Frequent Copy Gains of Viral LMP-1 Confers Radiosensitivity in Natural-Killer/T Cell Lymphoma

Introduction

Discovered from Burkitt’s lymphoma (BL) in 1964, the Epstein-Barr Virus (EBV) was the first virus to associate with human cancers. EBV associated malignancies of particular significance in Asia include natural-killer/T cell lymphoma (NKTCL) and nasopharyngeal carcinoma (NPC), where their neoplastic cells are ubiquitously infected with EBV. Recently, numerous studies have reported somatic mutations altering immune-evasion, JAK-STAT, NF-κB, epigenetic, and MAPK pathways in NKTCL. In addition, we have also reported germline single-nucleotide polymorphisms (SNPs) of HLA-DPB1, IL18RAP and HLA-DRB1 that conferred increased risk of NKTCL and prognostic models that could contribute to the progression of NKTCL. Although frequent copy-number variations (CNV) of chromosome 6q21 implicating PRDM1, ATG5 and AIM1 genes were reported in NKTCL, there is still a general lack of research of CNV on oncogenic EBV genes in NKTCL.

Materials

To detect non-reference CNV events within the EBV genomes, we used whole-genome sequencing (WGS) to comprehensively profile the genomic landscape of 77 high quality tumor-normal matched samples from patients with NKTCL and 10 NK/T lymphoma/leukaemia cell lines. We also included whole-transcriptome (n=36) sequencing data, with matching WGS data, to analyse dysregulated pathways within mutations-of-interest.

Results

We used a step-wise analytical methodology to detect viral copy-gains and discovered frequent copy-gains of LMP-1 in 22.1% (18/77) of NKTCL tumors and 10% (1/10) of cell lines. The recurrently gained locus encapsulated the viral LMP-1, BNLF2a, and BNLF2b genes, which led to increased expressions of LMP-1. This recurrence rate supersedes the frequencies of most previously reported mutated human-genes in NKTCL. We also further validated these genomic alterations with both Nanopore long-read sequencing and Sanger sequencing. Gene-set enrichment analysis also showed that the “P53_PATHWAY” gene-set was the most enriched (FDR<0.001) within the LMP-1^copy-gain samples. Importantly, we found that patients with tumors harbouring LMP-1^copy-gain were significantly associated with better progression-free survival (P=0.028) and overall survival (P=0.026), when treated with radiation, regardless of early or late tumor staging. Experimentally, we showed that overexpression of LMP-1 rendered radiosensitivity and, inversely, shLMP-1 rendered radioresistance in two NKTCL cell lines. Mechanistically, expression of LMP-1 increased double stranded breaks as evident by increased γH2AX and drove anti-tumoral activity by GADD45a-induced apoptosis.

Conclusion

In conclusion, this study reports on the frequent copy-gains of viral LMP-1 in NKTCL. The experiments also reports on the mechanistic link between an unexpected relationship between increased LMP-1 and better survivals from better sensitization to DNA damaging therapeutic agents.