New insights into ATR inhibition in muscle invasive bladder cancer: The role of apolipoprotein B mRNA editing catalytic subunit 3B.

Background: Apolipoprotein B mRNA editing catalytic polypeptide (APOBEC), an endogenous mutator, induces DNA damage and activates the ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase 1 (Chk1) pathway. Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer (MIBC), it has a poor survival rate. Therefore, this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B (APOBEC3B) expressing MIBC. Methods: Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC. The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis. Western blot analysis was performed to confirm differences in phosphorylated Chk1 (pChk1) expression according to the APOBEC3B expression. Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin. Conclusion: There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC. Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels. Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression. Compared to cisplatin single treatment, combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression. Conclusion: Our study shows that APOBEC3B's higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition. This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Introduction: Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Methods: Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. Results: DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Discussion: Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

DNA mismatch and damage patterns revealed by single-molecule sequencing.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

SUMO-specific protease 1 regulates germinal center B cell response through deSUMOylation of PAX5.

Humoral immunity depends on the germinal center (GC) reaction where B cells are tightly controlled for class-switch recombination and somatic hypermutation and finally generated into plasma and memory B cells. However, how protein SUMOylation regulates the process of the GC reaction remains largely unknown. Here, we show that the expression of SUMO-specific protease 1 (SENP1) is up-regulated in GC B cells. Selective ablation of SENP1 in GC B cells results in impaired GC dark and light zone organization and reduced IgG1-switched GC B cells, leading to diminished production of class-switched antibodies with high-affinity in response to a TD antigen challenge. Mechanistically, SENP1 directly binds to Paired box protein 5 (PAX5) to mediate PAX5 deSUMOylation, sustaining PAX5 protein stability to promote the transcription of activation-induced cytidine deaminase. In summary, our study uncovers SUMOylation as an important posttranslational mechanism regulating GC B cell response.

Loss of cytidine deaminase expression as a potential attempt to counteract the process of carcinogenesis by reducing basal PARP-1 activity and increasing tau levels.

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of free cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Our team discovered that CDA deficiency is associated with several aspects of genetic instability, such as increased sister chromatid exchange and ultrafine anaphase bridge frequencies. Based on these results, we sought (1) to determine how CDA deficiency contributes to genetic instability, (2) to explore the possible relationships between CDA deficiency and carcinogenesis, and (3) to develop a new anticancer treatment targeting CDA-deficient tumors. This review summarizes our major findings indicating that CDA deficiency is associated with a genetic instability that does not confer an increased cancer risk. In light of our results and published data, I propose a novel hypothesis that loss of CDA, by reducing basal PARP-1 activity and increasing Tau levels, may reflect an attempt to prevent, slow or reverse the process of carcinogenesis.

The implications of APOBEC3-mediated C-to-U RNA editing for human disease.

Intra-organism biodiversity is thought to arise from epigenetic modification of constituent genes and post-translational modifications of translated proteins. Here, we show that post-transcriptional modifications, like RNA editing, may also contribute. RNA editing enzymes APOBEC3A and APOBEC3G catalyze the deamination of cytosine to uracil. RNAsee (RNA site editing evaluation) is a computational tool developed to predict the cytosines edited by these enzymes. We find that 4.5% of non-synonymous DNA single nucleotide polymorphisms that result in cytosine to uracil changes in RNA are probable sites for APOBEC3A/G RNA editing; the variant proteins created by such polymorphisms may also result from transient RNA editing. These polymorphisms are associated with over 20% of Medical Subject Headings across ten categories of disease, including nutritional and metabolic, neoplastic, cardiovascular, and nervous system diseases. Because RNA editing is transient and not organism-wide, future work is necessary to confirm the extent and effects of such editing in humans.

Her2 amplification, Rel-A, and Bach1 can influence APOBEC3A expression in breast cancer cells.

