Activation-induced Cytidine Deaminase Activity Research Articles

Abstract A052: ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell

Abstract Objective: Ovulation is a major risk factor of epithelial ovarian cancer (EOC), presumably acts from the cancer initiation stage. High-grade serous carcinoma (HGSC) is the most prevalent and fatal EOC, mainly arising from fallopian tube fimbrial epithelium (FTE). TP53 missense mutations are considered the earliest event of carcinogenesis since they form “p53 signatures” in FTE and were found in virtually all HGSC. Previously, we discovered that a subset of ovulatory follicular fluids (FF) harbored high levels of ROS, which were both mutagenic and oncogenic, causing DNA double-strand breaks (DSB) and tumorigenesis. However, DSB is unlikely the cause of TP53 mutations which are mostly CG: TA base substitutions involving deamination. We hypothesized that during ovulation FF exposes the fallopian tube fimbria and causes TP53 mutations by activating the activation-induced cytidine deaminase (AID). Materials and Methods: By conducting an HPRT gene mutation assay, we determined the mutagenic activity of FF on FTE cells with or without AID-knockdown. AID gene activation and protein nuclear translocation were analyzed in human FTE tissues and cells with different degrees of transformation, as well as in the oviducts of mice under controlled ovulation. The binding of AID to the TP53 gene and the subsequent deamination were determined by ChIP-PCR and uracil-qPCR, respectively. Results: After FF exposure, the mutation frequency of HPRT in FTE cells increased by 4.1 folds, at levels 40% higher than the ethyl methane sulfonate control and could be diminished by antioxidants or knockdown of the AID gene. AID activation, including mRNA and protein inductions and nuclear translocation, was evident in FTE cells and tissue after FF exposure and in mouse oviducts after ovulation. Two waves of AID activations were noted, peaking at 45 minutes (early activity) and 8.5 hours (late activity) after ovulation or FF exposure. TNF-α and ROS were found to be responsible for the activities in FF. Mechanistically, the early activity worked through the MAPK, while the late activity worked through the NF-κB pathway. The induced AID in both activities physically binds to and biochemically deaminates the TP53 gene irrelative to the known mutational hotspots of HGSC. However, it did not act on PTEN, the other prevalent tumor suppressor gene in HGSC. Meanwhile, ovulatory FF also activates the putative tumor-initiating and stemness gene LGR6 in FTE at locations where AID was also activated. Conclusion: The serial studies characterized the mutagenic activities of ovulatory FF, which acts on the LGR6-expressing cells in the fallopian tube fimbria and targets the TP53 gene. The study also identified ROS as the mutagen and pointed to strategies of ovulation inhibition and antioxidation for the prevention of EOC. Citation Format: Tang-Yuan Chu, Sanchana Subramani, Hsuan-Shun Huang. ROS in ovulatory follicular fluid exert de novo mutagenic activities through activation-induced cytidine deaminase acting on the TP53 gene in the fallopian tube epithelial cell [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A052.

5-Chloro-2'-deoxycytidine Induces a Distinctive High-Resolution Mutational Spectrum of Transition Mutations In Vivo.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C → T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gptΔ C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG → TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA → CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

Cell Cycle-Mediated Regulation of Secondary Ig Diversification.

Secondary Ig diversification in B cells requires the deliberate introduction of DNA damage into the Ig genes by the enzyme activation-induced cytidine deaminase (AID) and the error-prone resolution of AID-induced lesions. These processes must be tightly regulated because they may lead to lymphomagenesis if they act on genes other than the Ig genes. Since B cells may limit secondary Ig diversification mechanisms during the cell cycle to minimize genomic instability, we restricted the activity of AID specifically to the G1 or S/G2 phase to investigate the cell cycle contribution to the regulation of somatic hypermutation, class switch recombination, and Ig gene conversion in human, murine, and avian B cells, respectively. The efficient induction of AID in different cell cycle phases allowed us for the first time, to our knowledge, to discriminate G1- from S/G2-specific events of regulation. We show that the processes of Ig gene conversion and C/G mutagenesis during somatic hypermutation can be achieved throughout the cell cycle, whereas A/T mutagenesis and class switch recombination require AID-mediated deamination in G1. Thus, AID activity in G1, but not in S/G2, leads to the efficient accomplishment of all mechanisms of secondary Ig diversification. Our findings refine the current state-of-the-art knowledge in the context of the regulation of secondary Ig diversification.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large Bcell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

