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Background/aim Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme which has an essential function in promoting antibody diversification. Its overexpression is strongly associated with B-cell derived malignancies including Burkitt lymphoma, where AID is required for the characteristic c-MYC/IGH translocation. This study aimed at defining AID's oncopathogenic role which is still poorly understood. Materials and methods We created over-expressing and knock-down cell culture models of AID, and used cellular assays to provide insight into its contribution to lymphomagenesis. Results We showed that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers and that ectopic overexpression of AID leads to rapid cell death. Using a knock-down model, we revealed that AID expression significantly impacts genomic stability, proliferation, migration and drug resistance. Conclusion AID is an important driver of lymphoma, impacting multiple cellular events, and is potentially a strong candidate for targeted therapy in lymphoma.

Activation Induced cytidine Deaminase (AID) is an essential enzyme of the adaptive immune system. Its canonical activity is restricted to B lymphocytes, playing an essential role in the diversification of antibodies by enhancing specificity and changing affinity. This is possible through its DNA deaminase function, leading to mutations in DNA. In the last decade, AID has been assigned an additional function: that of a powerful DNA demethylator. Adverse cellular conditions such as chronic inflammation can lead to its deregulation and overexpression. It is an important driver of B-cell lymphoma due to its natural ability to modify DNA through deamination, leading to mutations and epigenetic changes. However, the deregulation of AID is not restricted to lymphoid cells. Recent findings have provided new insights into the role that this protein plays in the development of non-lymphoid cancers, with some research shedding light on novel AID-driven mechanisms of cellular transformation. In this review, we provide an updated narrative of the normal physiological functions of AID. Additionally, we review and discuss the recent research studies that have implicated AID in carcinogenesis in varying tissue types including lymphoid and non-lymphoid cancers. We review the mechanisms, whereby AID promotes carcinogenesis and highlight important areas of future research.

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244GC; p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus; however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

Viruses and viral components have been shown to manipulate the expression of host microRNAs (miRNAs) to their advantage, and in some cases to play essential roles in cancer pathogenesis. Burkitt lymphoma (BL), a highly aggressive B-cell derived cancer, is significantly over-represented among people infected with HIV. This study adds to accumulating evidence demonstrating that the virus plays a direct role in promoting oncogenesis. A custom miRNA PCR was used to identify 32 miRNAs that were differently expressed in Burkitt lymphoma cells exposed to HIV-1, with a majority of these being associated with oncogenic processes. Of those, hsa-miR-200c-3p, a miRNA that plays a crucial role in cancer cell migration, was found to be significantly downregulated in both the array and in single-tube validation assays. Using an in vitro transwell system we found that this downregulation correlated with significantly enhanced migration of BL cells exposed to HIV-1. Furthermore, the expression of the ZEB1 and ZEB2 transcription factors, which are promotors of tumour invasion and metastasis, and which are direct targets of hsa-miR-200c-3p, were found to be enhanced in these cells. This study therefore identifies novel miRNAs as role players in the development of HIV-associated BL, with one of these miRNAs, hsa-miR-200c-3p, being a candidate for further clinical studies as a potential biomarker for prognosis in patients with Burkitt lymphoma, who are HIV positive.

HIV-1 infection often leads to the development of co-morbidities including cancer. Burkitt lymphoma (BL) is one of the most over-represented non-Hodgkin lymphoma among HIV-infected individuals, and displays a highly aggressive phenotype in this population group, with comparatively poorer outcomes, despite these patients being on anti-retroviral therapy. Accumulating evidence indicates that the molecular pathogenesis of HIV-associated malignancies is unique, with components of the virus playing an active role in driving oncogenesis, and in order to improve patient prognosis and treatment, a better understanding of disease pathobiology and progression is needed. In this study, we found HIV-1 Tat to be localized within the tumor cells of BL patients, and enhanced expression of oncogenic c-MYC in these cells. Using luciferase reporter assays we show that HIV-1 Tat enhances thec-MYCgene promoter activity and that this is partially mediated via two AP-1 binding elements located at positions -1128 and -1375 bp, as revealed by mutagenesis experiments. We further demonstrate, using pull-down assays, that Tat can exist within a protein complex with the AP-1 factor JunB, and that this complex can bind these AP-1 sites within thec-MYCpromoter, as shown byin vivochromatin immunoprecipitation assays. Therefore, these findings show that in HIV-infected individuals, Tat infiltrates B-cells, where it can enhance the expression of oncogenic factors, which contributes toward the more aggressive disease phenotype observed in these patients.

