title: Relevance of EBV infection for sensitivity of B-cell malignancies to anti-apoptotic protein inhibitors
Creators: name: Obeidat, Ahmed
Publisher: name: University of Liverpool Repository
Year: 2030

description: The Epstein Barr Virus (EBV) is a ubiquitous gamma-herpesvirus that ‎infects &gt;90% of the world population. In majority of infected subjects, it exists in a dormant/non-pathogenic state, after primary infection, but has variable ability to induce cancers especially of B-lymphocytes (B-cells). EBV positivity has been demonstrated in Burkitt’s lymphoma ‎‎ (BL), classical Hodgkin’s lymphoma (HL), chronic lymphocytic leukemia (CLL), and diffuse large B-‎cell lymphoma (DLBCL). 
Within non-malignant and cancerous host cells, EBV can exist in a latent or lytic phase based on its replication status and patterns of viral gene expression. ‎ To better understand the impact of EBV infection we assembled a panel of cell lines (EBV+ and -ve) that represent various B-cell malignancies (CLL, MCL, DLBCL and BL). Our initial studies focused on EBV gene expression patterns of viral protein coding genes and miRNAs and established variable levels of expression. The investigation of specific genes (EBNA1, EBNA2 and LMP1) by qRT-PCR and immunoblot analysis established that the studied cell lines (HG-3, MEC-1, EHEB, PCL-12, and RAJI) exhibited latency pattern III. Such variable expression likely impacts disease behaviour.
The EBV genome encodes two homologues (BHRF1 and BALF1) of the anti-apoptotic BCL-2 protein. Their expression likely helps host cell survival and in turn viral persistence. The EBV-BHRF1 protein, in particular has been shown to be expressed in both lytic and latent phases. All studied cell lines demonstrated good expression of BHRF1 a result that is consistent with reports that its expression is more common in latency III state. Our results demonstrate variable expression within cell lines. 
Of relevance, BCL2 inhibition is now an established treatment in haematological malignancies such as CLL, Acute Myeloid Leukaemia (AML), and Multiple Myeloma (MM). Drugs that are currently used to target BCL2 (e.g. venetoclax/ABT199) are small molecule inhibitors (BH3-domain mimics) that block protein function and induce apoptosis. As the EBV-BHRF1 protein also encodes a BH3 domain which although shares sequence similarities with BH3 domain of BCL2. Despite similarities it sufficiently diverse to escape inhibition by venetoclax and hence may impact sensitivity and treatment outcomes in EBV+ve B-cell cancers. 
The impact of BHRF1 expression on venetoclax (ABT-199) and indeed other anti-apoptotic inhibitors on sensitivity of malignant B-cells is not clearly established. The major aim of this project was to investigate the impact of BHRF1 expression on the sensitivity of B-malignant cells to BCL-2 antiapoptotic inhibitors that target BCL-2, BCL-XL, and MCL-1. 
We compared and contrasted the sensitivity of both EBV+ve and -ve cell lines to venetoclax/ABT199, A1331853, and S63845 that target BCL-2, BCL-XL, and MCL-1 respectively. Our results clearly demonstrate that EBV+ve cell lines have greater IC50s and tolerance to venetoclax. Interestingly the cell lines do exhibit greater sensitivity to approaches that combine the inhibitors which may have clinical relevance. At least some of these combinations were found to be synergistic and may overcome the protective effect of BHRF1.
To further elucidate and investigate the protective role of BHRF1 and its impact on venetoclax sensitivity, we used an siRNA approach to knockdown BHRF1 expression and show that its protective effect against anti-apoptotic inhibitors can be partly overcome. Conversely, to see if induced expression of BHRF1 in EBV-ve cells reduces sensitivity, we cloned FLAG-tagged BHRF1 into a lentiviral plasmid. Having confirmed expression of BHRF1 in stably transduced cell lines, we clearly demonstrate its protective effects against anti-apoptotic inhibitors and specifically venetoclax. 
Taken together, our results clearly show that BHRF1 expression in EBV+ve B-cell cancers can influence venetoclax sensitivity. The study provides a rationale for routine EBV (and BHRF1 expression) screening of patients who are being considered for treatment with venetoclax as this may impact treatment outcomes.