APOBEC3 Genes Research Articles

Abstract 2518: APOBEC3-mediated C-to-U RNA editing in non-small cell lung cancer tumors is increased in patients with smoking history and squamous tumor histology

Abstract Cytidine deaminase activity of APOBEC3 enzyme family is one of the causes of DNA mutations in many cancers. Members of this family are also RNA editors, capable of converting cytidine (C) bases at specific positions of RNAs to uracil (U). The prevalence and significance of APOBEC3-mediated site-specific C>U RNA editing in cancer is unknown. To study this in non-small cell lung cancer (NSCLC), we examined 1009 primary NSCLC tumors of The Cancer Genome Atlas project for RNA editing at 5,208 sites of 3,630 gene transcripts that are known to undergo APOBEC3-mediated RNA editing. A bioinformatics pipeline was developed to determine editing levels at these sites using whole exome (DNA) and RNA sequencing data. Cox regression, Spearman, and Welch's t tests were respectively used for analysis of survival, correlation, and group comparison. RNA editing could be determined for only some (mean 35, SD 64) of the examined sites because of poor sequencing coverage. For 333 sites (313 transcripts), editing was determinable for ≥10 tumors, with editing occurring at the sites in 2%-92% of tumors (mean 23%, SD 20%). Editing was most prevalent for SLC37A2 (92% of tumors), SERPIN1A (92%), and HPS1 (88%) transcripts. There were 366 tumors with editing determinable for ≥10 sites and occurring at ≥1 site. Among these tumors, editing occurred at 0.1%-100% of sites (mean 20%, SD 27%) at a level of 1%-18% (mean 6%, SD 3%). We summarized the site-specific editing determinations into an editing score, and used this measurement for further analyses. Editing score was on average 3.7x higher in tumors compared to adjacent normal tissues (P = 0.01). Tumors of squamous histology had 3.6x higher editing than adenocarcinoma (P <0.01). Patients with a history of smoking had 2x higher editing score than never-smokers (P = 0.02). There was no association of editing with pathologic tumor stage, but increased editing was associated with improved disease progression-free interval (HR = 0.4, P = 0.01). Correlation of tumor APOBEC3 gene expression levels with editing (r = 0.53 for APOBEC3A, 0.2-0.4 for others) was better than with APOBEC-mediated mutation burden (r = 0.29 for APOBEC3A, 0.07-0.27 for others). In conclusion, APOBEC3-mediated C>U RNA editing is prevalent in non-small cell lung cancer tumors, and reflects APOBEC3 gene expression better than APOBEC-mediated mutation burden. Additional research is needed to identify if editing occurs in cancer cells or in stromal cells of tumors and how it affects genesis and progression of cancer. Citation Format: Mariko Asaoka, Frank Zhang, Takashi Ishikawa, Kazuaki Takabe, Saikrishna Yendamuri, Santosh K. Patnaik. APOBEC3-mediated C-to-U RNA editing in non-small cell lung cancer tumors is increased in patients with smoking history and squamous tumor histology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2518.

HIV-1 restriction by endogenous APOBEC3G in the myeloid cell line THP-1.

HIV-1 replication in CD4-positive T lymphocytes requires counteraction of multiple different innate antiviral mechanisms. Macrophage cells are also thought to provide a reservoir for HIV-1 replication but less is known in this cell type about virus restriction and counteraction mechanisms. Many studies have combined to demonstrate roles for APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H in HIV-1 restriction and mutation in CD4-positive T lymphocytes, whereas the APOBEC enzymes involved in HIV-1 restriction in macrophages have yet to be delineated fully. We show that multiple APOBEC3 genes including APOBEC3G are expressed in myeloid cell lines such as THP-1. Vif-deficient HIV-1 produced from THP-1 is less infectious than Vif-proficient virus, and proviral DNA resulting from such Vif-deficient infections shows strong G to A mutation biases in the dinucleotide motif preferred by APOBEC3G. Moreover, Vif mutant viruses with selective sensitivity to APOBEC3G show Vif null-like infectivity levels and similarly strong APOBEC3G-biased mutation spectra. Importantly, APOBEC3G-null THP-1 cells yield Vif-deficient particles with significantly improved infectivities and proviral DNA with background levels of G to A hypermutation. These studies combine to indicate that APOBEC3G is the main HIV-1 restricting APOBEC3 family member in THP-1 cells.