APOBEC-induced mutations occur in 50% of sequenced human tumors, with APOBEC3A (A3A) being a major contributor to mutagenesis in breast cancer cells. The mechanisms that cause A3A activation and mutagenesis in breast cancers are still unknown. Here, we describe factors that influence basal A3A mRNA transcript levels in breast cancer cells. We found that basal A3A mRNA correlates with A3A protein levels and predicts the amount of APOBEC signature mutations in a panel of breast cancer cell lines, indicating that increased basal transcription may be one mechanism leading to breast cancer mutagenesis. We also show that alteration of ERBB2 expression can drive A3A mRNA levels, suggesting the enrichment of the APOBEC mutation signature in Her2-enriched breast cancer could in part result from elevated A3A transcription. Hierarchical clustering of transcripts in primary breast cancers determined that A3A mRNA was co-expressed with other genes functioning in viral restriction and interferon responses. However, reduction of STAT signaling via inhibitors or shRNA in breast cancer cell lines had only minor impact on A3A abundance. Analysis of single cell RNA-seq from primary tumors indicated that A3A mRNA was highest in infiltrating immune cells within the tumor, indicating that correlations of A3A with STAT signaling in primary tumors may be result from higher immune infiltrates and are not reflective of STAT signaling controlling A3A expression in breast cancer cells. Analysis of ATAC-seq data in multiple breast cancer cell lines identified two transcription factor sites in the APOBEC3A promoter region that could promote A3A transcription. We determined that Rel-A, and Bach1, which have binding sites in these peaks, elevated basal A3A expression. Our findings highlight a complex and variable set of transcriptional activators for A3A in breast cancer cells.

Targeting APOBECs in cancer: It's about timing.

APOBEC3 cytidine deaminases have emerged as key drivers of mutagenesis in a wide spectrum of tumor types and are now appreciated to play a causal role in driving tumor evolution and drug resistance. As efforts to develop APOBEC3 inhibitors progress, understanding the timing and consequences of APOBEC3-mediated mutagenesis in distinct clinical contexts will be critical for guiding the development of anti-cancer therapeutic strategies.

APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Targeted C•G-to-T•A base editing with TALE-cytosine deaminases in plants.

BACKGROUND: TALE-derived DddA-based cytosine base editors (TALE-DdCBEs) can perform efficient base editing of mitochondria and chloroplast genomes. They use transcription activator-like effector (TALE) arrays as programmable DNA-binding domains and a split version of the double-strand DNA cytidine deaminase (DddA) to catalyze C•G-to-T•A editing. This technology has not been optimized for use in plant cells. RESULTS: To systematically investigate TALE-DdCBE architectures and editing rules, we established a ß-glucuronidase reporter for transient assays in Nicotiana benthamiana. We show that TALE-DdCBEs function with distinct spacer lengths between the DNA-binding sites of their two TALE parts. Compared to canonical DddA, TALE-DdCBEs containing evolved DddA variants (DddA6 or DddA11) showed a significant improvement in editing efficiency in Nicotiana benthamiana and rice. Moreover, TALE-DdCBEs containing DddA11 have broader sequence compatibility for non-TC target editing. We have successfully regenerated rice with C•G-to-T•A conversions in their chloroplast genome, as well as N. benthamiana with C•G-to-T•A editing in the nuclear genome using TALE-DdCBE. We also found that the spontaneous assembly of split DddA halves can cause undesired editing by TALE-DdCBEs in plants. CONCLUSIONS: Altogether, our results refined the targeting scope of TALE-DdCBEs and successfully applied them to target the chloroplast and nuclear genomes. Our study expands the base editing toolbox in plants and further defines parameters to optimize TALE-DdCBEs for high-fidelity crop improvement.

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) - a new important player in fish immunology.

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.

Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Distinguishing preferences of human APOBEC3A and APOBEC3B for cytosines in hairpin loops, and reflection of these preferences in APOBEC-signature cancer genome mutations.

