Browsing by Author "Mowla, Shaheen"

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    Aqueous extracts of Dodonaea viscosa induce potent and selective cytotoxicity in DLBCL cells
    (2024) Yekelo, Babalwa; Mowla, Shaheen
    Cancer is a major cause of death globally, with approximately 10 million deaths in 2020. In South Africa, the number of new cancer cases is expected to double by 2030. Non-Hodgkin lymphoma (NHL), which represents a group of cancers originating from lymphoid tissues, was ranked the 11th most common cancer globally, accounting for 544,000 new cases and 260,000 deaths in 2020. In South Africa, NHL ranks among the top five invasive cancers among both males and females. Diffuse large B cell lymphoma (DLBCL), an aggressive B-cell derived cancer is the most prevalent cancer within the NHL group of cancers. DLBCL can be grouped into two main subtypes, namely the germinal-centre B cell (GCB) and the activated-B cell (ABC) subtypes, with the ABC subtype reported to have a more aggressive clinical course than the GCB subtype. Additionally, DLBCL is an HIV-associated cancer and is, thus, highly overrepresented among HIV-infected individuals. Currently, 30-40 % of DLBCL patients relapse or develop refractory disease following treatment with the standard DLBCL therapy. This figure is worse among HIV-infected DLBCL patients. There is therefore a need to develop more effective therapeutic regimens to treat this cancer. In recent years there has been increasing focus, by cancer sufferers, on the use of alternative therapies for treating their disease. An estimated 80% of the South African population seek health care from traditional healers. Medicinal plants form a major part of the repertoire of tools that these traditional healers use to treat their patients. Many plant species have already been the source of bioactive compounds used to develop currently approved cancer drugs. In the current research, the anti-cancer potential of aqueous extracts of Dodonaea viscosa (DVE), a plant commonly used by traditional healers in the Western Cape region of South Africa, against DLBCL cells, is being investigated. Previously published reports showed that extracts of Dodonaea viscosa can inhibit the growth of several types of cancer cells, including breast, prostate, and colon cancer. There are currently no published reports on the effects of DVE on DLBCL cells. Xv The IC50 of DVE against two DLBCL cell lines (HBL-1 and SU-DHL-4) was determined, relative to a non-cancerous lymphoblastoid cell line (LCL) (PB-LCL-B95-8H) using viability assays. Thereafter, the effect of DVE on proliferation was investigated using proliferation-tracking and colony formation in a semi-solid medium. The effect of DVE on the cell cycle was also investigated. Lastly, induction of apoptosis was determined using microscopy, Annexin V incorporation assay, caspase activity assay, and western blotting to assess the expression of apoptotic markers. Viability assays showed that DVE could potently and selectively inhibit the proliferation of two DLBCL cell lines, namely the GCB cell line SU-DHL-4, and the ABC cell line HBL-1, relative to the non-cancerous lymphoblastoid cell line PB-B95-8H. This converted into a favorable selectivity index of 2.25 for SU-DHL-4 and 3 for HBL-1, demonstrating that DVE preferentially triggers cell death in the cancer cells. The cell-Trace proliferation assay, which tracks live cell proliferation over time, showed a 2.3-fold and 1.3-fold reduction of daughter cells (P2 generation) in the DVE- treated SU-DHL-4 and HBL-1 cells relative to untreated SU-DHL-4 and HBL-1 cells respectively, with the non-cancerous cells being much less affected. The effect of proliferation was further confirmed through a colony-forming assay, which showed potent inhibition of colony formation over 7 days, by DVE, for both cancer cell lines. No notable changes in the phases of the cell cycle (G1, S, G2/M) were observed in all cell lines when exposed to DVE. However, an increase in the sub-G1 population, which is indicative of cell death, was evident in the DVE-treated DLBCL cells. The induction of apoptosis was investigated firstly through microscopy, to assess for the presence of cellular morphological features typical of this mode of cell death. Membrane blebbing, nuclear fragmentation, the presence of apoptotic bodies, and cell shrinkage were observed for both DLBCL cell lines while slight cell swelling was observed for the PB-B95-8H cells. Using the Annexin V incorporation assay, a majority of late apoptotic (64%) and non-viable/necrotic (15%) cells were detected in DVE-treated SU-DHL-4 cells, while mostly early apoptotic cells (49%) were observed for HBL-1. The non-cancerous cell lines were left mostly unaffected. These findings were further supported by the caspase-3/7 activity assay and western blot analysis, which demonstrated that DVE treatment induces the expression of the apoptotic markers PARP-1 and caspase-3 in DLBCL cells, with the SU-DHL-4 cells displaying traces of necrosis, as evidenced by smaller cleaved PARP-1 fragments (74 kDa and below). Xvi Overall, the study shows that aqueous extract of D. viscosa is selectively cytotoxicity towards DLBCL cells, and significantly less toxic towards a corresponding non-cancerous B cell line. Additionally, at the same concentration as DVE, and under the same treatment conditions, the GCB DLBCL cell line was more sensitive to DVE than the ABC DLBCL cell line. This is in line with reports on the more aggressive and resistant nature of the ABC subtype. While this research is the first to demonstrate that extracts of the medicinal plant D. viscosa are cytotoxic toward DLBCL cells, more research is needed to further understand the mechanisms of cell death, as well as to demonstrate these findings in an in vivo model. Additionally, biochemical studies should be done to identify the bioactive compounds responsible for the cytotoxic effects observed.
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    Bi-Allelic Novel Variants in CLIC5 Identified in a Cameroonian Multiplex Family with Non-Syndromic Hearing Impairment
    (2020-10-23) Wonkam-Tingang, Edmond; Schrauwen, Isabelle; Esoh, Kevin K; Bharadwaj, Thashi; Nouel-Saied, Liz M; Acharya, Anushree; Nasir, Abdul; Adadey, Samuel M; Mowla, Shaheen; Leal, Suzanne M; Wonkam, Ambroise
    DNA samples from five members of a multiplex non-consanguineous Cameroonian family, segregating prelingual and progressive autosomal recessive non-syndromic sensorineural hearing impairment, underwent whole exome sequencing. We identified novel bi-allelic compound heterozygous pathogenic variants in CLIC5. The variants identified, i.e., the missense [NM_016929.5:c.224T>C; p.(L75P)] and the splicing (NM_016929.5:c.63+1G>A), were validated using Sanger sequencing in all seven available family members and co-segregated with hearing impairment (HI) in the three hearing impaired family members. The three affected individuals were compound heterozygous for both variants, and all unaffected individuals were heterozygous for one of the two variants. Both variants were absent from the genome aggregation database (gnomAD), the Single Nucleotide Polymorphism Database (dbSNP), and the UK10K and Greater Middle East (GME) databases, as well as from 122 apparently healthy controls from Cameroon. We also did not identify these pathogenic variants in 118 unrelated sporadic cases of non-syndromic hearing impairment (NSHI) from Cameroon. In silico analysis showed that the missense variant CLIC5-p.(L75P) substitutes a highly conserved amino acid residue (leucine), and is expected to alter the stability, the structure, and the function of the CLIC5 protein, while the splicing variant CLIC5-(c.63+1G>A) is predicted to disrupt a consensus donor splice site and alter the splicing of the pre-mRNA. This study is the second report, worldwide, to describe CLIC5 involvement in human hearing impairment, and thus confirms CLIC5 as a novel non-syndromic hearing impairment gene that should be included in targeted diagnostic gene panels.
