DNA Cytosine Deaminase APOBEC3B Research Articles

Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma.

Two APOBEC DNA cytosine deaminase enzymes, APOBEC3A and APOBEC3B, generate somatic mutations in cancer, thereby driving tumour development and drug resistance. Here, we used single-cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell-cycle stage associated with APOBEC-mediated mutagenesis. In contrast, in squamous cell carcinoma we find that, there is expansion of GRHL3expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings suggest that APOBEC3A may play a functional role during keratinocyte differentiation, and offer a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.

Human APOBEC3B promotes tumor development in vivo including signature mutations and metastases

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many cancers. However, despite years of work, a causal relationship has yet to be established invivo. Here, we report a murine model that expresses tumor-like levels of human APOBEC3B. Animals expressing full-body APOBEC3B appear to develop normally. However, adult males manifest infertility, and older animals of both sexes show accelerated rates of carcinogenesis, visual and molecular tumor heterogeneity, and metastasis. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Enrichment for APOBEC3B-attributable single base substitution mutations also associates with elevated levels of insertion-deletion mutations and structural variations. APOBEC3B catalytic activity is required for all of these phenotypes. Together, these studies provide a cause-and-effect demonstration that human APOBEC3B is capable of driving both tumor initiation and evolution invivo.

Primary mucosal melanomas of the head and neck are characterised by overexpression of the DNA mutating enzyme APOBEC3B.

Primary head/neck mucosal melanomas (MMs) are rare and exhibit aggressive biologic behaviour and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in head/neck MMs remain elusive. The DNA cytosine deaminase APOBEC3B (A3B) constitutes a major endogenous source of mutation in human cancer. A3B-related mutations are identified through C-to-T/-G base substitutions in 5'-TCA/T motifs. Herein, we present immunohistochemical and genomic data supportive of a role for A3B in head/neck MMs. A3B protein levels were assessed in oral (n =13) and sinonasal (n =13) melanomas, and oral melanocytic nevi (n =13) by immunohistochemistry using a custom rabbit α-A3B mAb (5210-87-13). Heterogeneous, selective-to-diffuse, nuclear only, A3B immunopositivity was observed in 12 of 13 (92.3%) oral melanomas (H-score range=9-72, median=40) and 8 of 13 (62%) sinonasal melanomas (H-score range=1-110, median=24). Two cases negative for A3B showed prominent cytoplasmic staining consistent with A3G. A3B protein levels were significantly higher in oral and sinonasal MMs than intraoral melanocytic nevi (P < 0.0001 and P=0.0022, respectively), which were A3B-negative (H-score range=1-8, median=4). A3B levels, however, did not differ significantly between oral and sinonasal tumours (P > 0.99). NGS performed in 10 sinonasal MMs revealed missense NRAS mutations in 50% of the studied cases and one each KIT and HRAS mutations. Publicly available whole-genome sequencing (WGS) data disclosed that the number of C-to-T mutations and APOBEC3 enrichment score were markedly elevated in head/neck MMs (n=2). The above data strongly indicate a possible role for the mutagenic enzyme A3B in head/neck melanomagenesis, but not benign melanocytic neoplasms.

Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large >1000-Å2 binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.

Regulatory Mechanisms of Endogenous APOBEC3B Expression in HPV(+) and (–) Oral Premalignancies and Head and Neck Cancer

