An investigation of the impact of the B cell lymphoma factor Activation Induced Cytidine Deaminase (AICDA/AID) on epigenetic plasticity

Makofane, Lincon Kgasha

An investigation of the impact of the B cell lymphoma factor Activation Induced Cytidine Deaminase (AICDA/AID) on epigenetic plasticity

Thesis / Dissertation

2025

Publisher

University of Cape Town

Department

Department of Pathology

Faculty

Faculty of Health Sciences

Abstract

Cancer is a major cause of both mortality and morbidity globally, with 10 million deaths reported in 2020 by the Global Cancer Observatory (GLOBOCAN). To address this burden, it is crucial to deepen our understanding of the biology of the disease to create opportunities for addressing therapeutic challenges. Epigenetic regulation has recently emerged as a prominent factor in cancer progression, and aberrant epigenetic alterations have been reported in many key cancer-driving genes. The overexpression of the DNA modifying enzyme Activation-induced cytidine deaminase (AICDA) is a factor linked to cancer progression, particularly B-cell derived non-Hodgkin lymphomas, but has also been associated with other inflammation-associated cancers, including colorectal cancer (CRC). The widely recognised normal role of AICDA is in antibody-producing B cells, where its cytosine deaminating ability within DNA is essential for the production of a diverse array of antibodies. In high grade B cell lymphomas, such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL), overexpression of AICDA is particularly associated with oncogenic genomic translocations, thought to be driven by aberrant DNA repair, following the deamination events. More recently, the oncogenic function of AICDA has been expanded to include modulation of the epigenetic landscape, through the enzyme's ability to alter CpG sites/islands, because of its deamination function. Additionally, a few studies have shown that AICDA can partner with other regulatory proteins to modulate gene methylation. However, such a direct involvement of AICDA in the alteration of gene expression by disrupting DNA methylation remains uncertain due to a lack of scientific evidence. The current study therefore sought to investigate the impact of AICDA on the transcription of key oncogenes, with the view to focus on the role of AICDA in their methylation status in future studies. The human AICDA open reading frame (ORF) was cloned into a lentiviral backbone vector, and AICDA overexpression was verified in HEK293 cells using western blotting. A third-generation Lentiviral transduction system was used to produce lentiviral particles (AICDA expressing and empty-vector control) to transduce a non-cancerous lymphoblastoid cell line (PB-B95-8H) and a DLBCL cell line (HBL 1). Flow cytometry was used to sort successfully transduced cells. However, the AICDA-overexpressing cells did not survive in culture for longer than 72 hours post-sorting. This is likely due to oncogene induced death, as these cells already express AICDA at relatively high levels. An alternate CRC model was chosen and was justified by the published literature as well as in silico analyses using the Cancer Genome Atlas (TCGA) CRC dataset which indicated that AICDA expression in CRC correlated with poor survival. Two CRC cell lines stably expressing AICDA were developed and were found to tolerate high levels of AICDA. Thereafter, proliferation was assessed using growth curve analysis, and WST-1 assay. Growth curves showed that expressing AICDA significantly enhanced cell proliferation. In addition, the WST-1 cell proliferation assay also showed similar results, indicating that AICDA expression promotes cell proliferation. A wound healing assay was performed to investigate the impact of AICDA expression on the migration, and the results indicated that AICDA expression in CRC cells enhances the migratory ability if the cells, relative to the control. CRC cells expressing AICDA had higher IC50 values compared to control cells, when treated with increasing doses of the chemotherapeutic drug 5-Fluorouracil (5-FU), indicating a resistance to cell death. However, this was not reflected in western blot analyses where the expressions of apoptotic markers PARP-1 and Caspase-3 were assessed. The cell cycle profiling showed that AICDA provides mild protection from cell death induced by 5-FU, and delays S-phase, in CRC cells. Quantitative PCR (qPCR) was performed to investigate the impact of AICDA expression on the transcriptional regulation of seven key oncogenes, namely PIM1, FANCA, DNMT1, DNMT3A, ZEB1, CD274, and MYC. The results showed that AICDA expression in CRC significantly impacted the expression level of the selected oncogenes. Variations could be observed between the two CRC cell models, which imply that the cellular context is important in mediating the impact of AICDA on changes in gene transcription. Additionally, both upregulation and downregulation of genes were observed, indicating diversity in the mechanism via which AICDA modulates gene expression. The findings of this study therefore demonstrate that AICDA functions as an oncogene in CRC and provides the cells with a significant proliferative advantage. Crucially, it shows that AICDA expression impacts the transcription of key oncogenes. Future work will focus on investigating the role of methylation in the transcriptional changes observed, and how AICDA is involved in this process.