The APOBEC3 genes and their role in cancer: insights from human papillomavirus.

The interaction between human papillomaviruses (HPV) and the apolipoprotein-B mRNA-editing catalytic polypeptide-like (APOBEC)3 (A3) genes has garnered increasing attention in recent years, with considerable efforts focused on understanding their apparent roles in both viral editing and in HPV-driven carcinogenesis. Here, we review these developments and highlight several outstanding questions in the field. We consider whether editing of the virus and mutagenesis of the host are linked or whether both are essentially separate events, coincidentally mediated by a common or distinct A3 enzymes. We discuss the viral mechanisms and cellular signalling pathways implicated in A3 induction in virally infected cells and examine which of the A3 enzymes might play the major role in HPV-associated carcinogenesis and in the development of therapeutic resistance. We consider the parallels between A3 induction in HPV-infected cells and what might be causing aberrant A3 activity in HPV-independent cancers such as those arising in the bladder, lung and breast. Finally, we discuss the implications of ongoing A3 activity in tumours under treatment and the therapeutic opportunities that this may present.

PYHIN genes as potential biomarkers for prognosis of human papillomavirus-positive or -negative head and neck squamous cell carcinomas.

The aim of the present study is to determine the expression levels of PYHIN (IFI16 and AIM2) and APOBEC3 (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) gene family members in a cohort of patients with head and neck squamous cell carcinoma (HNSCC) and assess their potential correlation with human papillomavirus (HPV) infection status, clinical characteristics, and survival. For this purpose, 34 HNSCC tissue specimens along with healthy surrounding mucosa were collected from patients surgically treated for HNSCC. Nucleic acids were isolated to assess the presence of HPV and the expression levels of selected molecular markers. Survival analysis was carried out using the Kaplan-Meier method. In HPV-negative (HPV-) HNSCCs, we detected low mRNA expression levels of IFI16, A3A, and A3B, whereas these genes were upregulated of 2-100 folds in HPV-positive (HPV+) tumors (p < 0.05). Interestingly, AIM2 gene expression levels were predominantly unchanged in HPV+ HNSCCs compared to their HPV- counterparts, in which AIM2 was predominantly upregulated (10% vs. 50% of patients). In HPV- tumors, upregulation of TP53, NOTCH1, PD-L1, and IFI16 correlated with lower occurrence of nodal metastases. On the other hand, the expression of APOBEC family members did not correlate with clinical characteristics. Regarding survival, patients with upregulated A3F gene expression had a worse prognosis, while patients without changes in A3H expression had a lower survival rate. In conclusion, our findings indicate that the innate immune sensors IFI16 and AIM2 and some APOBEC family members could be potentially used as biomarkers for disease outcome in HNSCC patients regardless of HPV presence.

Pan-cancer transcriptomic analysis dissects immune and proliferative functions of APOBEC3 cytidine deaminases.

APOBEC3 cytidine deaminases are largely known for their innate immune protection from viral infections. Recently, members of the family have been associated with a distinct mutational activity in some cancer types. We report a pan-tissue, pan-cancer analysis of RNA-seq data specific to the APOBEC3 genes in 8,951 tumours, 786 cancer cell lines and 6,119 normal tissues. By deconvolution of levels of different cell types in tumour admixtures, we demonstrate that APOBEC3B (A3B), the primary candidate as a cancer mutagen, shows little association with immune cell types compared to its paralogues. We present a pipeline called RESPECTEx (REconstituting SPecific Cell-Type Expression) and use it to deconvolute cell-type specific expression levels in a given cohort of tumour samples. We functionally annotate APOBEC3 co-expressing genes, and create an interactive visualization tool which ‘barcodes’ the functional enrichment (http://fraternalilab.kcl.ac.uk/apobec-barcodes/). These analyses reveal that A3B expression correlates with cell cycle and DNA repair genes, whereas the other APOBEC3 members display specificity for immune processes and immune cell populations. We offer molecular insights into the functions of individual APOBEC3 proteins in antiviral and proliferative contexts, and demonstrate the diversification this family of enzymes displays at the transcriptomic level, despite their high similarity in protein sequences and structures.