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.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2(UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels ofUNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Normal B Cell-Derived iPSCs Capable of Inducing RAS Mutants and Aid to Explore Myeloma-Initiating Cells

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and induce the expression of activation-induced cytidine deaminase (AID) by the Tet-off system (Sci Rep, 2017). Subsequently, we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5′-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14) carrying the DJ-rearranged allele of IgH that reciprocally translocated with CCND1 (G28). All these BiPSCs differentiated into hematopoietic progenitor cells (HPCs) (Sci Res, 2021). Next, we created AID fused with the hormone-binding domain of the estrogen receptor T2 mutant (AID-ER) to analyze the function of AID. The ER sequence of pCAG-ERT2CreERT2 (Addgene # 13777) was amplified by PCR and cloned into the downstream part of the TREtight promoter of pRetroX-Tight-Hyg as an open reading frame using an In-Fusion HD cloning kit. The ER sequence was also cloned into the same vector as a negative control for the analysis of AID function. These vectors were packaged into a retrovirus using gp293 and pMD2.G, and the viruses were infected into HEK293 cells expressing Tet-off transcription factor and fluorescence-deficient EGFP, in which the artificially-introduced stop codon was at the 106 thcodon of the EGFP gene. The expression of AID-ER fusion protein was induced by removal of doxycycline three days before analysis. AID activity was induced by the addition of 4-hydroxytamoxifen, an agonist of ER mutants, one day before analysis. Therefore, AID activity was monitored by detecting the frequency of EGFP-positive cells, i.e., the recovery of fluorescence, by flow-cytometry. Mutation of the artificial stop codon (from TAG to TAC; from stop codon to Tyrosine) was confirmed by sequencing the EGFP DNA amplified by PCR from the sorted EGFP-positive cells. Subsequently, we examined the introduction of mutated KRAS and NRAS genes into G28, because the most frequently occurring mutations in patients with MM are in KRAS and NRAS. Mutated KRAS (G12A) and NRAS (G13D) were amplified by RT-PCR from poly (A) mRNA of MM.1S and KMM1 of MM cell lines, respectively. After confirming the sequence of these mutants, the mutant cDNA of KRAS or NRAS was cloned into pRetroX-Tight-Hyg in combination with AID-ER. These two vectors were packaged into retroviruses, following which the viruses were infected into BiPSC13 with t(11;14) expressing Tet-off transcription factors, respectively. These BiPSCs also differentiated into HPCs. However, unlike cord blood, these HPCs did not differentiate into B lymphocytes by co-culture with BM stromal cell (MS-5). Therefore, further ingenuity is required to differentiate the BiPSC-derived HPCs into B lymphocytes. DisclosuresNo relevant conflicts of interest to declare.

“Snapshotting” Somatic Hypermutation in Single Follicular Lymphoma Cells

IntroductionFollicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity.Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies.The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL.MethodsTumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5).ResultsWe analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of “SHM snapshot events” when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples.ConclusionHere we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression. DisclosuresNo relevant conflicts of interest to declare.

B Cell-Derived Induced Pluripotent Stem Cells Are Useful for Determining the Cellular Origin of Abnormal B Cells in Multiple Myeloma

Induced pluripotent stem cells (iPSCs) are generated by the genetic reprogramming of somatic cells. In addition to regenerative medicine, these cells have been used in studies on the pathogenesis of inherited genetic diseases or neoplasms. If B cell-derived iPSCs (BiPSCs) could be developed from mature B cells or plasma cells, the genomic rearrangement of the B cell receptor of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous introduction of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) with CCAAT/enhancer-binding protein-alpha using a Sendai virus vector (Bueno C, et al. Leukemia 2016). In this study, we were also able to generate BiPSCs, albeit using different method. The BiPSCs had immunoglobulin heavy chain (IgH) gene rearrangement but no class-switch in both alleles from normal B cells purified from lymph nodes and peripheral blood; therefore, we postulated that they were most likely established from naive B cells or IgM-expressing mature B cells. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells using a retrovirus vector, which were pre-stimulated with interleukin (IL)-21 and CD40 ligand (CD40L), combined with consecutive centrifugations of the cells after their activation by IL-4, IL-2, and CD40L on a retronectin coated plate. We also established doxycycline-controlled (Tet-off system) activation-induced cytidine deaminase (AID)-induced BiPSCs (BiPSCs-A). AID is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in B cells. Furthermore, it has been noted recently that AID has similar effects on other than immunoglobulin genes. We hypothesized that myeloma cells are derived from reprogrammed mature B cells, in which reciprocal chromosome translocation occurs by double-strand DNA breaks induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14 and in another chromosome containing genes such as cyclin D1, Maf. To test our hypothesis, we used the comet assay to confirm that DNA cleavage was induced by AID in the BiPSCs-A. Next, we analyzed the ability of these BiPSCs to differentiate into hematopoietic progenitor cells (HPCs), and found that both parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs, as determined by evaluating CD34 expression and the colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45 expression, indicating that they may be hematoendothelial cells as Maxim proposed previously (Vodyanik MA et al. Blood 2006). Based on our findings regarding the differentiation of BiPSCs-A into HPCs and their retention of the IgH gene rearrangement, it is possible that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HPCs and further into mature or antibody-producing B cells in experiments using mouse. Because the BiPSCs had teratoma-forming ability, if a chromosome break were to occur in either an allele with IgH gene rearrangement (productive allele) or in another nonproductive IgH allele followed by reciprocal translocation with another chromosome and oncogene activation, BiPSCs reactivation and differentiation into B cells might be a cause of multiple myeloma in vivo . Thus, these BiPSCs might be useful for elucidating the origin of abnormal B cells in this disease. DisclosuresNoji:Alexion Pharma GK: Honoraria, Research Funding.