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    Chemotherapeutic drugs, 5-fluorouracil and cisplatin, differentially affect exprssion of drug metabolising enzyme genes in an oesophageal cancer cell line
    (2014) Hassen, Naseeha; Dandara, Collet; Mowla, Shaheen; Parker, M I
    Cancer is a leading cause of death worldwide. Oesophageal cancer in particular is the sixth most common cause of cancer deaths globally and its incidence and mortality rates in Southern Africa are among the highest in the world. One of the major challenges with cancer treatment is the vast variability in patient response to chemotherapy, which is predominantly due to genetic variability. The most relevant genes in this context encode the CYP and GST drug metabolising enzymes (DMEs) as these enzymes metabolise up to 90% of clinically-prescribed medication. Patients are also exposed to a variety of other compounds that along with chemotherapeutic drugs may alter DME gene expression. Changes in DME gene expression influence the therapeutic outcomes for patients; thus, understanding the effects of drugs and compounds on the expression of DMEs is crucial for the advancement of personalised medicine. The aim of this study was to determine the effects of two commonly-used chemotherapeutic drugs, as well as a CYP-inducing compound, on the differential expression of four pharmacogenetically relevant DME-encoding genes, CYP1A1, 1A2, 1B1 and GSTP1, in a human oesophageal cancer cell line.
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    Development of a rapid diagnostic screen for telomerase mutations associated with immunosuppressive therapy failure in patients with aplastic anaemia
    (2013) Xulu, Khethelo Richman; Shires, Karen; Mowla, Shaheen
    Includes abstract. Includes bibliographical references.
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    Genetic aetiology of autosomal recessive non-syndromic hearing loss in sub-Saharan African patients: evaluation using targeted and whole exome sequencing
    (2019) Lebeko, Kamogelo; Wonkam, Ambroise; Dandara, Collet; Mowla, Shaheen
    Hearing Loss (HL) is one of the highest contributors to disability worldwide. The highest incidence of the disease is seen in developing countries, such as those in subSaharan Africa (SSA). Patients affected with disabling HL are reported to be more than 466 million worldwide. The causes of HL can either be environmental or genetic with each contributing about 50% towards all cases, in many settings. In developing countries, the environment might contribute more due to poor health services and infrastructure available to the population. In the absence of environmental causes, there is a genetic component at play, that is largely unknown in African populations. Up to 70% of HL of genetic origin are non-syndromic (NS). The mode of inheritance is recessive in nearly 77% of non-syndromic HL. Up to date, more than 100 genes have been associated with HL harbouring more than 1000 causative variants. In many populations of European and Asian descent, pathogenic variants in GJB2 (connexin gene 26) and GJB6 (connexin gene 30) are a major contributor to autosomal recessive non-syndromic hearing loss (ARNSHL). Comprehensive hearing health care programs should cover genetic causes by providing molecular testing, and genetic counselling, specifically SSA where genes and mutations causing HL remain largely unknown. The aim of this project was thus to uncover the genetic causes of HL among patients’ cohorts from Cameroon and South Africa. This was addressed by 1) sequencing common variants in the most relevant genes in other populations (GJB2 and GJB6), 2) using a targeted gene panel to resolve HL in 10 multiplex families from Cameroon presenting with ARNSHL and negative for GJB2 and GJB6 mutations screening, 3) screening novel variants found in known genes in a cohort of 82 singleplex HL cases from Cameroon and South Africa, and lastly, 4) using Whole Exome sequencing to explore the two unresolved multiplex cases with and subsequent findings confirmed by functional studies, and also screened in 80 singleplex HL cases. The following findings are reported: GJB6, GJA1 mutations screening and literature review No GJA1 or GJB6 mutation was not found in multiplex and simplex cases of HL in both Cameroonians and South Africans. The review of the literature confirms that the prevalence of GJB2- or GJB6-related NSHL is approximating to zero in most subSaharan African populations. Targeted Exome Sequencing (OtoSCOPE) The targeted genes, panel that included 116 genes, was able to resolve 7 of 9 families (77.8%) which were successfully sequenced, with one family failing to be sequenced. The causative variants identified in the 7 resolved families were : 1) compound heterozygous c.5806_5808delCTC and c.5880_5882delCTT in MYO7A; 2) compound heterozygous c.646T>A (p.Phe216Ile) and c.38G>A (p.Arg13His) in LOXHD1; 3) homozygous c.766-2A>G in OTOF; 4) a deletion and a complex copy number variation in STRC; 5) compound heterozygous c.1678G>A (p.Asp560Asn) and c.2007C>A(p.Asp669Glu) in SLC26A4; 6) Homozygous c.1996C>T(p.Arg666Stop) in MYO7A; 7) compound heterozygous c.6399C>A(p.Asp2133Glu) and c.2000T>C (p.Met667Thr) in CDH23. Five out of 12 variants were novel. Screening of these causative variants in known genes, in 82 singleplex HL cases from Cameroon and South Africa was unable to resolve any of the cases: the variants were in either heterozygous in low frequency or absent. Bioinformatic pathways exploration of SNP data of known HL genes revealed an extensive network within the HL genes, with 10 identified as important nodes, including MYO7A. Most HL genes were found to be involved in two biological processes which were sensory perception of mechanical stimulus (GO: 0050954, p= 1.430e-8) and sound (GO: 0007605, p = 1.246e-8). The molecular functions of variants found within these genes were found to mostly fall within the binding (GO: 0005488) and/or structural molecule activity (GO: 0005198). Whole Exome sequencing Whole exome sequencing was performed on four of the nine multiplex families: the two families that were unresolved by targeted panel sequencing, and two previously resolved families that were used as positive controls for the variant annotation and filtering pipeline. The results were the resolution of 3/4 families, including the two- positive control. The previously unresolved “family 8” was found to harbour a novel variant within the GRXCR2 gene, a gene only associated with HL once before. The c.251delC variant was revealed through in silico studies to cause a premature stop codon at position 116 due to its frameshift effect. The screening of this variant in our cohort of 80 singleplex cases revealed one other unrelated HL patient harbouring this causative variant. Due to the limited literature on the gene and its protein, in silico studies were used to show the predicted secondary structure folding of the protein as well as potential protein binding regions. Analysis showed that the predicted loss of a stable region of the protein as well as that of a putative binding domain could explain the pathogenic nature of the variant. In vitro studies showed that the variant hindered the detection of the protein by way of a DDK tag downstream