Single Cell Transcriptome of Epstein Barr Virus-Associated Burkitt Lymphoma from People Living with Human Immunodeficiency Virus

Non-Hodgkin lymphoma (NHL) is the most common type of cancer in people living with human immunodeficiency virus (PLWH) in economically developed countries. The most common subtypes of NHL among PLWH are diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Importantly, EBV is present in >90% in endemic BL and ~30% in other epidemiologic groups. After infection, EBV persists in memory B cells in a latent state, where it can express viral oncogenic proteins that contribute to cellular transformation and thus to lymphoma development. There are three different latency programs (I, II and III) with varied oncogenic potencies depending on the viral oncoproteins expressed: 1 (EBNA-1), 4 (EBNA-1, LMP-1, LMP-2A, LMP-2B) or 9 (EBNA-1, LMP-1, LMP-2a, LMP-2b, EBNA2-6), respectively. All latency programs express small non-coding RNA: EBER-1. The expression of each latency program varies depending on the lymphoma subtype and the immune surveillance of the host. Thus PLWH, in the pre-CART era, tended to show latency state III whereas immunocompetent individuals tend to exhibit latency I state. In the post-CART era, latency I-II and mixed states (IIa and IIb) have been frequently reported; however, it remains incompletely understood which viral latency programs are present in BL and how they impact its biology. Taking advantage of the 5´expression library single cell RNA sequencing technology, we added custom primers targeting EBV genes to characterize viral gene expression in single cells. The datasets of two EBV+ BL cases from PLWH were aggregated for the scRNAseq analysis. Here we found at least one EBV transcript in 76% of B cells. Thus, although the majority showed only EBER-1 (latency I), 18% of B cells showed latency II or III. The analysis of differentially expressed genes in EBER1+ cells expressing LMP1 and/or LMP2 versus EBER1+ cells that are negative for LMP1 and/orLMP-2, showed upregulation of 29 genes. Among them, we found already reported genes such as Jun which results in the activation of AMP-1 and is independent of NFKB pathway activation by LMP-1. As expected, the overexpression of the gene myc was observed in the whole tumor; however, it showed a slightly augmented expression in the LMP1 and/or LMP2 positive cells thus indicating that LMP-1 and myc can be simultaneously expressed in human BL cells contrary to what has been observed in in vitro culture. Other upregulated genes were those involved in the ribosomal 40S subunit structure (RPS4Y1) and its binding to transfer RNA (EIF1AY) as well as genes encoding class I MHC and class II MHC. Interestingly, we also found overexpression of the class I MHC and class II MHC genes in EBV-infected BL cell lines belonging to latency III (Mutu-III and Kem-III) when compared to their correspondent latency I states (Mutu-I and Kem-I) at the RNA level (bulk-RNAseq). Our findings suggest the presence of a mixture of latency states in EBV+ BL and a transcriptional signature distinctive of LMP1 and/or LMP2 positive cells that is related to an increase B cell activation, translational activity and cellular growth. Importantly, it gives evidence towards the expression of myc and LMP-1 in vivo in PLWH.

Roth:Merck: Consultancy; Roche: Consultancy.