Stepwise generation of AID knock-in and conditional knockout mice from a single gene-targeting event.

Activation-induced cytidine deaminase (AID) encoded by the Aicda gene initiates class-switch recombination and somatic hypermutation of immunoglobulin genes. In addition to this function, AID is also implicated in the epigenetic regulation in pluripotent stem cells and in the oncogenesis of lymphoid and non-lymphoid origins. To examine AID's role in specific cell types, we developed mouse strains of conditional knockout (Aicda-FL) and knock-in with a red fluorescent protein gene (RFP) inserted into the Aicda locus (Aicda-RFP). These two strains were obtained from a single targeting event in embryonic stem cells by a three-loxP or tri-lox strategy. Partial and complete recombination among the three loxP sites in the Aicda-RFP locus gave rise to Aicda-FL and AID-deficient loci (Aicda-KO), respectively, after mating Aicda-RFP mice with Cre-expressing mice driven by tissue-non-specific alkaline phosphate promoter. We confirmed RFP expression in B cells of germinal centers of intestine-associated lymphoid tissue. Mice homozygous for each allele were obtained and were checked for AID activity by class-switch and hypermutation assays. AID activity was normal for Aicda-FL but partially and completely absent for Aicda-RFP and Aicda-KO, respectively. Aicda-FL and Aicda-RFP mice would be useful for studying AID function in subpopulations of B cells and in non-lymphoid cells.

HIV-1 Tat Activates Akt/mTORC1 Pathway and AICDA Expression by Downregulating Its Transcriptional Inhibitors in B Cells.

HIV-1 infects T cells, but the most frequent AIDS-related lymphomas are of B-cell origin. Molecular mechanisms of HIV-1-induced oncogenic transformation of B cells remain largely unknown. HIV-1 Tat protein may participate in this process by penetrating and regulating gene expression in B cells. Both immune and cancer cells can reprogram communications between extracellular signals and intracellular signaling pathways via the Akt/mTORC1 pathway, which plays a key role in the cellular response to various stimuli including viral infection. Here, we investigated the role of HIV-1 Tat on the modulation of the Akt/mTORC1 pathway in B cells. We found that HIV-1 Tat activated the Akt/mTORC1 signaling pathway; this leads to aberrant activation of activation-induced cytidine deaminase (AICDA) due to inhibition of the AICDA transcriptional repressors c-Myb and E2F8. These perturbations may ultimately lead to an increased genomic instability and proliferation that might cause B cell malignancies.

A Hyper-IgM Syndrome Mutation in Activation-Induced Cytidine Deaminase Disrupts G-Quadruplex Binding and Genome-wide Chromatin Localization

Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting invivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.