in the plasmid. Additionally, GFP-Tagged GRXCR2 showed altered expression pattern in the variant when compared to the wildtype. In summary, our data has revealed the efficacy of using next generation sequencing tools in resolving HL among sub-Saharan African patients as opposed to the single candidate gene approach. In our quest, we have employed two widely used strategies, targeted panel and whole exome sequencing (WES), both of which have had great successes in various populations. The targeted approach was able to resolve 77.8% of our families but did not detect variants for two of the families revealing the presence of other variants harboured in rarely associated gene not captured or included on the panel. This prompted for the use of a more comprehensive approach such as WES. These results corroborated with those of two families previously resolved by targeted exome sequencing. Additionally, one of the previously unresolved family was now resolved. This showed that WES was sensitive enough to detect variants in known HL genes but comprehensive enough to detect variants in other regions of the exome which have not been associated with HL or rarely associated with HL. The benefit of WES also extends to the contribution of exomic data from patients of African descent as there is an underrepresentation of this group in exome repositories as well as genomic or SNP databases. To the best of our knowledge, this is the first study to use WES to resolve HL in patients of African descent. The other benefit of such a venture is the use of this data not only for patients in SSA but also those in the diaspora. In conclusion, we have successfully demonstrated the feasibility of using NGS tools in identifying causative variants in HL patients in SSA. Additionally, we have shown that WES is a more suitable approach to trying to resolve HL in Africa. Therefore, the data strongly support that genetic studies on families segregating HL in SSA could be the next frontier of HL genetic research, of global importance through discovering novel variants in known genes, and potentially novel genes. These studies will improve HL genetic diagnosis, retrospective counselling and testing, prevention and care including future prediction of treatment outcomes in sub-Saharan Africans, and in people of African descent.
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    HIV alters the expression of miRNA hsa-miR-200c-3p in B-cells, leading to enhanced migration of lymphoma cells
    (2018) Ramorola, Beatrice Relebogile; Mowla, Shaheen; Shires, Karen
    Background: The sub-Saharan African region is one that is affected most by the HIV/AIDS pandemic, with South Africa being the country with the highest number of infected individuals at 7.06 million. Infection with HIV is often associated with co-morbidities, including HIV-associated Non-Hodgkin’s Lymphomas (HIV-NHLs). Burkitt’s lymphoma (BL), a highly aggressive cancer, is one of the most common NHLs associated with HIV infection. Despite receiving highly active anti-retroviral therapy, the prognosis for this HIV-associated lymphoma remains poor and the incidence keeps on increasing in this group of patients. Recent studies have shown that microRNA (miRNA) dysregulation play essential roles in the pathogenesis of many cancers, including NHLs. While several human pathogenic viruses have been shown to deregulate cellular miRNAs, to date, no comprehensive studies have been carried out to determine whether HIV infection can lead to miRNA dysregulation in B-cells, which may contribute to the development of HIV-associated lymphomas. Objective: This research project aimed to validate the differential expression of selected miRNAs which were identified as potentially important in a PCR array, and characterise their roles in Burkitt’s lymphoma cells exposed to an attenuated strain of HIV-1, compared to control cells. Methods: Single-tube TaqMan miRNA assays were used to validate the previously observed differential expression of four selected miRNAs in Burkitt’s lymphoma cell lines (Ramos and BL41) exposed to HIV-1 compared to matched-microvesicle treated (control) cells. Following validation, the role of miRNA hsa-miR-200c-3p in the development of HIV-associated BL was investigated. This was done by using online bioinformatic prediction tools, as well as literature searches, to identify gene targets. Thereafter, the differential expression of a selected gene target was investigated by qPCR and western blotting. The functional significance of the observed changes in miRNA and gene expression was investigated by performing cell viability and migration assays. Results: Three upregulated (hsa-miR-575, hsa-miR-363-3p and hsa-miR-222-3p) and one downregulated (hsa-miR-200c-3p) miRNAs that were significantly deregulated by 2-fold or more (p< 0.05) in the PCR array were selected for validation. Thereafter, the miRNA hsa-miR200c-3p was selected for further analysis. Upon exposure to attenuated HIV-1, hsa-miR-200c3p was downregulated in the BL cell line Ramos, and this was reproducible in a second BL cell line BL41. The transcription factors ZEB1 and ZEB2, which are involved in cancer cell migration, were identified as targets of hsa-miR-200c-3p. Contrary to what is expected, the mRNA expression of both genes was found to be significantly downregulated in Ramos and BL41 exposed to attenuated HIV-1. At the protein level, in the Ramos cells, ZEB1 and ZEB2 matched what was observed for the mRNA. In contrast, both ZEB1 and ZEB2 protein were upregulated in BL41 cells under the same treatment conditions. At the functional level, the migration of both cell lines was enhanced when exposed to attenuated HIV-1, compared to control cells. Conclusions: The present study has demonstrated that HIV-1 has the ability to modulate cellular miRNA expression in Burkitt’s lymphoma cells. Of these miRNAs, hsa-miR-200c-3p is consistently downregulated when two BL cell lines were exposed to HIV. The ZEB transcription factors ZEB1 and ZEB2, which promote Epithelial-to-Mesenchymal Transition (EMT) through enhancing cellular migration, were investigated as hsa-miR-200c-3p targets. The mRNA levels of ZEB1 and ZEB2 were downregulated in both cell lines under the same experimental conditions. This is contrary to what is expected, since miRNAs lead to the attenuation of transcription or translation of their target genes and a downregulation of a miRNA should lead to an upregulation of its target. However, protein expression rather than mRNA expression has been described as a more accurate indication of target validation for miRNAs. The protein expression levels for ZEB1 and ZEB2 correlated with the mRNA expression results observed in the Ramos cells. In the BL41 cells, ZEB1 and ZEB2 protein levels were upregulated. Furthermore, in both cell lines, an increase in migratory ability was observed when cells were exposed to attenuated HIV-1. These results demonstrate that exposure to HIV enhances the cancer phenotype and that this is potentially due to changes in cellular miRNA expression brought about by the virus or viral components. Future studies should focus on gain-offunction and loss-of-function studies to determine whether the increase in cell migration is specifically due to a decrease in hsa-miR-200c-3p.