Recurrent Loss of APOBEC3H Activity during Primate Evolution.

Genes in the APOBEC3 family encode cytidine deaminases that provide a barrier against viral infection and retrotransposition. Of all the APOBEC3 genes in humans, APOBEC3H (A3H) is the most polymorphic: some genes encode stable and active A3H proteins, while others are unstable and poorly antiviral. Such variation in human A3H affects interactions with the lentiviral antagonist Vif, which counteracts A3H via proteasomal degradation. In order to broaden our understanding of A3H-Vif interactions, as well as its evolution in Old World monkeys, we characterized A3H variation within four African green monkey (AGM) subspecies. We found that A3H is highly polymorphic in AGMs and has lost antiviral activity in multiple Old World monkeys. This loss of function was partially related to protein expression levels but was also influenced by amino acid mutations in the N terminus. Moreover, we demonstrate that the evolution of A3H in the primate lineages leading to AGMs was not driven by Vif. Our work suggests that the activity of A3H is evolutionarily dynamic and may have a negative effect on host fitness, resulting in its recurrent loss in primates.IMPORTANCE Adaptation of viruses to their hosts is critical for viral transmission between different species. Previous studies had identified changes in a protein from the APOBEC3 family that influenced the species specificity of simian immunodeficiency viruses (SIVs) in African green monkeys. We studied the evolution of a related protein in the same system, APOBEC3H, which has experienced a loss of function in humans. This evolutionary approach revealed that recurrent loss of APOBEC3H activity has taken place during primate evolution, suggesting that APOBEC3H places a fitness cost on hosts. The variability of APOBEC3H activity between different primates highlights the differential selective pressures on the APOBEC3 gene family.

Abstract 389: Integrative genomic analyses of mutation signatures, immunogenicity and germline copy number variation of the APOBEC genes in multiple cancer types

Abstract The APOBEC-signature mutation has been frequently observed in multiple cancer types, including bladder, breast, and lung adenocarcinoma. Increased expression of the APOBEC3A and APOBEC3B genes is known to induce APOBEC-signature mutations in these cancer types. Limited studies have investigated whether the mutation patterns are associated with individual isoforms of the APOBEC3A and APOBEC3B genes, particularly the isoform uc011aoc from an APOBEC3A/3B chimera that is primarily generated by a common germline deletion. Previous studies have shown that somatic missense mutations contribute to neoantigen load and consequently may affect cancer immunogenic abilities such as attracting tumor infiltration lymphocytes (TILs). However, the influence of APOBEC-signature mutations on tumor immunology remains largely unknown. In this study, we systematically analyzed the APOBEC isoform expression and mutation signature, neoantigen load, and copy number variations in ~4000 tumor samples across 10 cancer types using data from The Cancer Genome Atlas. Using multiple regression analyses that included all isoforms of both APOBEC3A and APOBEC3B, we found that the isoforms uc003awn (APOBEC3A) and uc011awo (APOBEC3B) were significantly associated with APOBEC-signature mutations (defined as TCW change to either TTW or TGW mutations) in multiple cancer types, while the association for uc011aoc was observed only in breast cancer. We also observed that APOBEC-signature mutations were positively associated with neoantigen load in all cancer types except lung adenocarcinoma and kidney carcinoma (P &amp;lt; 0.05). To further investigate the effects of these mutations on TILs, we used gene expression data and the tool CIBERSORT to predict the relative compositions of immune cells for each cancer type. We found that APOBEC-signature mutations were significantly associated with the compositions of CD8+ and CD4 memory-activated T cells only in breast and bladder cancer types. Furthermore, we found that neoantigen load was significantly associated with these cell compositions in multiple cancer types, but was not observed in breast cancer. Lastly, our results showed that a germline common deletion in APOBEC3A/B was associated with a significantly increased expression level of uc011aoc isoform, but decreased expression levels of both uc003awn and uc011awo across cancer types. In addition, we found that the deletion was associated with the evaluated neoantigen load and the composition of T cells (CD8+) in breast cancer. The results suggest potential mechanisms for the association of APOBEC3A/B gene deletion with increased breast cancer risk reported from previous genome wide association studies. Our study provides novel insights into understanding the biological and immunological mechanisms through which APOBEC genes may be involved in carcinogenesis. Citation Format: Xingyi Guo, Xiao-ou Shu, Jiandong Bao, Wanqing Wen, Qiuyin Cai, Jirong Long, Wei Zheng. Integrative genomic analyses of mutation signatures, immunogenicity and germline copy number variation of the APOBEC genes in multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 389.