Optimized high-fidelity 3DPCR to assess potential mitochondrial targeting by activation-induced cytidine deaminase.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of immunoglobulin genes in B cells, whereas off-targeted AID activity contributes to oncogenic mutations and chromosomal translocations associated with B cell malignancies. Paradoxically, only a minority of AID is allowed to access the nuclear genome, but the majority of AID is retained in the cytoplasm. It is unknown whether cytoplasmic AID can access and target the mitochondrial genome [mitochondrial DNA (mtDNA)]. To address this issue, we developed high-fidelity differential DNA denaturation PCR, which allowed the enrichment of genuine mtDNA mutations and therefore the identification of endogenous mtDNA mutation signatures invitro. With this approach, we showed that AID targeting to mtDNA is a rare event in AID-expressing lymphoma lines. Further biochemical and microscopic analysis revealed that a fraction of cytosol AID is associated with the outer membrane of mitochondria but unable to access the mitochondrial matrix. Together, our data suggested that the mitochondrial genome is protected from AID-mediated mutagenesis by physical segregation of AID from accessing mtDNA within the mitochondrial matrix.

Abstract A49: Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides

Abstract Many studies have drawn associations between pesticide exposures and lymphomagenesis, both in occupational settings and in the general population. The specific compounds responsible for an increased risk of non-Hodgkin lymphoma in pesticide-exposed individuals have not been entirely established. Furthermore, the biologic mechanisms by which pesticides act as lymphomagens are unknown. One mechanism of B-cell genomic instability that plays a role in lymphomas derived from germinal center (GC)-type B cells, including follicular lymphoma, diffuse large B cell lymphoma, and Burkitt lymphoma, is the off-target/aberrant activity of activation-induced cytidine deaminase (AID). AID damages DNA through deamination of cytidine. This occurs on a physiologic basis within GC B cells and is responsible for both class switch recombination (CSR) and somatic hypermutation (SHM). AID can also target nonimmunoglobulin loci, including several proto-oncogenes, resulting in both mutations (aberrant somatic hypermutation; aSHM) and chromosomal translocations. Overexpression of AID has previously been found to increase B-cell genomic instability. In this study, GC-type B cell lines (Ramos, Raji, and CL-01) were treated with pesticides, including herbicides and insecticides from various classes, to determine the effect on AID expression. Real-time RT-PCR was used to determine the degree of AID mRNA expression after chemical treatment. Two representative phenylurea herbicides, linuron and diuron, were found to induce AID expression, in a time- and dose-dependent manner, at concentrations ranging from 50-200 micromolar, with maximal induction of 2- to 6-fold depending on the compound and time point. Additional pesticides that were tested but not found to induce AID expression, at concentrations that were minimally toxic in vitro, included representative organochlorine, organophosphate, phenoxy, and phosphanoglycine pesticides. The findings raise the possibility of increased AID-mediated genomic instability in GC type B cells exposed to phenylurea herbicides. Future studies will investigate the possible link between the observed phenylurea herbicide-related induction of AID, B-cell genomic instability, and lymphomagenesis. Citation Format: Rebecca J. Leeman-Neill, Uttiya Basu. Induction of the lymphomagenic enzyme activation-induced cytidine deaminase (AID) upon in vitro exposure to phenylurea herbicides [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A49.

Activation‐induced cytidine deaminase can target multiple topologies of double‐stranded DNA in a transcription‐independent manner

Activation-induced cytidine deaminase (AID) mutates immunoglobulin genes and acts genome-wide. AID targets robustly transcribed genes, and purified AID acts on single-stranded (ss) but not double-stranded (ds) DNA oligonucleotides. Thus, it is believed that transcription is the generator of ssDNA for AID. Previous cell-free studies examining the relationship between transcription and AID targeting have employed a bacterial colony count assay wherein AID reverts an antibiotic resistance stop codon in plasmid substrates, leading to colony formation. Here, we established a novel assay where kb-long dsDNA of varying topologies is incubated with AID, with or without transcription, followed by direct sequencing. This assay allows for an unselected and in-depth comparison of mutation frequency and pattern of AID targeting in the absence of transcription or across a range of transcription dynamics. We found that without transcription, AID targets breathing ssDNA in supercoiled and, to a lesser extent, in relaxed dsDNA. The most optimal transcription only modestly enhanced AID action on supercoiled dsDNA in a manner dependent on RNA polymerase speed. These data suggest that the correlation between transcription and AID targeting may reflect transcription leading to AID-accessible breathing ssDNA patches naturally occurring in de-chromatinized dsDNA, as much as being due to transcription directly generating ssDNA.

Splicing regulator SRSF1-3 that controls somatic hypermutation of IgV genes interacts with topoisomerase 1 and AID

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.