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    Investigation of the role of the DNA-modifying enzyme, activation-induced cytidine deaminase (AID), in cancer
    (2016) Godsmark, Grant Kenneth; Mowla, Shaheen
    The DNA-editing enzyme, Activation-Induced cytidine Deaminase (AID) is essential for antibody diversification and plays an important role in immunity. AID is specifically expressed in activated B-cells and mutates targeted DNA sites, diversifying the antibody repertoire. Due to its mutagenic nature, AID expression is tightly regulated, however, its overexpression has been associated with translocation of the c-MYC oncogene, a characteristic of the B-cell derived cancer, Burkitt's lymphoma (BL). Although currently uncharacterised, AID overexpression has also been implicated in non-lymphoid cancers including prostate, liver and colon. The function of AID in the oncogenic process is not well defined and therefore this project is aimed at using cell culture models to study the function of AID in cancer. AID mRNA and protein expression levels were determined in five cell lines of B-cell origin, and 16 epithelial cell lines (colon, prostate, head and neck and oesophageal). While AID expression was easily detected in the B-cell derived cell lines, no significant expression of both AID mRNA and protein expression was found in all the epithelial derived cell lines. The B lymphoblastoid cell line, L1439A, which is derived from a healthy donor, and harboured relatively low AID expression, was originally selected for ectopic expression of AID. Transfection of this cell line using conventional methods and lipid and polymer based transfection reagents was not successful, and therefore, nucleofection was used, which caused successful uptake of the AID-expressing plasmids as well as corresponding controls. However, this method was too harsh for the L1439A cell line as it did not survive post-nucleofection. Based on this result, the BL cell line Ramos, which expresses relatively low AID compared to the other BL cell lines used in the screen, was selected as an alternative. Plasmids constitutively expressing AID, as well as their corresponding empty vector, were successfully introduced into these cells using an established nucleofection protocol. While cells containing the empty vectors could be selected and expanded in culture, cells overexpressing AID underwent apoptosis approximately three days post-transfection. This is likely due to the highly mutagenic nature of the enzyme. As an alternative approach to studying the function of AID in cancer, a knockdown approach was taken, using siRNA.
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    Molecular Characterisation of Diffuse Large B-cell Lymphoma in South Africa
    (2022) Ramorola, Beatrice Relebogile; Mowla, Shaheen; Antel, Katherine; Chetty, Dharshnee
    Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous and aggressive disease and is the most common subtype of non-Hodgkin lymphoma (NHL) in adults. Additionally, this subtype of lymphoma is also the most common in people infected with the Human Immunodeficiency virus (HIV), and the incidence has remained high despite the advent of Antiretroviral therapy (ART). About 30-40% of DLBCL patients' relapse, or are refractory to standard first-line therapy, and this is attributed to the high genetic and clinicopathological heterogeneity of the disease. Reports indicate that, among HIV-positive DLBCL patients, the response rate is even poorer. In low resourced settings, this is further aggravated by multiple factors including access to health facilities and gaps in communication. Recent genetic studies of DLBCL tumours allowed for the further subclassification of the ABC- and GCB- subtypes into at least 5 new groups, each with distinct genetic, molecular and clinicopathologic features, revealing potentially novel drivers of the disease. There is therefore a need to further understand the molecular pathology of these distinct subtypes, including within the context of HIV. The latter formed the basis of this study and used multiple approaches and methodologies, including immunophenotyping and mutational analysis of samples from local patient cohorts, as well as gene set enrichment analyses of publicly available DLBCL datasets. An analysis and comparison of immune cell populations in peripheral blood mononuclear cells, of HIV negative and HIV positive DLBCL patients was performed using flow cytometry (Chapter 3). The participants were newly diagnosed, chemotherapy naïve DLBCL patients. Some of the key observations were as follows: HIV-positive patients were diagnosed with DLBCL at significantly younger ages (75% under the age of 50 years), compared to the HIV negative DLBCL group. Furthermore, more extranodal disease and EBV infection were observed in the HIV-infected group, and both these factors are known to be indicative of more aggressive, advanced-stage disease. In general, cytopenias were observed in the DLBCL patient cohort, regardless of HIV status. Since most of the HIV infected patients were not adequately receiving antiretroviral therapy at the time of DLBCL diagnosis, the CD4+ helper T-cell population within this group was significantly reduced, in comparison to the HIVuninfected group. Interestingly, there was a significant difference in monocyte count between the two groups, with lower counts observed among the HIV-infected DLBCL patients. Additionally, increased activation of cytotoxic T-cells (CD8+CD38+), as well as lower mature cytolytic CD56dimCD16+ Natural Killer (NK) cells were observed in the HIV-positive DLBCL group. The prevalence of Myeloid differentiation primary response factor 88 (MYD88) L265P and Cluster of Differentiation 79B (CD79B) Y196 activating mutations were analysed in a cohort of archived ABC-DLBCL tumours (consisting of both HIV positives and negatives) (Chapter 4). Genomic DNA was isolated, the relevant genomic regions amplified by PCR, subjected to Sanger sequencing and then analysed and confirmed. The co-occurrence of both these mutations is characteristic of a newly described subset of DLBCL shown to have an inferior outcome. The prevalence of these mutations within an African population has as yet not been reported. The MYD88L265P mutation was detected in 26% of the amplified ABC-DLBCL tumours, while mutations at CD79BY196 were observed in 12% of the tumours. Co-occurrence of both these MYD88 and CD79B mutations were present in only 3 tumours, all of which were HIVnegative. Analysis of patient survival in relation to the mutations highlighted a trend showing that patients harbouring both mutations had worse overall survival. Interestingly, this pathogenic effect was more prominent for CD79B mutations, and this was comparable to the survival probability observed for HIV-positive patients. For the MYD88L265P mutation, an HIV positive status further decreased the survival probability. The final study presented in this thesis focused on investigating the expression and regulation of the Suppressor of cytokine signalling 1 (SOCS1) gene. SOCS1 has been recently reported to be a frequently altered gene in DLBCL, but molecular pathological studies on the role of SOCS1 in DLBCL are scarce. Using cell line models, basal SOCS1 gene and protein expression levels were assessed. In two DLBCL cell lines (SUDHL-4/GCB-DLBCL subtype and HBL-1/ABC-DLBCL subtype), SOCS1 expression was reduced at both the mRNA and protein levels when compared to control lymphoblastoid cell lines (LCLs), while in a third ABC-DLBCL cell line (U2932), results were varied. In silico analysis using the Cancer Genome Atlas (TCGA) database confirmed that SOCS1 is in the top 20 frequently mutated genes in DLBCL. Furthermore, these frequent mutations, which lead to reduced or low SOCS1 expression, are associated with DLBCL disease in its early stages (stages I and II) and with better survival estimates. In contrast, high expression of SOCS1 was associated with poor overall survival. This was further corroborated by gene set and pathway enrichment analyses, which highlighted factors involved in the enhancement of cancer-promoting processes such as proliferation, migration, invasion, and metastasis. Additionally, a previously reported association between the expression of methylation-related genes and SOCS1 expression was confirmed. Overall, these studies have uncovered novel insights into the pathology of DLBCL, including features unique to HIV-associated DLBCL. The implementation of a differential approach to the management of the disease, based on specific genetic and molecular features, should be explored. These findings support the importance of more studies, incorporating comprehensive genomic and molecular technologies, to continue to unravel the complex disease that is DLBCL.