PO-303 Germline determinants of the somatic mutation landscape in 2642 cancer genomes

IntroductionCancers develop through somatic mutations; however, germline genetic variation contributes to cancer risk via diverse mechanisms including by modulating mutational processes.Material and methodsWithin the Pan Cancer Analysis of Whole Genomes (PCAWG) project, we discovered and phased 88 million single nucleotide variants, short insertions/deletions, and large structural variants in whole genomes from 2642 cancer patients, and employed this resource to investigate germline determinants of somatic mutation across 39 cancer types.Results and discussionsWe describe over 100 germline L1 retrotransposons mediating somatic retrotransposition activity in cancer. Furthermore, rare damaging germline mutations in genes involved in DNA repair, DNA replication, and cell cycle associate with a variety of somatic mutation processes. We implicate mutations in the DNA glycosylase MBD4 with an elevated rate of C>T mutations at CpG dinucleotides, resulting in the genetic modulation of a widespread mutational process. Genome-wide association analysis reveals common genetic variation within the APOBEC3 gene cluster modulating mutations attributed to APOBEC cytidine deaminases in multiple cancer types. Analysis of somatic structural variation additionally exposed complex rearrangement patterns including duplications and template insertion cycles in BRCA1-deficient cancers.ConclusionOur study underscores the notable impact rare and common germline variants have on cancer mutational landscapes.

Correlation of gene expression and associated mutation profiles of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT with chemosensitivity of cancer cell lines to drug treatment

BackgroundThe APOBEC gene family of cytidine deaminases plays important roles in DNA repair and mRNA editing. In many cancers, APOBEC3B increases the mutation load, generating clusters of closely spaced, single-strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested a possible involvement of APOBEC3A, REV1, UNG, and FHIT in molecular processes affecting APOBEC mutagenesis. It is important to understand how mutagenic processes linked to the activity of these genes may affect sensitivity of cancer cells to treatment.ResultsWe used information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine associations of the prevalence of APOBEC-like motifs and mutational loads with expression of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT and with cell line chemosensitivity to 255 antitumor drugs. Among the five genes, APOBEC3B expression levels were bimodally distributed, whereas expression of APOBEC3A, REV1, UNG, and FHIT was unimodally distributed. The majority of the cell lines had low levels of APOBEC3A expression. The strongest correlations of gene expression levels with mutational loads or with measures of prevalence of APOBEC-like motif counts and kataegis clusters were observed for REV1, UNG, and APOBEC3A. Sensitivity or resistance of cell lines to JQ1, palbociclib, bicalutamide, 17-AAG, TAE684, MEK inhibitors refametinib, PD-0325901, and trametinib and a number of other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancers or across cancer types.ConclusionsWe observed correlations of expression levels of the five candidate genes in cell line models with sensitivity to cancer drug treatment. We also noted suggestive correlations between measures of abundance of APOBEC-like sequence motifs with drug sensitivity in small samples of cell lines from individual cancer categories, which require further validation in larger datasets. Molecular mechanisms underlying the links between the activities of the products of each of the five genes, the resulting mutagenic processes, and sensitivity to each category of antitumor agents require further investigation.