The IL ‐6 signaling complex is a critical driver, negative prognostic factor, and therapeutic target in diffuse large B‐cell lymphoma

Interleukin‐6 (IL‐6) is a growth factor for normal B cells and plasma cell‐derived malignancies. Here, we show that the IL‐6 signaling pathway is also active in a subset of diffuse large B‐cell lymphoma (DLBCL) patients with particularly poor prognosis. Primary DLBCL cells and DLBCL cell lines expressing IL‐6R engraft and form orthotopic lymphomas in humanized mice that ectopically produce human IL‐6, and in mice reconstituted with a human immune system. We show that a subset of DLBCL cases have evolved mechanisms that ensure constitutive activation of the IL‐6 signaling pathway, i.e., the expression of both chains of the IL‐6R, the expression of the cytokine itself, and the mutational inactivation of a negative regulator of IL‐6 signaling, SOCS1. IL‐6 signaling promotes MYC‐driven lymphomagenesis in a genetically engineered model, and treatment with the IL‐6R‐specific antibody tocilizumab reduces growth of primary DLBCL cells and of DLBCL cell lines in various therapeutic settings. The combined results uncover the IL‐6 signaling pathway as a driver and negative prognosticator in aggressive DLBCL that can be targeted with a safe and well‐tolerated biologic.

DSB structure impacts DNA recombination leading to class switching and chromosomal translocations in human B cells.

Class switch recombination (CSR) requires activation-induced cytidine deaminase (AID) to trigger DNA double strand breaks (DSBs) at the immunoglobulin heavy chain (IGH) in B cells. Joining of AID-dependent DSBs within IGH facilitate CSR and effective humoral immunity, but ligation to DSBs in non-IGH chromosomes leads to chromosomal translocations. Thus, the mechanism by which AID-dependent DSBs are repaired requires careful examination. The random activity of AID in IGH leads to a spectrum of DSB structures. In this report, we investigated how DSB structure impacts end-joining leading to CSR and chromosomal translocations in human B cells, for which models of CSR are inefficient and not readily available. Using CRISPR/Cas9 to model AID-dependent DSBs in IGH and non-IGH genes, we found that DSBs with 5’ and 3’ overhangs led to increased processing during end-joining compared to blunt DSBs. We observed that 5’ overhangs were removed and 3’ overhangs were filled in at recombination junctions, suggesting that different subsets of enzymes are required for repair based on DSB polarity. Surprisingly, while Cas9-mediated switching preferentially utilized NHEJ regardless of DSB structure, A-EJ strongly preferred repairing blunt DSBs leading to translocations in the absence of NHEJ. We found that DSB polarity influenced frequency of Cas9-mediated switching and translocations more than overhang length. Lastly, recombination junctions from staggered DSBs exhibited templated insertions, suggesting iterative resection and filling in during repair. Our results demonstrate that DSB structure biases repair towards NHEJ or A-EJ to complete recombination leading to CSR and translocations, thus helping to elucidate the mechanism of genome rearrangements in human B cells.

Design and Application of a Mutational Sensor for Characterizing AID Activity

Activation‐induced cytidine deaminase (AID) is a mutator enzyme that introduces uracil:guanine mismatches in Immunoglobulin (Igh) genes during the antibody diversification processes of class switch recombination and somatic hypermutation in B lymphocytes. When this happens, one of three pathways is possible. Firstly, the DNA can be directly replicated with the mutation, causing a transition (C → T) mutation. Secondly, during genomic mutation, the repair enzyme Uracil N‐Glycosylase (UNG) can act to base excise the mutation in either an error free fashion, replacing the original cytidine in the mutated spot, or thirdly, an error prone fashion, in which a transversion mutation (C → G/A) will then be introduced. Certain “hotspots” in the DNA sequence increase mutational frequency: cytosine residues are strongly favored when they are part of an RGYW sequence, or its reverse complement, sequence WRCY (where W=A/T, R=A/G, and Y=C/T). However, AID can also produce off target damage to genes, including oncogenes, that result in lymphomas. Therefore, tight regulation of AID mutator activity is necessary for genomic stability. In this study, we designed a cellular mutation reporter assay to determine the role of AID in regulating mutational activity. Mutations introduced by AID were measured with the use of constructs containing a green fluorescent protein (GFP) with premature STOP codons which‐ upon mutation‐ will “turn on” GFP and can be screened by flow cytometry. The use of this construct allows for mutational screening to be done in any cell type in which AID is naturally occurring, such as lymphomas and leukemias, rather than having one cell type with the reporter gene built into the cell line (current method). Once the constructs were successfully created via PCR SOEing reactions, we verified their ability to serve as mutational sensors. To achieve this, cells were co‐transfected with constructs and AID into BOSC cells and analyzed for rates of nucleotide reversion via flow cytometry. AID is shown to reproducibly revert two constructs with premature stop codons in contextual hotspots from GFP− to GFP+, as seen by the increased presence of GFP when analyzed via flow cytometry. The data show that the GFP mutational sensor works and serves as a functional system for determining mutational analysis among BOSC cells via flow cytometry analysis.Support or Funding Information1. Allison Woods is an undergraduate scholar supported in part by the MARC Program at St. Mary's University and the Summer Program in Cancer Research at MD Anderson Cancer Center support by National Cancer Institute (R25CA181004).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Genome-Wide Mutational Patterns in Indolent B-Cell Lymphomas