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    A Monoallelic Variant in REST Is Associated with Non-Syndromic Autosomal Dominant Hearing Impairment in a South African Family
    (2021-11-06) Manyisa, Noluthando; Schrauwen, Isabelle; de Souza Rios, Leonardo Alves; Mowla, Shaheen; Tekendo-Ngongang, Cedrik; Popel, Kalinka; Esoh, Kevin; Bharadwaj, Thashi; Nouel-Saied, Liz M.; Acharya, Anushree; Nasir, Abdul; Wonkam-Tingang, Edmond; Kock, Carmen de; Dandara, Collet; Leal, Suzanne M.; Wonkam, Ambroise
    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.
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    Pharmacogenomics of sickle cell disease therapeutics: pain and drug metabolism associated gene variants and hydroxyurea-induced post-transcriptional expression of miRNAs
    (2020) Mnika,Khuthala; Wonkam, Ambroise; Dandara, Collet; Mazandu, Gaston; Mowla, Shaheen; Chimusa, Emile
    Sickle cell disease (SCD) is a common blood disease caused by a single nucleotide substitution (c.20T>A, p.Glu6Val) in the beta globin gene on chromosome 11. The prevalence of the disease is high throughout large areas in sub-Saharan Africa, the Mediterranean basin, the Middle East, and India due to the level of protection that the sickle cell trait, provides against severe malaria. Approximately 300,000 infants are born per year with sickle cell anemia, which is defined as homozygosity for the sickle hemoglobin (HbS). The majority (nearly 75%) of these births occur in sub-Saharan Africa, particularly in two countries: Nigeria, and the Democratic Republic of the Congo where there are poorly resourced healthcare systems. Early diagnosis, penicillin prophylaxis, blood transfusions, hydroxyurea, and hematopoietic stem-cell transplantation can dramatically improve survival and quality of life for patients with SCD. However, our understanding of the role of genetic and clinical factors in explaining the complex phenotypic diversity of this disease is still limited. Early prediction of the severity, and patients' responses to specific therapeutics of SCD could lead to more precise treatment and management. Beyond well-known modifiers of disease severity, such as fetal hemoglobin (HbF) levels and αthalassemia, other genetic variants might influence specific sub-phenotypes. New treatments and management strategies accounting for these genetic and nongenetic factors could substantially and rapidly improve the quality of life and reduce health care costs for patients with SCD. Patients with SCD are subjected to long term administration of drugs and there is a limited data on pharmacogenomics of SCD therapeutics. Vaso-occlusive crisis (VOC) are the main clinical events of SCD and are associated with recurrent and long-term use of antalgics/opioids and HU. This project aimed to investigate the clinical and genetic predictors of painful vaso-occlusive crisis (VOC) among SCD Cameroon patients by exploring pharmacokinetic determinants of treatment responses as well as post-transcriptional signatures triggered by hydroxyurea treatment, particularly, miRNA expression. SCD patients were recruited from Yaounde Central Hospital and Laquintinie Hospital in Douala (Wonkam et al., 2018, Mnika et al., 2019 (b)), and recent migrants SCD patients from the DRC, recruited at the Haematology Clinic, Groote Schuur Hospital in Cape Town, South Africa (Mnika et al., 2019 (a) and Mnika et al., 2019 (b)). Sociodemographic and clinical data were collected by means of a structured questionnaire. Patients' medical records were reviewed to extract their clinical features over the past 3 years. Specifically, the occurrences of VOC, hematological parameters, hospital outpatient visits, hospitalisation, overt strokes, blood transfusions, and administration of hydroxyurea were recorded. Height, weight, body mass index (BMI), systolic and diastolic blood pressures (SBP and DBP) were measured. Detailed descriptions of patients and sampling methods used in the Cameroonian patients have been reported previously (Wonkam et al., 2018 Mnika et al., 2019 (a) and Mnika et al., 2019 (b)). For the purpose of comparing frequencies of variants, ethnically matched Cameroonian controls were randomly recruited from apparently healthy blood donors in Yaounde for participation in the study. All blood samples were collected for genomic characterisation and analysis. DNA was extracted from peripheral blood, following instructions on the available commercial kit [QIAamp DNA Blood Maxi Kit ® (Qiagen, United States)]. Genotyping (TaqMan and MassArray) was performed for 40 variants in 17 pain-related genes, three fetal haemoglobin (HbF)-promoting loci, two kidney dysfunction-related genes, and HBA1/HBA2 genes for 436 patients. A subset of these samples was also genotyped to analyse 32 core and 267 extended pharmacogenes using commercially available PharmacoScan® platform for characterisation of pharmacokinetic determinant of response. We also compared the pharmacogenes variants from these African groups, to data extracted from the 1000 genomes Project. Moreover, association studies were carried out on pharmacogenes variants with SCD clinical variability. Additionally, protein-protein interaction (PPI) network and enriched biological processes and pathways were investigated. For association studies, statistical models using regression frameworks to analyse 40 variants were performed in R®. For miRNA expression, total RNA was isolated using the miRNeasy kit according to protocol of the Manufacturer (QIAGEN, Hilden, Germany); and sequenced by the Genomic and RNA Profiling Core at Baylor College of Medicine, United States, using the NanoString Platform (NanoString Technologies, Inc., Seattle, WA, United States), according to manufacturer's instructions. Genes with statistically significant changes in expression were analysed using the significance analyses of microarrays (SAM) tools. Female sex, body mass index, Hb/HbF, blood transfusions, leucocytosis and consultation or hospitalisation rates significantly correlated with VOC. Three painrelated gene variants correlated with VOC (CACNA2D3-rs6777055, P = 0·025; DRD2- rs4274224, P = 0·037; KCNS1-rs734784, P= 0·01). Five pain-related gene variants correlated with hospitalization/consultation rates (COMT-rs6269, P = 0·027; FAAHrs4141964, P = 0·003; OPRM1- rs1799971, P = 0·031; ADRB2-rs1042713; P < 0·001; UGT2B7-rs7438135, P = 0·037). The 3·7 kb HBA1/HBA2 deletion correlated with increased VOC (P = 0·002). HbF-promoting loci variants correlated with decreased hospitalisation (BCL11A-rs4671393, P = 0·026; HBS1L-MYB-rs28384513, P = 0·01). APOL1 G1/G2 correlated with increased hospitalisation (P = 0·048). A commercial genotyping array platform (PharmacoScan®) with 4627 markers located in 1191 genes was used to investigate 299 pharmacogenes (32 ADME core and 267 extended pharmacogenes). Based on the PharmacoScan analyses, no statistically significant differences in allele frequencies were detected between SCD cases and controls from Cameroon. A principal component analysis (PCA) revealed that Cameroonians' data clustered with other Africans, but this population is significantly distinct from American, European and Asian populations data. Variant allele frequencies in 21/32 core pharmacogenes were significantly different between the two SCD groups (Cameroon vs. Congo). No correlation between clinical variability and variants in the core genes was detected for both populations under study. An association study of the core and extended PharmacoScan variants to VOC identified statistically significant associations between two single nucleotide polymorphisms (SNPs) to VOC after correction of multiple testing. These two SNPs mapped to 50 genes, with two SNPs located in core pharmacogenes (SLCO4A1- rs118042746, p=1.21e-07; UGT1A10, UGT1A8- rs10176426, p=1.22e-07). Functional enrichment analyses revealed that these 50 genes are involved in three biological processes and four pathways relevant to SCD pathophysiology, including xenobiotic glucuronidation (GO:0052697, p = 2.3e-03), and drug metabolism - other enzymes (p = 2.1e-02). Further analyses of the 50 genes, identified key genes in human proteinprotein networks: NTSR1, LRMDA, SMAD SMAD4 and CDH2. These four genes also interacted with three core pharmacogenes associated with VOC: UGT1A8, UGT1A10 and SLCO4A1. We found 22/798 miRNAs to be differentially expressed under HU treatment, with the majority (13/22) being functionally associated with HbF-regulatory genes, including BCL11A (miR-148b-3p, miR-32-5p, miR-340-5p, miR-29c-3p), MYB (miR-105-5p), KLF-3 (miR-106b-5), and SP1 (miR-29b-3p, miR-625-5p, miR-324-5p, miR-125a-5p, miR-99b-5p, miR-374b-5p, miR-145-5p). The present thesis started by highlighting the scarcity of studies investigating variable responses to pain in SCD patients and then proceeded to addressing this research gap. To our knowledge this is the first body of from Africa to provide evidence supporting the possible development of a genetic risk model for pain in SCD. This is also the first body of work to report an association between these two SNPs and VOC in core and extended pharmacogenes. Our data reveals that the commercial pharmacogenes arrays investigated might need additional evidence for appropriateness among Africans. Therefore, it advocates the need to invest in research exploring population-specific arrays, drug design, targeting, and efficacy, for improved clinical management of patients of African descent. Previous studies have investigated various mechanisms to understand the genomic variations affecting responses to HU, but full understanding of the variable HU-mediated HbF production among individuals affected by SCD remains elusive. The present study showed that mechanisms of HbF production in response to HU, could particularly be mediated through miRNA regulation. The data reveals some alternative perspectives and routes towards identifying new therapeutic targets and approaches for SCD. However, this study needs to be replicated in larger samples in multiple African populations.
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    Programmed cell death-ligand 1 (PD-L1) expression in HIV-associated diffuse large B-cell lymphoma – role and regulation
    (2025) Latib, Zahra; Mowla, Shaheen; Dharshnee, Chetty; Estelle, Verburgh
    Diffuse Large B cell Lymphoma (DLBCL) is an aggressive disease that displays striking heterogeneity at both the molecular and clinical levels; and as a result, up to 40% of patients relapse or are refractory to standard first-line therapy. DLBCL is the most common subtype of lymphoma affecting people living with HIV (approximately 50% of all lymphomas seen in this group), and while the introduction of Highly Active Antiretroviral Therapy (HAART) has improved patient outcome, the incidence of DLBCL in this group remains disproportionately high, especially in resource-limited settings. Emerging data indicate that HIV-associated DLBCL, although highly heterogeneous, has distinct clinical, morphological and molecular features from non-HIV-related DLBCL. The complex biology that underpins HIV-associated DLBCL remains largely undefined, necessitating clinical and molecular investigations to identify biomarkers which can be targeted for therapy, specifically within those populations most affected, such as in Sub-Saharan Africa. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signalling pathway is an important immunoregulatory mechanism that dampens the immune response by inhibiting T cell activity, playing a central role in controlling and maintaining tolerance to self-antigens. However, cancer cells have hijacked this mechanism by overexpressing PD-L1 to impair T cell functioning and ultimately escape immune recognition and destruction. Additionally, impairment of this pathway impacts the broader tumour microenvironment (TME), for instance through inhibition of T cell function, creating favourable conditions for tumour progression. In a previous report, PD-L1 levels were shown to be elevated in HIV-positive patients prior to a lymphoma diagnosis, with those harbouring the highest levels of PD-L1 progressing to develop malignancies, suggesting that PD-L1 is a key factor in the onset of lymphoma among HIV-infected individuals. While blockade of the PD-1/PD-L1 pathway with monoclonal antibodies has achieved considerable success in several cancer types, the results remain suboptimal in DLBCL, where the status and relevance of the deregulation of the pathway, and particularly the role and status of PD-L1, remains unclear, and even more so, within an HIV-positive background. This warrants a deeper exploration into the significance of PD-L1 overexpression, as well as the mechanisms influencing PD-L1 expression, in