Background: Non-Hodgkin lymphoma is of high prevalence among HIV-infected people. In particular, the incidence of HIV-associated Burkitt lymphoma (BL) remains high despite the advent of Highly Active Anti-Retroviral Therapy. Recent evidence shows that serum-soluble HIV proteins can enhance oncogenesis, particularly in lymphoid tissues. This study sought to define the role of HIV protein Negative regulatory factor (Nef) in BL development by assessing its effect on key lymphoma driver genes. Methods: A recombinant Nef protein was used to assess changes in expressions of activation-induced cytidine deaminase (AICDA/AID) and c-MYC in B lymphocytes exposed extracellularly to the protein. Additionally, changes in the promoter activities of these genes were measured using a Nef-expressing cellular model and reporter assays. Confocal microscopy was used to observe c-MYC and AID expression and localization, and genomic integrity via the recruitment of phosphorylated γ-H2AX, in Nef-exposed cells. Results: mRNA transcription of c-MYC and AICDA were significantly enhanced in lymphoma cells, up to 2-fold for c-MYC and up to 4-fold for AICDA, when exposed to varying concentrations of Nef (0-1000 ng/ml) and for different periods of time (3, 6 and 12 h). The protein expressions of AID and c-MYC followed a similar pattern and these effects were specific to BL but not lymphoblastoid cells. While the promoter activity of c-MYC was enhanced in the presence of Nef in a dose-dependent manner, the same was not observed for AICDA. Both AID and c-MYC accumulated within the cytoplasmic and nuclear spaces of Nef-exposed lymphoma cells, with a concomitant increase in DNA double strand breaks within the genome. Conclusions: Exposure to HIV Nef leads to significant increases in AID and c-MYC, leading to genomic instability, potentially enhancing the oncogenic potential of Burkitt lymphoma. Our findings align with that of others to show that HIV proteins can directly contribute to the development and pathogenesis of HIV-associated lymphoma and accounts for the elevated incidence of BL observed in the HIV-infected population.

Background Mechanisms underlying an association between HIV or antiretroviral therapy (ART) during pregnancy with risk of preterm delivery (PTD) and small-for-gestational-age (SGA) remain unclear. We explored the association between cellular immune activation and PTD or SGA in HIV-infected women initiating ART during or before pregnancy. Methods HIV infected women enrolled at median 15 weeks gestation; were analyzed for immune markers, matched on ART initiation timing (15 women initiated pre- and 15 during pregnancy). There were 30 PTD (delivery <37 weeks), 30 SGA (weight for age ≤10 th centile) cases and 30 controls (term, weight for gestational age >25 th centile) as outcomes. Lymphocytes, monocytes and dendritic cell populations, their activation status or functionality were enumerated by flow cytometry. Results PTD cases initiating ART in pregnancy showed decreased CD8 + T cell, monocyte and dendritic cell activation, increased classical (CD14 +CD16 -) and intermediate (CD14 +CD16 +) monocyte frequencies, and decreased inflammatory monocytes (CD14 dimCD16 +) compared to SGA cases and term controls (all p<0.05). Allowing for baseline viral load, the immune markers remained significantly associated with PTD but only in women initiating ART in pregnancy. Lower monocyte activation was predictive of PTD. TLR ligand-induced IFN-α and MIP-1β levels in monocytes were significantly lower in PTD women initiating ART in pregnancy. Conclusion Low immune activation, skewing towards anti-inflammatory monocytes and lower monocyte cytokine production in response to TLR ligand stimulation were associated with PTD but not SGA among women initiating ART in, but not before, pregnancy suggesting immune anergy to microbial stimulation as a possible underlying mechanism for PTD in women initiating ART in pregnancy.

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 the c-MYC gene 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 the c-MYC promoter, as shown by in vivo chromatin 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.

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Introduction: The pathogenesis HIV-associated lymphomas involve a complex interplay of biological factors, including the immerging role of HIV encoded proteins. These proteins have been shown to have oncogenic potential and to enhance the pathogenesis of HIV-associated malignancies. However, very little is known about the molecular mechanisms involved in this interaction. Here, we investigate the effect of HIV protein Negative Factor (Nef) on the expression of c-MYC and activation-induced cytidine deaminase (AID) in lymphoblasts and lymphoma cells. Methods: Native histidine-tagged HIV Nef was produced in a bacterial system and used to treat (1) an EBV-immortalized B lymphoblastoid cell line (L1439A) and (2) the established lymphoma cell line Ramos, at various concentrations and for various time points. Changes in the expressions of c-MYC and AID were measured at both the mRNA and proteins levels using qPCR and western blotting respectively. The ability of Nef to regulate the promoter of these two genes was determined using luciferase reporter assays. Furthermore, confocal microscopy was used to investigate the downstream effects of Nef exposure. Results: In L1439A cells, a general decrease in the expression of c-MYC and AID was observed. After 1 and 3 hours of exposure to HIV-Nef both c-MYC and AID mRNA levels reduced by approximately 0.5-fold. In contrast, the expressions of both genes increased significantly upon these treatments in the lymphoma cell line Ramos. After 3 hours of exposure, c-MYC expression increased by ~1.9-fold, while AID expression increased up to 4.6-fold. The protein expression followed a similar pattern, as indicated by western blotting. Furthermore, luciferase reporter assays showed that the effect on c-MYC expression is likely to be direct since the activity of the c-MYC promoter increased in a dose-dependent manner in the presence of Nef. c-MYC translocated to the nucleus, which is indicative of an active protein. Furthermore, an increase in gamma H2AX foci on DNA, indicative of double strand breaks, shows that the increase in AID lead to genomic instability. Conclusion: This study demonstrates a differential effect of HIV Nef on normal immortalized lymphoblasts versus lymphoma cells. More importantly, it shows for the first time that HIV Nef may enhance the pathogenesis of lymphomas by triggering pathways and mechanisms which increase the expression of c-MYC and AID. Citation Format: Shaheen Mowla, Nontlantla Mdletshe. The role of HIV protein Nef in the development of HIV/AIDS-associated lymphomas [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B31.