutations and non-Ig SHM hotspots.

A p53 Axis Regulates B Cell Receptor-Triggered, Innate Immune System-Driven B Cell Clonal Expansion

Resting mature human B cells undergo a dynamic process of clonal expansion, followed by clonal contraction, during an in vitro response to surrogate C3d-coated Ag and innate immune system cytokines, IL-4 and BAFF. In this study, we explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures. Several findings, involving both human and mouse B cells, show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high activation-induced cell death (AICD) susceptibility of replicating blasts. Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of proapoptotic molecules known to be under direct p53 transcriptional control, Bax, Bad, Puma, Bid, and procaspase 6, accompanied by reduced anti-apoptotic Bcl-2. Under these conditions, Bim levels were not increased. The finding that full-length Bid protein significantly declines in AICD-susceptible replicating blasts, whereas Bid mRNA does not, suggests that Bid is actively cleaved to short-lived, proapoptotic truncated Bid. AICD was diminished, albeit not eliminated, by p53 small interfering RNA transfection, genetic deletion of p53, or Bcl-2 overexpression. DNA damage is a likely trigger for p53-dependent AICD because susceptible lymphoblasts expressed significantly elevated levels of both phosphorylated ataxia telangiectasia mutated-Ser(1980) and phospho-H2AX-Ser(139). Deficiency in activation-induced cytosine deaminase diminishes but does not ablate murine B cell AICD, indicating that activation-induced cytosine deaminase-induced DNA damage is only in part responsible. Evidence for p53-influenced AICD during this route of T cell-independent clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of IL-4 and BAFF.