HIV-associated DLBCL. In the current study, three approaches were taken to explore this. Firstly, PD-L1 levels were evaluated and compared within the peripheral blood cell populations of a cohort of newly diagnosed, treatment naïve, HIV-positive and HIV-negative DLBCL patients, using flow cytometry. As per previous observations, HIV-positive DLBCL patients were typically diagnosed at a younger age (64% below the age of 50 years) compared to their HIV-negative counterparts (36% under the age of 50 years). The GCB subtype was the major DLBCL subtype (64%) within the HIV-positive DLBCL group, and a striking 82% of cases had extranodal involvement, reflecting an aggressive/advanced-stage disease. Flow cytometric analysis revealed a significantly higher proportion of PD-L1-positivity overall (CD274+; median: 0.44%; p ≤ 0.01), as well as PD-L1-positive B cells (CD19+ CD274+; median: 6.62%; p ≤ 0.001), in DLBCL patients (irrespective of HIV status), compared to healthy controls. When comparing within the DLBCL patient groups based on HIV status, the HIV-positive cohort displayed a significantly higher proportion of PD-L1-positive cells overall (CD274+; median: 0.65%; p ≤ 0.05), and PD-L1-positive B cells (CD19+ CD274+; median: 10.9%; p ≤ 0.05). No noticeable differences were observed regarding the population of regulatory B cells (CD19+ CD24+ CD38+; median: 57.5% vs 61.2%; p = 0.5344) and PD-L1- positivity within this subset of B cells (CD19+ CD24+ CD38+ CD274+; median: 1.74% vs 0.83%; p = 0.3551) between HIV-negative and HIV-positive DLBCL patients. In the second approach, the status of PD-L1, and T cells and macrophages were evaluated in the TME of HIV-positive and HIV-negative DLBCL tissues, using immunohistochemistry. In concordance with what was observed in the peripheral blood, a higher proportion of PD-L1+ cells were present in the TMEs of HIV-positive DLBCL patients, relative to HIV-negative ones (HIV-positive vs HIV-negative; median: 0.47% vs 0.09%; p ≤ 0.0.5). This was accompanied by reduced CD8+ cytotoxic T cell infiltration (HIV-positive vs HIV-negative; median: 1.25% vs 2.12%; p ≤ 0.0.5), and enhanced CD68+ tumour-associated macrophage infiltration (HIV-positive vs HIV-negative; median: 2.69% vs 1.56%; p ≤ 0.0.5), suggesting differential impairment of the TME in these two groups, and representing a heightened immunosuppressive environment in DLBCL patients infected with HIV. The third approach in this study attempted to delineate the complex relationship between PD-L1, c-MYC, EBNA2 and HIV, using a combination of in silico tools, as well as in vitro analyses using established DLBCL cell lines and models. Using DLBCL gene expression data publicly available on The Cancer Genome Atlas, we found highest expression of PD-L1 to be associated with the ABC subtype. Additionally, an inverse (negative) correlation between PD-L1 and c-MYC expression was observed in patients with this specific subtype of DLBCL. The regulation of PD-L1 by c-MYC was assessed in DLBCL cell lines through an experimental approach, taking HIV and EBV infections into account. In vitro analyses using qPCR and western blotting experiments confirmed this inverse correlation within ABC-derived DLBCL cell lines, as well as within a c-MYC knock-down DLBCL cell model. To investigate the effect of HIV-1 on PD-L1 expression in DLBCL cells, two independent experimental laboratory HIV-1 variants were used, namely aldrithiol-2 inactivated HIV-1, and HIV-1 pseudovirus. Exposure to the virus led to reduced expression of both PD-L1 and c-MYC protein levels, even in the presence of EBNA2, a result which contrasts with our observations using patient derived blood, and tumours. This indicated the importance of studying complex interactions using the appropriate experimental systems. Overall, this study provided novel insights into the status, role and regulation of PD-L1 in DLBCL, specifically within the context of HIV-infection. These findings further confirm that HIV-associated lymphomas harbour unique pathobiological features and provides directions for future basic and clinical research aimed at improving therapeutic approaches specifically tailored for this patient group.
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    Study of genetic modifiers of fetal hemoglobin and mechanisms of hydroxyurea-induced γ-globin expression in sickle cell disease
    (2016) Pule, Gift Dineo; Wonkam, Ambroise; Mowla, Shaheen; Novitzky, Nicolas
    Sickle Cell Disease (SCD) is a growing global problem with firm roots in sub-Saharan Africa (SSA) representing over 3/4 of the global burden of the disease. The prevalence of the sickle mutation (HbS) in SSA has been amplified by the partial resistance to Plasmodium falciparum malaria, which is endemic along tropical equatorial Africa. Several genetic variants have since been associated with fetal hemoglobin (HbF), the disease-ameliorating globin protein, including variants at three principal loci; BCL11A, HBS1L-MYB intergenic polymorphisms (HMIP1/2) and the β-globin gene cluster, which together account for 10 - 20% HbF variance in SCD patients. Similarly, numerous signalling pathways have been implicated in the regulation of γ-globin expression, however, a complete understanding of the regulation of HbF remains elusive. The overall aims of this project were: 1a) to investigate the known variants in key HbF-promoting loci such as BCL11A erythroid-specific enhancer, BCL11A, HBS1L-MYB intergenic polymorphism (HMIP1/2), the β-globin gene cluster, as well as the influence of the co-inheritance of 3.7kb alpha globin gene deletion in a cohort of SCD patients from Cameroon; and 1b) to validate novel HbF-promoting loci reported in 2 genome-wide association studies (GWAS) carried out in a population of Sardinians (Italy) and SCD patients from Tanzania and explore the influence of known promoter variants in SAR1 associated with HbF in African American patients amongst Cameroonian SCD patients; 2) to investigate the molecular mechanisms of hydroxyurea (HU)-induced production of HbF using a primary erythroid cell model from hematopoietic stem cells (HSCs) derived from umbilical cord blood and lastly, 3) to investigate the prevalence of SCD-related polymorphisms; β-globin gene haplotype, HbS mutation and malaria-resistance variants in 3 SCD-unaffected (HbAA) cohorts from South Africa, Zimbabwe and Malawi.