Introduction: In recent years, strategies have been developed to identify specific mutation patterns within next-generation sequencing data. Distinct mutational patterns can be linked to underlying mutagenic processes in human cancer. One approach analyzes single base substitutions in the context of their neighboring bases as trinucleotides. The relative prevalence of all possible 96 altered trinucleotides defines distinctive mutational signatures. The activity of activation-induced cytidine deaminase (AID) initiates a specific mutational process in B cells. AID induces deamination of deoxycytidine into deoxyuridine. Subsequent mechanisms to repair the resulting mismatch lead to different genomic alterations that can be assigned to three mutational signatures: a canonical signature characterized by C>T/G transitions at WRCY motifs, a non-canonical signature defined by A>C transversions at WAN motifs, and a third AID signature characterized by C>T transitions at RCG motifs with preference for methylated CpG (W: A or T; R: purine; Y: pyrimidine, N: any nucleotide). The latter signature has specifically been designated as AID-mediated CpG-methylation-dependent mutagenesis. AID activity has been linked to the pathogenesis of several B-cell lymphomas, including follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). Therefore, we searched for the contribution of different AID signatures in these B-cell malignancies.Methods: We analyzed the mutational landscape in whole exome (WES) and whole genome (WGS) sequencing data from 41 FL, 30 CLL, 2 MBL, and 43 MCL cases. Somatic variants were called by comparison of tumor and germline DNA with an in-house developed pipeline. Mutational signatures were defined according to the 96-base substitution model (Alexandrov et al. 2013) by an unsupervised machine learning with implementation of the SomaticSignatures R package (Gehring et al. 2015). In addition, MutationalPattern R package (Blokzijl et al. 2018) was executed for comparison to mutational signatures defined in COSMIC.Results: In unsupervised analyses of FL, CLL/MBL, and MCL cases, 77% of the mutation spectrum variance was attributable to four signatures (S1-4). In FL, the mutational landscape was dominated by S4 characterized by mutations in both canonical and non-canonical AID motifs (40%, 95% CI: 35-76%). The second most frequent signature (S2; 27%, 21-49%) was characterized by C>A transitions in the context of the non-canonical AID and the CpG hotspot motifs (RCG). The mutational landscape of CLL and MBL was strongly dominated by signature S3 (50%, 45-95%). S3 contains mutations in RCG motifs as well as mutations in non-canonical AID motifs (NTW), but with a lower contribution that in S4. In contrast, the mutational landscape of MCL was dominated by S1 (31%, 24-55%) characterized by C>T transitions in the RCG motif in addition to a striking prevalence of the TCT>TTT transition that is known to be associated with the activity of APOBEC enzymes. In comparison to the mutational signatures in COSMIC, the lymphomas analyzed here carry a strong similarity to the COSMIC signatures 1, 5, and 25. These signatures are observed across a wide spectrum of cancer types and are either of unknown etiology (S5 and S25) or associated with age (S1).Conclusions: The most common point mutations in CLL/MBL and FL are C>T transitions and indicate a strong influence of AID on their mutational landscape. In the indolent B-cell malignancies, all three known AID-related signatures, i.e. canonical, non-canonical, and CpG-methylation-dependent can be found. In contrast, the genomic landscape of MCL is dominated by variants in CpG-methylation-dependent mutagenesis sites and by an APOBEC-related motif. In addition to AID-related signatures, we also found consensus signatures described in COSMIC such as the age-related spontaneous deamination signature 1. Our work independently confirms the role of AID in B-cell lymphoma pathogenesis but points to disease-specific mechanisms that modulate AID in the respective lymphoma cell of origin. In addition, our data suggest that distinctive repair mechanisms operate in different entities. DisclosuresNo relevant conflicts of interest to declare.