The human immune response to RSV includes a loop between the tumor suppressor p53 and expression of APOBEC3 genes

BackgroundRespiratory syncytial virus (RSV) infects nearly all children by theirsecond year of life with re‐infections occurring throughout adolescence andadulthood. RSV infection typicallyresults in mild host symptoms. However, susceptible individuals can develop severe lower respiratory tract infectionsand bronchiolitis, sometimes leading to hospitalization or death. Recently we established that in addition toits protective role against cancer development, the tumor suppressor p53 alsocontributes to host immune responses regulating the expression of several genesincluding the APOBEC3 family of DNA cytidine deaminases (A3), consideredsentinels in innate immune response against viral infections.ObjectiveUsinghuman lung cancer cells, we tested the role of p53 in response to RSV infectionand its replication through the regulation of the A3 gene family as well asother components of the immune response.MethodsA549 and H1299 cells were infected with RSV (Strain A2). Cells were thenharvested 72–120 hours post‐infection and qPCR and Nanostring arrays? were usedto evaluate mRNA expression of RSV‐g, P53, A3s and other immune targets. Expression of RSV was assessed? by ConfocalMicroscopy and cytofluorometry while Annexin V/PI assay was used to evaluateapoptosis.ResultsIn response to acute and chronic RSV infection in human lung cancer cells, p53 was activated and had an impact on RSV replication, which involves theexpression in a p53 dependent manner of several members of the A3 gene familyas well as other innate immune genes. APOBEC3G (A3G) was highly induced in a dose‐ and time‐dependent mannerafter RSV only in lung cancer cells harboring WT p53. Accordingly, overexpression of A3G in lungcancer cells reduced RSV replication. Furthermore, a Nanotstring transcriptome analysis (immune code set) after RSV infectionrevealed a strong p53 dependent regulation of immune response genes, includingseveral components of the innate immune antiviral response. In addition, RSV infection induced p53phosphorylation at serine 15 and triggered p53‐dependent apoptosis in p53 functional cells while p53 deficiency correlated with increased viralreplication and increased syncytia phenotype.ConclusionsOverall, our findings establish that the “guardian of the genome” role ascribed to p53 also extends to a unique component of the immune system – the A3 genes – revealing novel components in the antiviralimmune response against RSV infection.Support or Funding InformationSupported by NIEHS Division of Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity.

Bats have attracted attention in recent years as important reservoirs of viruses deadly to humans and other mammals. These infections are typically nonpathogenic in bats raising questions about innate immune differences that might exist between bats and other mammals. The APOBEC3 gene family encodes antiviral DNA cytosine deaminases with important roles in the suppression of diverse viruses and genomic parasites. Here, we characterize pteropid APOBEC3 genes and show that species within the genus Pteropus possess the largest and most diverse array of APOBEC3 genes identified in any mammal reported to date. Several bat APOBEC3 proteins are antiviral as demonstrated by restriction of retroviral infectivity using HIV-1 as a model, and recombinant A3Z1 subtypes possess strong DNA deaminase activity. These genes represent the first group of antiviral restriction factors identified in bats with extensive diversification relative to homologues in other mammals.

Expansions, diversification, and interindividual copy number variations of AID/APOBEC family cytidine deaminase genes in lampreys

Cytidine deaminases of the AID/APOBEC family catalyze C-to-U nucleotide transitions in mRNA or DNA. Members of the APOBEC3 branch are involved in antiviral defense, whereas AID contributes to diversification of antibody repertoires in jawed vertebrates via somatic hypermutation, gene conversion, and class switch recombination. In the extant jawless vertebrate, the lamprey, two members of the AID/APOBEC family are implicated in the generation of somatic diversity of the variable lymphocyte receptors (VLRs). Expression studies linked CDA1 and CDA2 genes to the assembly of VLRA/C genes in T-like cells and the VLRB genes in B-like cells, respectively. Here, we identify and characterize several CDA1-like genes in the larvae of different lamprey species and demonstrate that these encode active cytidine deaminases. Structural comparisons of the CDA1 variants highlighted substantial differences in surface charge; this observation is supported by our finding that the enzymes require different conditions and substrates for optimal activity in vitro. Strikingly, we also found that the number of CDA-like genes present in individuals of the same species is variable. Nevertheless, irrespective of the number of different CDA1-like genes present, all lamprey larvae have at least one functional CDA1-related gene encoding an enzyme with predicted structural and chemical features generally comparable to jawed vertebrate AID. Our findings suggest that, similar to APOBEC3 branch expansion in jawed vertebrates, the AID/APOBEC family has undergone substantial diversification in lamprey, possibly indicative of multiple distinct biological roles.

Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5.

Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif.IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

Abstract PD8-11: APOBEC3 contributes to mutational load in breast cancer

Abstract Breast cancer results in large part from the accumulation of multiple mutations in premalignant cells, which provide a molecular basis for genetic diversity. This genetic diversity in premalignant cells allows selection for increased proliferation and survival and ultimately leads to invasion, metastasis, and therapeutic resistance. Recent genome-wide sequencing data showed that APOBEC3B (A3B) contributes to mutational load in breast cancer. A3B, a DNA cytosine deaminase, is overexpressed in more than 50% of breast tumors and more than 75% of breast cancer cell lines. Its overexpression and aberrant activation lead to unexpected clusters of mutations in the majority of breast cancers. This phenomenon of clustered mutations, termed kataegis (shower in Greek) forms a unique mutation signature in breast cancer. On the basis of the finding that A3B is a key molecular determinant of the mutator phenotype in breast cancer, the goal of our research is to utilize informatics tools to systematically characterize genetic alterations of APOBEC3 family proteins in breast cancer genomic data and define the molecular impact of altered APOBEC3 family proteins on mutability and anti-tumor immunity. Our data showed that the mutation rate and copy number amplification/deletion of APOBEC3 genes are low. The levels of APOBEC3A (A3A) and A3B are highly correlated and are highest in Basal subtype and lowest in Luminal A tumors, in concordance with the proliferation of subtypes. Additionally, A3A and A3B are significantly correlated with total mutational load as well as with TP53 mutation, and with somatic copy number alterations (SCNA), especially focal SCNA. Among APOBEC3 genes, A3B is significantly associated DNA replication, DNA damage repair, cell cycle and proteasome signatures, and shows predictive and prognostic capacity in ER-positive patients. Interestingly, A3G expression is strongly associated with immune response signature genes in all breast tumors. Consequently, A3G is highly associated with tumor-infiltrating lymphocytes in breast and several other disease types. In summary, our data demonstrate distinct expression pattern of APOBEC3 genes in different breast cancer subpopulations. Overexpression of different APOBEC3 family members leads to distinct molecular consequences. These data provide new molecular insights into pathophysiological functions of APOBEC3 genes in breast cancer and provide therapeutic opportunities for the breast cancer patients whose tumors have altered APOBEC3 expression levels and potentially are driven by APOBEC3 genes. Importantly, APOBEC3G is associated with evidence of immune activation that may signal responsiveness to immune checkpoint inhibitors. Citation Format: Zhao W, Peng Y, Mills GB, Peng G. APOBEC3 contributes to mutational load in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-11.

Molecular cloning and characterization of APOBEC3 family in tree shrew

The APOBEC3 family is a series antiviral factors that inhibit the replication of many viruses, such as HIV-1 and HBV. Tree shrews (Tupaia belangeri) possess great potential as an animal model for human diseases and therapeutic responses. However, the APOBEC3 family is unknown in tree shrews. Recent work has showed the presence of the APOBEC3 family in tree shrews. In this work, the cDNA sequences of five APOBEC3 members were identified in tree shrews, namely, tsAPOBEC3A, -3C, -3F, -3G and -3H. The results showed that their sequences encoded a zinc (Z)-coordinating-domain as a characteristic of APOBEC3 proteins. Phylogenetic analysis revealed that the tree shrew APOBEC3 (tsAPOBEC3) genes have occurred independently and that they are clustered with other mammalian APOBEC3 members. Transcript expression analysis indicated that tsAPOBEC3 genes are constitutively expressed, and high in immune-related tissues. tsAPOBEC3 gene expression was up-regulated in hepatocytes and PBMCs by IFN-α stimulation. Finally, tsAPOBEC3 proteins could edit both sides of DNA by inserting G→A and C→T hypermutations. Overall, the results suggest that the tsAPOBEC3 family could play a key role in defense immunity through distinct editing mechanisms. Our results provided insights into the genetic basis for the development of a tree shrew model for studying viral infection. Future studies will focus on deepening our understanding on the antiviral functions of these editing enzymes in tree shrew.

Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide-Like Gene Expression, RNA Editing, and MicroRNAs Regulation.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) protein family is encoded by eleven genes located in human genome. APOBECs are a family of evolutionarily conserved cytidine deaminases in vertebrates, and particularly in mammals. APOBECs play key roles in innate immunity against viral infection and retrotransposons. Subtypes of APOBEC3 can cause specific mutations in RNA and DNA at distinct preferred nucleotide contexts in human cancer. The pervasive APOBEC3s activation in the host genome converts cytosine to uracile on single-stranded DNA, which has been suggested to depend on ATR/chk1 pathways. In this chapter, we review the expression profiling of APOBEC expression in normal and disease states, discuss how microRNAs interact with APOBEC gene family, and post-transcriptionally regulate APOBEC gene expression in the APOBECA-B fusion allele and APOBEC-mediated RNA editing. It is reasonable to speculate targeting specific microRNAs may reduce host genome mutagenesis via inactivation of APOBEC deaminases.

IFN-\u03b1-mediated Base Excision Repair Pathway Correlates with Antiviral Response Against Hepatitis B Virus Infection

Previous studies identified APOBEC deaminases as enzymes targeting hepatitis B virus (HBV) DNA in the nucleus thus affecting its persistence. Interferon (IFN)-α treated chimpanzees and hepatitis C patients showed elevated APOBEC expression. We thus hypothesized that the responses to IFN-α treatment of chronic hepatitis B (CHB) patients is influenced by IFN-induced base excision repair (BER). CHB-treatment naïve patients, patients treated with PEGylated IFN-α, and patients with sequential treatment of Entecavior and PEGylated IFN-α were recruited. Blood and liver biopsy samples were collected before treatment and at treatment endpoint. BER genes were assessed by quantitative RT-PCR. BER gene expression levels and IFN treatment responses were correlated in patient liver biopsies. APOBEC3A, -B, -C, -D/E, and-G mRNA levels were up-regulated in IFN-treated patients. APOBEC3A expression was significantly higher in IFN-responders than in non-responders. BER genes NEIL3 was down-regulated in IFN-treated patients. APOBEC3 and BER gene expression at treatment endpoints partially correlated with the corresponding absolute DNA level or degree of HBsAg and HBV DNA decline. Our study suggests that the expression of APOBEC3A positively correlates with IFN-treatment responses in CHB patients, while NEIL3 shows negative correlation. These genes may involve to IFN mediated viral suppression and serve as biomarkers for CHB disease management.

APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism

Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B−/−:89A3B+/−:27A3B+/+), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.

Abstract 3700: Prognostic value of the expression of NKG2D and CD96 in early stage non-small cell lung cancer (NSCLC)

Abstract Background: Immune checkpoint inhibitors showed impressive activity in advanced NSCLC and are expected to be effective in postoperative treatment of early disease. New therapies aimed at T and NK cell activation are in development to expand treatments options. It is conceivable that markers of ‘inflamed’, ‘immune system ignorant’ and ‘immune system excluding’ tumor phenotypes may be predictive for specific immune-targeting therapies. Aim: We assessed prognostic value of expression of CD8(+) T and NK cell markers: CD96 and NKG2D in early stage NSCLC. In addition, we analyzed expression of 11 transcripts of APOBEC genes with hypothesis that APOBEC induced mutation burden may affect tumor immunogenicity or host adaptive immunity responsiveness. Methods: mRNA levels were measured by RT-PCR in frozen tumor tissue from 65 NSCLC stage I-IIIA patients who underwent pulmonary resection (75% lung adenocarcinoma, 33% never-smokers, 44% with subsequent dissemination), and in 11 NSCLC cell-lines. The relative gene expression (vs. 5 normalization genes) was compared between groups that did and did not disseminate, and in relation to clinico-pathological features. Results: After Bonferroni correction for multiple testing, NKG2D and CD96 mRNA expression was significantly lower in tumors with subsequent dissemination (p.adj.=0.045). Out of 11 APOBEC genes, expression of AICDA, APOBEC3A and APOBEC3G was lower in tumors with subsequent dissemination (p.unadj.=0.025). The NSCLC lines did not express NKG2D, CD96, AICDA or APOBEC3A. The Spearman corr. coeff. between the expression of NKG2D, CD96 and APOBEC3G were ≥0.8. AICDA expression was correlated with CD96, NKG2D and APOBEC3G with Spearman corr. coeff. in the range of 0.5-0.62. Expression of CD96 and NKG2D was significantly lower in tumors with vs. without subsequent brain metastasis (p.unadj.=0.048). The negative prognostic impact of low NKG2D and CD96 expression was independent from patient smoking status (FC &amp;lt;1, irrespective of smoking status). In the subset of never-smokers, the fold difference of NKG2D expression between tumors with vs. without subsequent dissemination was particularly pronounced (HR=0.37; p.unadj.=0.023). In multivariate Cox analysis including stage, CD96 and NKG2D expression was significantly correlated with distant metastasis free survival (p=0.028). The expression of CD96, NKG2D and APOBEC genes was not related to gender or NSCLC histological type. IHC co-localization and mRNA expression experiments at the background of other more established markers are planned. Conclusions: Low expression of CD96 and NKG2D in early stage NSCLC may be predictive for high propensity to dissemination, including metastasis to the brain. The expression of APOBEC transcripts does not seem to correlate with metastatic potential. Citation Format: Marcin Skrzypski, Michał Marczyk, Amelia Szymanowska, Anna Kowalczyk, Agnieszka Maciejewska, Ryszard Pawlowski, Wojciech Biernat, Joanna Polańska, Jacek Jassem. Prognostic value of the expression of NKG2D and CD96 in early stage non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3700. doi:10.1158/1538-7445.AM2017-3700