<h3>Introduction</h3> HPV(+) oral and oropharyngeal squamous cell carcinoma (OOPSCC) shows strong association with high-risk HPV16 and 18. The DNA cytosine deaminase APOBEC3B constitutes a major endogenous source of muta- tions in a wide variety of cancers. APOBEC3B causes C-to-T/-G base substitutions in 5'-TCA/T trinucleotide motifs and is overexpressed in head and neck cancer (HNC). High-risk HPV infection and dysregulation of the RB/E2F axis regu- late <i>APOBEC3B in vitro</i>. Here, we investigate changes of APOBEC3B levels in HPV(+) and (–) oral epithelial dysplasia (OED) and OOPSCC, and probe whether APOBEC3B upregulation correlates with cellular proliferation. <h3>Materials and Methods</h3> APOBEC3B and Ki67 expression was assessed by immunohistochemistry in HPV(+) OED (n=10) and OOPSCC (n=28), and HPV(–) low-grade (n=42) and high-grade OED (n=38), and OSCC (n=46). Epithelial hyperplasia (OEH, n=14) and normal adjacent epithelium (n=10) served as controls. APOBEC3B H-scores and per- centage of Ki67(+) cells were calculated using the Aperio ScanScope XT platform. Public NGS datasets available through TCGA were mined to identify molecular mechanisms associated with APOBEC3B upregulation in HNC. <h3>Results</h3> High-risk HPV significantly upregulated APOBEC3B in OED (Kruskal-Wallis test, p<0.05) and OOPSCC (p<0.01) compared to OEH. HPV(+) OEDs and tumors showed strong and diffuse, nuclear APOBEC3B immunore- activity which mirrored p16 staining and coincided with disrupted p53 expression. Progressive APOBEC3B up- regulation was observed in HPV(–) lesions; high-grade premalignancies and OSCCs showed significantly elevated APOBEC3B H-scores compared to low-grade OED (p<0.01) and controls (p<0.01). High mRNA and protein APOBEC3B levels strongly associated with advanced histologic grading in HPV(–) OSCC (p<0.01). A positive linear correlation was evident between APOBEC3B and Ki67 expression (Pearson's r=0.5), while RNAseq analysis suggests cell cycle regulation of APOBEC3B in HPV(–) HNC. <h3>Conclusions</h3> High-risk HPV drives upregulation of APOBEC3B in viral-related OED and HNC. In non-viral lesions, APOBEC3B levels also increase in a mechanism associated in part with cellular proliferation.

Head and Neck Mucosal Melanomas are Characterized by Overexpression of the DNA Mutating Enzyme APOBEC3B

<h3>Introduction</h3> Primary head and neck mucosal melanomas (HNMMs) are rare and exhibit aggressive biologic behavior, dismal prognosis, and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in HNMMs remain elusive. The DNA cytosine deaminase APOBEC3B constitutes a major endogenous source of mutation in a plethora of human cancers. APOBEC3B-related mutations are identified through C-to-T/-G base substitutions in 5'-TCA/T trinucleotide motifs. Herein, we provide immunohistochemical and genomic data strongly supportive of the role of APOBEC3B in HNMMs. <h3>Materials and Methods</h3> APOBEC3B protein levels were assessed in oral (N=16) and sinonasal (N=15) melanomas by immunohistochemistry using a custom rabbit monoclonal antibody (5210-87-13). Benign oral melanocytic nevi (N=13) served as a control group. Nuclear APOBEC3B immunoreactivity was visualized and quantified with the Aperio ScanScope XT platform. Statistical differences between groups were calculated using Kruskal-Wallis oneway non-parametric tests. Publicly available genomic data were further analyzed to assess the impact of APOBEC3B expression in mucosal melanomas. <h3>Results</h3> Focal to diffuse, nuclear APOBEC3B immunopositivity was observed in all oral melanomas (H-score range=9-72, median=39) and 12 of 15 sinonasal melanomas (H-score range=1-110, median=24). The intensity of immunohistochemical positivity demonstrated a wide range of inter- and intra-tumor heterogeneity. APOBEC3B protein levels were significantly higher in oral and sinonasal melanomas than oral nevi (p<0.0001 and p=0.0032, respectively), which were overall negative for APOBEC3B (H-score range=1-12, median=6). APOBEC3B levels, however, did not differ significantly between oral and sinonasal tumors (p>0.99). Genomic analysis showed that overall mutation load, number of C-to-T transitions, and proportion of APOBEC3B-related mutation signatures 2 and 13 were higher in HNMMs than melanomas developing in other mucosal sites, and cutaneous melanomas. <h3>Conclusions</h3> The mutagenic enzyme APOBEC3B is overexpressed in HNMMs but not benign mucosal melanotic neoplasms. APOBEC3B may be a novel biomarker and therapeutic target for this clinically aggressive type of human malignancy.

ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

R-Loop Editing by DNA Cytosine Deaminase APOBEC3B Determines the Activity of Estrogen Receptor Enhancers

R-Loop Editing by DNA Cytosine Deaminase APOBEC3B Determines the Activity of Estrogen Receptor Enhancers

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Host-Pathogen Interactions in Human Polyomavirus 7‒Associated Pruritic Skin Eruption

Abstract 286: Regulation of APOBEC3B gene expression through the Rb/E2F pathway

Abstract The propensity of tumors to increase genomic diversity allows for a bottleneck-type adaptation to anti-cancer treatments. The genomic alterations fueling these adaptations in many different tumor types are, in part, brought about by ongoing endogenous activity of the DNA cytosine deaminase APOBEC3B (A3B). The ability of A3B to convert DNA cytosine bases into uracils has been shown to facilitate C-to-T transitions, C-to-G transversions, and DNA breakage. While low in healthy tissues, A3B expression and activity is elevated in tumors and may increase further throughout metastatic disease. However, molecular mechanisms responsible for transcriptional upregulation of A3B in tumor cells are poorly understood. Here, we investigate the possibility that the Rb/E2F pathway, which is often dysregulated in breast cancer, contributes to A3B gene expression. First, we cloned the A3B promoter upstream of a luciferase reporter and used a combination of deletion and site-directed mutagenesis to demonstrate that only one out of five in silico predicted E2F binding sites is functionally relevant, as disrupting this site caused constitutive reporter activation. Moreover, expression of the BK polyomavirus T-antigen, which inactivates Rb, induced the reporter and additional activation was not observed by mutating the functional E2F binding site. Second, SILAC-labelled nuclear protein extracts were subjected to affinity purification coupled to quantitative mass spectrometry to compare transcriptional complexes that bound wild-type promoter sequences, but not sequences with mutant E2F binding site promoter elements. These proteomics experiments showed that proteins belonging to the DREAM and PRC1.6-complexes strongly bind the wild-type but not the mutant E2F binding sites. Third, RNAi-mediated knock-down of candidate E2F family members implicated E2F6, and to a lesser extend E2F4, which are components of the PRC1.6 and DREAM complexes, respectively, in A3B gene repression. Taken together, these studies indicate that A3B expression may be suppressed by the concerted effort of two different E2F binding complexes and that Rb inactivation, by viral T-antigen here and genetic mutation as often seen in breast cancer, is able to upregulate A3B gene expression, promote genomic DNA mutagenesis, and fuel tumor evolution. Citation Format: Pieter A. Roelofs, William L. Brown, Paul N. Span, John W. Martens, Chai Y. Goh, Boon H. Chua, Dennis Kappei, Reuben S. Harris. Regulation of APOBEC3B gene expression through the Rb/E2F pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 286.

Endogenous APOBEC3B overexpression characterizes HPV-positive and HPV-negative oral epithelial dysplasias and head and neck cancers.

The DNA cytosine deaminase APOBEC3B (A3B) is a newly recognized endogenous source of mutations in a range of human tumors, including head/neck cancer. A3B inflicts C-to-T and C-to-G base substitutions in 5'-TCA/T trinucleotide motifs, contributes to accelerated rates of tumor development, and affects clinical outcomes in a variety of cancer types. High-risk human papillomavirus (HPV) infection causes A3B overexpression, and HPV-positive cervical and head/neck cancers are among tumor types with the highest degree of APOBEC signature mutations. A3B overexpression in HPV-positive tumor types is caused by the viral E6/E7 oncoproteins and may be an early off-to-on switch in tumorigenesis. In comparison, less is known about the molecular mechanisms responsible for A3B overexpression in HPV-negative head/neck cancers. Here, we utilize an immunohistochemical approach to determine whether A3B is turned from off-to-on or if it undergoes a more gradual transition to overexpression in HPV-negative head/neck cancers. As positive controls, almost all HPV-positive oral epithelial dysplasias and oropharyngeal cancers showed high levels of nuclear A3B staining regardless of diagnosis. As negative controls, A3B levels were low in phenotypically normal epithelium adjacent to cancer and oral epithelial hyperplasias. Interestingly, HPV-negative and low-grade oral epithelial dysplasias showed intermediate A3B levels, while high-grade oral dysplasias showed high A3B levels similar to oral squamous cell carcinomas. A3B levels were highest in grade 2 and grade 3 oral squamous cell carcinomas. In addition, a strong positive association was found between nuclear A3B and Ki67 scores suggesting a linkage to the cell cycle. Overall, these results support a model in which gradual activation of A3B expression occurs during HPV-negative tumor development and suggest that A3B overexpression may provide a marker for advanced grade oral dysplasia and cancer.