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244G&gt, C, p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus, however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

To investigate the functional effect of implicated variants within BGN and COL5A1 on gene expression of components of the extracellular matrix (ECM) in a TGF-β-stimulated risk model for musculoskeletal soft tissue injuries.Experimental research, laboratory study.Skin biopsies were obtained from nine healthy participants with either a combined increased or reduced risk profile for COL5A1 rs12722 CT and BGN rs1126499 CT - rs1042103 GA, and primary fibroblast cell lines were established. Total RNA was extracted at baseline (10% FBS), after serum starvation (1% FBS) and TGF-β1 treatment (1% FBS, 10ng/mL TGF-1β). Relative mRNA levels of BGN, COL5A1, DCN and VEGFA was quantified using Taqman® array pre-spotted plate assays (Applied Biosystems, Foster city, CA, USA).At baseline, the reduced risk group had 2.5, 1.9 and 2 fold increases (p0.001) in relative BGN, COL5A1 and VEGFA mRNA levels respectively. In the serum starved experiments, except for a significant 1.5 fold (p=0.017) increase in relative DCN mRNA expression in the reduced risk group, similar observations were noted for the other three genes. After TGF-1β treatment, the reduced risk group had 1.3 (p=0.011) and 1.4 fold (p=0.001) increases in the relative COL5A1 and VEGFA mRNA levels, respectively.Altered mRNA levels associated with genetic risk profiles for musculoskeletal soft injury risk at baseline (BGN, COL5A1 and VEGFA), with serum starvation (DCN) and after TGF-β1 treatment (COL5A1 and VEGFA) provide additional functional evidence to support the association of implicated genetic loci with several musculoskeletal soft tissue injuries. Implication of altered gene expression profiles underpinning these genetic risk associated loci potentially highlight key therapeutic targets for management of these injuries.

Burkitt lymphoma (BL) is one of the most common HIV-associated lymphomas. This cancer represents one of the most frequent causes of mortality among HIV+ people in Southern Africa, which has the highest incidence of HIV/AIDS worldwide. As is the case for Kaposi sarcoma, recent reports associate an oncogenic function with HIV in lymphoma development. This study explores the oncogenic potential of HIV-1 protein Transactivator of transcription (Tat) in BL via its ability to manipulate the expression of c-MYC and AID, two key drivers of disease in BL. The ability of HIV-1 Tat to influence the activity of the full-length (FL) (2861 bp) c-MYC promoter was assessed using Dual Luciferase Reporter (DLR) assays. Using sequential deletions, the minimal promoter region mediating the effect was identified. Site-directed mutagenesis (SDM) was used to identify promoter elements mediating the response. Co-immunoprecipitation assays (Co-IP) were used to assess interactions between Tat and the AP-1 factor JunB. Chromatin Immunoprecipitation (ChIP) assays were done to confirm that JunB bound the promoter in vivo. To assess the ability of Tat to mediate the expression of miRNA-181b-5p, a known repressor of AICDA, Ramos cells were transfected with Tat followed by qRT-PCR. Corresponding changes in AID protein levels were determined using Western blot. To assess whether miRNA-181b-5p targets AICDA in B cells, the FL AICDA 3'UTR was cloned into the pGL3-Promoter vector and DLR assays were performed in the presence of miRNA-181b-5p mimic. Our data reveal that Tat enhances c-MYC promoter activity in a dose-dependent manner, with a maximum activity of 2.6-fold recorded when 500ng of pcDNA-Tat was used on 400ng of pGL3-c-MYC plasmid harboring the FL promoter. A loss of 35% activity was observed [1.7 (±0.05) fold] upon promoter deletion and two AP-1 sites (positions -1128 bp and -1376 bp) within the deleted region were found to mediate the Tat-dependent increase. A loss in activity of 20.6% was recorded when AP-1 site 1 was mutated, and 25% when AP-1 site 2 was mutated. A double mutant decreased the activity by 29%. Co-IP revealed a strong interaction between Tat and AP-1 factor JunB, and ChIP data showed that JunB was strongly recruited to those two AP-1 sites in the presence of Tat. Our result also reveals that the expression of the AICDA specific miRNA181b-5p is significantly reduced in the presence of Tat, which correlates with an increase in AID protein levels. We also confirm that the AICDA FL 3'UTR, which contains putative miRNA-181b-5p binding sites, is partially repressed by a miRNA-181b-5p mimic. Our study reveals that Tat potentiates oncogenesis by enhancing the expression of c-MYC and AID, two key lymphoma drivers. We show that Tat can enhance c-MYC expression by enhancing the recruitment of cellular TFs to the promoter. We also show that the same HIV protein can enhance the expression of AID by modulating the expression of miRNA-181b-5p. Citation Format: Leonardo Alves de Souza Rios, Nontlantla Mdletshe, Shaheen Mowla. HIV-1 transactivator of transcription deregulates key Burkitt lymphoma associated oncogenes at both the transcriptional and post-transcriptional level [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-31.