Abstract 2590: Analysis of APOBEC3A and APOBEC3B mutational signatures using next-generation sequencing data from cancer cell lines

Abstract The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) gene family of cytidine deaminases includes evolutionarily conserved genes that play important roles in DNA repair and mRNA editing. Activity of at least two APOBEC family members, APOBEC3A and APOBEC3B, can lead to kataegis, a mutagenic process in cancer cells that generates clusters of closely spaced, single strand specific C-&amp;gt;T DNA substitutions. APOBEC mutagenesis has a characteristic signature, most commonly represented by the 5’-Tp(C-&amp;gt;T)pW-3’ sequence motif, with additional substitutions also reported. This hypermutation signature and high mRNA expression of APOBEC3A and APOBEC3B have been associated with several cancer types. Most previous studies of APOBEC signatures have examined tumor sequence data from clinical samples, for which limited or no information about drug response was available. We investigated the presence of the mutational signature and mRNA expression patterns of the APOBEC3A and APOBEC3B genes in extensively characterized cell lines, in order to identify those cell lines that carry mutations generated by kataegis, with the aim of establishing associations between the APOBEC mutational signature, individual cancer types, and the patterns of sensitivity to antitumor agents. For this purpose, we analyzed whole exome sequencing (WES) data and mRNA expression of the APOBEC3A and APOBEC3B genes in two resources with extensive drug response data: the NCI-60 cell line panel, which includes 59 human cancer cell lines representing 9 cancer types and drug response information for thousands of anticancer agents, and the Cancer Cell Line Encyclopedia (CCLE), which provides WES, whole genome, and RNA-seq sequence information on hundreds of cancer cell lines and drug response data to over 200 agents. We analyzed WES data of 325 CCLE cell lines and 59 NCI-60 cell lines, with variants identified using GATK pipeline and Varscan2 software. The variants in each cell line were filtered to remove common polymorphisms in dbSNP and 1000 Genome Project databases. We searched the discovered variants for the presence of APOBEC signatures, 5’-Tp(C-&amp;gt;AGT)pN-3’, 5’-Tp(C-&amp;gt;AGT)pD-3’, and 5’-Tp(C-&amp;gt;AGT)pW-3’ in closely spaced (1000 and 10,000 bp) windows that appeared on the same DNA strand. We will discuss the use of optimal filters for detecting APOBEC mutational signatures and will present the analyses of associations between APOBEC signatures, mutational load of the tumor cell lines, APOBEC gene expression, and chemosensitivity to treatment. These results contribute to additional characterization of available cell lines by providing information about specific mutational signatures in different categories of cancer. Our findings may assist with identifying antitumor agents that would be appropriate for treatment of cancer cells with specific signature patterns generated by APOBEC mutagenesis. Citation Format: Suleyman Vural, Julia Krushkal, Richard Simon. Analysis of APOBEC3A and APOBEC3B mutational signatures using next-generation sequencing data from cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2017-2590