The DNA Cytosine Deaminase APOBEC3B is a Molecular Determinant of Platinum Responsiveness in Clear Cell Ovarian Cancer.

Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

Active site plasticity and possible modes of chemical inhibition of the human DNA deaminase APOBEC3B.

The single‐stranded DNA cytosine deaminase APOBEC3B (A3B) functions in innate immunity against viruses, but it is also strongly implicated in eliciting mutations in cancer genomes. Because of the critical role of A3B in promoting virus and tumor evolution, small molecule inhibitors are desirable. However, there is no reported structure for any of the APOBEC3‐family enzymes in complex with a small molecule bound in the active site, which hampers the development of small molecules targeting A3B. Here we report high‐resolution structures of an active A3B catalytic domain chimera with loop 7 residues exchanged with those from the corresponding region of APOBEC3G (A3G). The structures reveal novel open conformations lacking the catalytically essential zinc ion, with the highly conserved active site residues extensively rearranged. These inactive conformations are stabilized by 2‐pyrimidone or an iodide ion bound in the active site. Molecular dynamics simulations corroborate the remarkable plasticity of the engineered active site and identify key interactions that stabilize the native A3B active site. These data provide insights into A3B active site dynamics and suggest possible modes of its inhibition by small molecules, which would aid in rational design of selective A3B inhibitors for constraining virus and tumor evolution.

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

Upregulation of MTA1 expression by human papillomavirus infection promotes CDDP resistance in cervical cancer cells via modulation of NF-κB/APOBEC3B cascade.

Compelling evidence establishes the etiological role of viral proteins E6 and E7 of high-risk human papillomaviruses (HPV) in cervical carcinogenesis, but their contribution in chemoresistance that leads to advanced metastatic lesions remains poorly defined. Since metastasis-associated protein 1 (MTA1) upregulation and augmentation of APOBEC3B expression are both strongly associated with cervical cancer (CCa) development, and both molecules have been shown to be functionally associated with NF-κB pathway, we therefore sought to investigate the potential mechanistic link between MTA1, APOBEC3B and NF-κB during the pathogenesis of cisplatin (CDDP) resistance in HPV-positive CCa cells. MTA1 expression was assessed in HPV-transfected CCa cells using quantitative RT-PCR and immunoblotting. Effects of MTA1 deregulation on CDDP chemosensitivity in CCa cells were determined by measuring cell viability, apoptosis and in vivo oncogenic capacity. Finally, we studied the transcriptional regulation of the antiviral DNA cytosine deaminase APOBEC3B by MTA1 using multiple approaches including DNA deaminase activity assay, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation and transient/stable transfection, at the molecular and functional levels. Expression levels of MTA1 were significantly induced in HPV-positive CCa cells. Transduction experiments showed that the E6 oncoprotein alone was sufficient to cause MTA1 upregulation. Moreover, MTA1 knockdown potentiated CDDP sensitivity in highly metastatic CCa cells. Mechanistically, MTA1 acted as an indirect upstream modulator of APOBEC3B transcription during the pathogenesis of CDDP chemoresistance. HPV-mediated stimulation of APOBEC3B expression was accompanied by the enhanced recruitment of Iκκ α/β and p65 to the NF-κB consensus sites in the APOBEC3B promoter, and this recruitment was substantially abrogated by MTA1 siRNA treatment. These findings reveal an obligatory coregulatory role of MTA1 in the indirect regulation of APOBEC3B expression via classical NF-κB pathway, and also suggest that inhibition of MTA1/NF-κB/APOBEC3B cascade may be repositioned to suppress cancer mutagenesis, dampen tumor evolution, and decrease the probability of adverse outcomes from CDDP resistance in CCa.