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    The biological effects of HIV-1 Nef on the development of B-cell Lymphoma
    (2022) Ahmed, Riyaadh; Mowla, Shaheen
    The incidence of HIV-associated cancers is significantly higher within the South African population compared to elsewhere in the world due to South Africa having one of the highest HIV burdens compared to the rest of the world. Burkitt lymphoma (BL) is an extremely aggressive cancer that is considered to be a highly prevalent subtype of Non-Hodgkin Lymphoma (NHL) affiliated with chronic HIV infection. While the immunosuppressive aspect of HIV remains a primary cause for the increased occurrence of cancer amongst HIV positive patients, new research demonstrates that the virus can have direct oncogenic effects, often through the action of specific virally-encoded proteins. The latter can act alone or collaboratively with cellular proteins, as well as with oncoproteins of established oncogenic viruses such as the Kaposi's Sarcoma-associated Herpes Virus (KSHV), or with Epstein-Barr Virus (EBV). To date, convincing evidence assign oncogenic activity to the HIV viral proteins Trans-activator of Transcription (Tat) and p17 in the progression of B-cell lymphomagenesis. Of particular interest to this study is the HIV-1 viral protein Nef (Negative Factor) which has been reported to have an oncogenic role in several cancer types including Kaposi's Sarcoma (KS) and Non-Small Cell Lung Cancer (NSCLC). However, the role of HIV-1 Nef in B-cell lymphoma, including BL, remains largely unexplored. Previous work performed in our research laboratory demonstrated that HIV-1 Nef protein could enhance the expression of two key lymphoma promoting factors, c-MYC and Activation Induced Cytidine Deaminase (AID), in BL cells and promoted genomic instability. The current study aimed to further explore the oncogenic effects of HIV-1 protein Nef in the development of BL. Furthermore, the potential internalization of recombinant Nef protein by B-cells during extracellular exposure was examined. Herein, we utilized cellular-based assays to examine alterations in the proliferation, the cell cycle and apoptosis of BL cells that have been extracellularly exposed to recombinant Nef protein. Our findings reveal that the proliferation of BL cells was enhanced in response to Nef exposure. Furthermore, the expression of the cyclin proteins A, B1 and E2 were found to be increased in Nef-exposed BL cells, which could account for the enhanced proliferation. No major changes in the cell cycle profile of BL cells were noted upon exposure to Nef. While a sub-G1 peak was noted during cell cycle analysis, Annexin V/7-Amino-actinomycin (7-AAD) staining confirmed that this observation was an anomaly, confirming that the Nef protein did not enhance apoptosis in BL cells. Additionally, the Nef protein did not provide any protective effect against apoptosis in BL cells exposed to the chemotherapeutic agent Doxorubicin. Finally, investigation of the potential internalization of the Nef protein by B-cells indicated that Nef may be trafficked to both the cytoplasm and the nucleus. However, this remains inconclusive due to Nef being detected in negative control samples. Overall, this study generated novel data on the oncogenic role of HIV-1 protein Nef in the development of BL, demonstrating that this viral protein has the ability to enhance proliferation of BL cells. Additionally, Nef was shown to alter the expression of cellular cyclin proteins, which could be one of the mechanisms via which proliferation is enhanced. This data allows for a better understanding of the oncogenic role of Nef in the development of B-cell lymphoma, and contributes to our observation of enhanced disease severity and progression in HIV infected people who develop BL. Future studies will focus on further defining the oncogenic potential of Nef in aggressive B-cell lymphomas by examining its effect on other oncogenic processes/pathways which define hallmarks of cancer, including cell migration and invasion, autophagy and angiogenesis, as well as its effect on oncogenic signalling pathways. In addition to this, further optimization of the experimental design used to assess the potential internalization of the Nef protein by BL-cells is recommended for future work. Ultimately, more research must be undertaken to further elucidate the oncogenic role of HIV-1 Nef protein in HIV-associated lymphomas such as BL.
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    Understanding the molecular pathogenesis of HIV-associated Burkitt Lymphoma – the impact of HIV-1 protein Tat on lymphoma driver genes
    (2021) Alves, de Souza Rios Leonardo; Mowla, Shaheen
    Burkitt Lymphoma (BL) is a B cell non-Hodgkin lymphoma that occurs as three distinct subtypes, namely: endemic, sporadic, and immunodeficiency/HIV-associated. This cancer represents a frequent cause of mortality among HIV+ people in Southern Africa which has the highest incidence of HIV/AIDS worldwide. Recent reports associate a direct oncogenic function of HIV in BL development. However, the molecular mechanisms underlying this HIV-associated malignancy are not well understood. This study explores the oncogenic potential of HIV-1 protein Tat in BL via its ability to manipulate the expression of c-MYC and activation-induced cytidine deaminase (AID), two key drivers of BL progression. Using dual-luciferase reporter assays, HIV-1 Tat was shown to enhance the activity of the cMYC promoter (-2324 bp - +537 bp), which corresponded with elevated c-MYC protein levels in BL cells (Ramos) expressing HIV-1 Tat. By generating sequential promoter deletions, the minimal promoter region mediating HIV-1 Tat induced activation was identified. Site-directed mutagenesis indicated that this response was mediated by AP-1 binding elements, and coimmunoprecipitation assays revealed that HIV-1 Tat and the AP-1 factor JunB interacted within the same complex. Chromatin immunoprecipitation assays confirmed that JunB bound the c-MYC promoter in vivo under the influence of HIV-1 Tat. The effect of HIV-1 Tat on the expression of the DNA editing enzyme AID was also investigated. Dual-luciferase assays revealed that HIV-1 Tat could enhance the activity of the three regulatory regions of the AICDA gene, namely R1, R2 and R4. This translated into elevated AID protein expression in Ramos cells expressing HIV-1 Tat, which was also reflected in an increase in genomic instability as shown by enhanced phosphorylated H2AX expression. Sequential promoter deletions of the R1 promoter did not lead to a loss in HIV-1 Tat-mediated activation, pointing to potential post-transcriptional regulation. Indeed, HIV-1 Tat was found to downregulate the expression of hsa-miRNA-181b-5p, a known repressor of the murine Aicda gene. Furthermore, using reporter assays, we show that an hsa-miRNA-181b5p mimic could repress AICDA via the full-length 3'UTR which contains three putative binding elements for the miRNA. To date, this study reveals that HIV-1 Tat induced c-MYC promoter activation is mediated by two AP-1 binding sites. HIV-1 Tat was shown to couple with JunB and bind to both AP-1 sites on the c-MYC promoter inducing promoter activation. Furthermore, this study reveals a novel mechanism of AID deregulation via HIV-1 Tat-mediated miRNA perturbation. Lastly, we show that HIV-1 Tat interferes with hsa-miRNA-181b-5p expression in B cells, alleviating AICDA 3'UTR repression.