The APOBEC3 enzymes convert cytosines in single-stranded DNA to uracils to protect against viruses and retrotransposons but can contribute to mutations that diversify tumors. To understand the mechanism of mutagenesis, we map the uracils resulting from expression of APOBEC3B or its catalytic carboxy-terminal domain (CTD) in Escherichia coli. Like APOBEC3A, the uracilomes of A3B and A3B-CTD show a preference to deaminate cytosines near transcription start sites and the lagging-strand replication templates and in hairpin loops. Both biochemical activities of the enzymes and genomic uracil distribution show that A3A prefers 3 nt loops the best, while A3B prefers 4 nt loops. Reanalysis of hairpin loop mutations in human tumors finds intrinsic characteristics of both the enzymes, with a much stronger contribution from A3A. We apply Hairpin Signatures 1 and 2, which define A3A and A3B preferences respectively and are orthogonal to published methods, to evaluate their contribution to human tumor mutations.

Engineered CBEs based on Macaca fascicularis A3A with improved properties for precise genome editing.

Cytidine deaminase defines the properties of cytosine base editors (CBEs) for C-to-T conversion. Replacing the cytidine deaminase rat APOBEC1 (rA1) in CBEs with a human APOBEC3A (hA3A) improves CBE properties. However, the potential CBE application of macaque A3A orthologs remains undetermined. Our current study develops and evaluates engineered CBEs based on Macaca fascicularis A3A (mA3A). Here, we demonstrate that BE4-mA3A and its RNA-editing-derived variants exhibit improved CBE properties, except for DNA off-target activity, compared to BE3-rA1 and BE4-rA1. Unexpectedly, deleting Ser-Val-Arg (SVR) in BE4-mA3A dramatically reduces DNA and RNA off-target activities and improves editing accuracy, with on-target efficiency unaffected. In contrast, a chimeric BE4-hA3A-SVR+ shows editing efficiency increased by about 50%, with other properties unaffected. Our findings demonstrate that mA3A-based CBEs could provide prototype options with advantages over rA1- and hA3A-based CBEs for further optimization, highlighting the importance of the SVR motif in defining CBE intrinsic properties.

Whole-Genome Mapping of Epigenetic Modification of 5-Formylcytosine at Single-Base Resolution by Chemical Labeling Enrichment and Deamination Sequencing.

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays a critical role in a variety of biological and pathological processes in mammals. In active DNA demethylation, the 10-11 translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution is still highly desirable. Herein, we propose a chemical labeling enrichment and deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (APOBEC3A or A3A) and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mESCs. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution.

The RNA tether model for human chromosomal translocation fragile zones.

One of the two chromosomal breakage events in recurring translocations in B cell neoplasms is often due to the recombination-activating gene complex (RAG complex) releasing DNA ends before end joining. The other break occurs in a fragile zone of 20-600 bp in a non-antigen receptor gene locus, with a more complex and intriguing set of mechanistic factors underlying such narrow fragile zones. These factors include activation-induced deaminase (AID), which acts only at regions of single-stranded DNA (ssDNA). Recent work leads to a model involving the tethering of AID to the nascent RNA as it emerges from the RNA polymerase. This mechanism may have relevance in class switch recombination (CSR) and somatic hypermutation (SHM), as well as broader relevance for other DNA enzymes.

Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function.

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

Cytidine deaminases APOBEC3C and APOBEC3D promote DNA replication stress resistance in pancreatic cancer cells.

Gemcitabine is a potent inhibitor of DNA replication and is a mainstay therapeutic for diverse cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, most tumors remain refractory to gemcitabine therapies. Here, to define the cancer cell response to gemcitabine, we performed genome-scale CRISPR-Cas9 chemical-genetic screens in PDAC cells and found selective loss of cell fitness upon disruption of the cytidine deaminases APOBEC3C and APOBEC3D. Following gemcitabine treatment, APOBEC3C and APOBEC3D promote DNA replication stress resistance and cell survival by deaminating cytidines in the nuclear genome to ensure DNA replication fork restart and repair in PDAC cells. We provide evidence that the chemical-genetic interaction between APOBEC3C or APOBEC3D and gemcitabine is absent in nontransformed cells but is recapitulated across different PDAC cell lines, in PDAC organoids and in PDAC xenografts. Thus, we uncover roles for APOBEC3C and APOBEC3D in DNA replication stress resistance and offer plausible targets for improving gemcitabine-based therapies for PDAC.