Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma

Abstract Epstein-Barr virus (EBV) infection is one of the main causative factors of nasopharyngeal carcinoma (NPC), which is prevalent in southern China and some Southeast Asian countries. EBV infection in NPC cells is characteristically type II latency, where very few viral proteins (EBNA1, LMP1 and LMP2A) but high levels of noncoding RNAs (especially BARTs: BamHI-A rightward transcripts) are expressed. Our previous study found that BART lncRNAs modulate transcription of IKZF3, which encodes Aiolos, through epigenetic pathways in NPC cells. However, the biological significance functional mechanism of IKZF3/Aiolos are not yet clearly defined. We now further show that IKZF3/Aiolos expression levels are associated with copy numbers of EBV episomes in the EBV-harboring NPC cell line, NPC43, and specifically correlate to the level of lnc-BARTs, as demonstrated using CRISPR systems. Transcriptomic analysis of IKZF3-knockout C666-1 NPC cells revealed that 17 EBV-encoded microRNAs and 53 lytic genes were significantly downregulated or upregulated (FDR p value <0.05 and posterior fold changes >1.5). Immunoblot testing showed that the EBV lytic reactivator Zta and antigen BMRF1 are upregulated following knockout of IKZF3 in C666-1 cells, suggesting involvement of IKZF3/Aiolos in maintaining EBV latency in NPC. Gene ontology and KEGG (Kyoto Encyclopaedia of Genes and Genomes) analysis of IKZF3-knockout C666-1 cells found that most of the host genes with differential expression that are enriched in Epstein-Barr virus infection are associated with apoptosis, focal adhesion, cancer cell growth and mammary neoplasm (p value <0.01). In addition, a soft agar assay demonstrated that oncogenic transformation of C666-1 cells was attenuated after knocking down expression of lnc-BARTs, knocking out IKZF3, or treatment with the IKZF3 inhibitor lenalidomide. Immunoprecipitation (IP) testing showed that Aiolos interacted with core elements (HDAC1 and MTA2) of the Mi-2/NuRD (nucleosome remodelling deacetylase) complex in NPC cells. Our findings suggest that Aiolos may recruit Mi-2/NuRD complexes for deacetylation of histone lysine residues bordering promoter regions of EBV lytic genes and host tumour suppressor genes, leading to inhibition of reactivation of EBV lytic infection, thereby preventing apoptosis of NPC cells. Citation Format: Songtao HE, Jiayan Liu, Honglin Chen. Epstein-Barr virus BART lncRNAs induce IKZF3/Aiolos to maintain EBV latency and promote tumorigenicity in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB211.