The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer.

Breast tumors often display extreme genetic heterogeneity characterized by hundreds of gross chromosomal aberrations and tens of thousands of somatic mutations. Tumor evolution is thought to be ongoing and driven by multiple mutagenic processes. A major outstanding question is whether primary tumors have preexisting mutations for therapy resistance or whether additional DNA damage and mutagenesis are necessary. Drug resistance is a key measure of tumor evolvability. If a resistance mutation preexists at the time of primary tumor presentation, then the intended therapy is likely to fail. However, if resistance does not preexist, then ongoing mutational processes still have the potential to undermine therapeutic efficacy. The antiviral enzyme APOBEC3B (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B) preferentially deaminates DNA C-to-U, which results in signature C-to-T and C-to-G mutations commonly observed in breast tumors. We use clinical data and xenograft experiments to ask whether APOBEC3B contributes to ongoing breast tumor evolution and resistance to the selective estrogen receptor modulator, tamoxifen. First, APOBEC3B levels in primary estrogen receptor-positive (ER+) breast tumors inversely correlate with the clinical benefit of tamoxifen in the treatment of metastatic ER+ disease. Second, APOBEC3B depletion in an ER+ breast cancer cell line results in prolonged tamoxifen responses in murine xenograft experiments. Third, APOBEC3B overexpression accelerates the development of tamoxifen resistance in murine xenograft experiments by a mechanism that requires the enzyme's catalytic activity. These studies combine to indicate that APOBEC3B promotes drug resistance in breast cancer and that inhibiting APOBEC3B-dependent tumor evolvability may be an effective strategy to improve efficacies of targeted cancer therapies.

The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis

Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of ‘APOBEC signature' mutations in cancer.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

Functional Upregulation of the DNA Cytosine Deaminase APOBEC3B by Polyomaviruses

The APOBEC3 family of DNA cytosine deaminases has important roles in innate immunity and cancer. It is unclear how DNA tumor viruses regulate these enzymes and how these interactions, in turn, impact the integrity of both the viral and cellular genomes. Polyomavirus (PyVs) are small DNA pathogens that contain oncogenic potentials. In this study, we examined the effects of PyV infection on APOBEC3 expression and activity. We demonstrate that APOBEC3B is specifically upregulated by BK polyomavirus (BKPyV) infection in primary kidney cells and that the upregulated enzyme is active. We further show that the BKPyV large T antigen, as well as large T antigens from related polyomaviruses, is alone capable of upregulating APOBEC3B expression and activity. Furthermore, we assessed the impact of A3B on productive BKPyV infection and viral genome evolution. Although the specific knockdown of APOBEC3B has little short-term effect on productive BKPyV infection, our informatics analyses indicate that the preferred target sequences of APOBEC3B are depleted in BKPyV genomes and that this motif underrepresentation is enriched on the nontranscribed stand of the viral genome, which is also the lagging strand during viral DNA replication. Our results suggest that PyV infection upregulates APOBEC3B activity to influence virus sequence composition over longer evolutionary periods. These findings also imply that the increased activity of APOBEC3B may contribute to PyV-mediated tumorigenesis. Polyomaviruses (PyVs) are a group of emerging pathogens that can cause severe diseases, including cancers in immunosuppressed individuals. Here we describe the finding that PyV infection specifically induces the innate immune DNA cytosine deaminase APOBEC3B. The induced APOBEC3B enzyme is fully functional and therefore may exert mutational effects on both viral and host cell DNA. We provide bioinformatic evidence that, consistent with this idea, BK polyomavirus genomes are depleted of APOBEC3B-preferred target motifs and enriched for the corresponding predicted reaction products. These data imply that the interplay between PyV infection and APOBEC proteins may have significant impact on both viral evolution and virus-induced tumorigenesis.

OC-0443: Radiotherapy sensitivity in breast cancer is influenced by the DNA cytosine deaminase APOBEC3B

OC-0443: Radiotherapy sensitivity in breast cancer is influenced by the DNA cytosine deaminase APOBEC3B