Super-Enhancer-Driven TOX2 Mediates Oncogenesis in Natural Killer⁄T Cell Lymphoma

Background: Extranodal natural killer/T-cell lymphoma (NKTL) is an EBV associated, aggressive malignancy. Activation of JAK/STAT pathway, overexpression of EZH2, and alternations in epigenetic program have been implicated in the pathogenesis of NKTL. Combined chemotherapy-radiotherapy is standard treatment for NKTL patients, but often associated with high relapse rate and serious side effects. New drugs, including anti-PD1 antibody pembrolizumab, have been explored. Overall, treatment for NKTL patients remains challenge in clinic. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac), coactivators and transcription factors. SE associated oncogenes have been demonstrated to play key roles in various types of cancers. In this study, we aim to define the SE landscapes of NKTL for a better understanding of the molecular pathogenesis of NKTL and to identify novel therapeutic targets.

Methods: We performed H3K27Ac ChIP-seq and RNA-seq on primary NKTL tumor samples and paired normal tonsil tissues as controls, then the Rank Ordering of Super Enhancers (ROSE) analysis was used to systematically annotate SEs and their associated genes between tumors and controls. A combination of RNA interference (RNAi), CRISPRi (dCas9-KRAB) technologies and overexpression experiments, followed by several functional assays were performed to determine the effects of candidate SE-genes on NKTL cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) experiments which examine the direct contact of SE and its promoter are ongoing.

Results: A total of 1266 SEs were identified in more than 2 out of 3 primary NKTL tumors but not in their normal tonsils. RNA-seq revealed overexpression of 1478 genes (false discovery rate <0.001, log2 fold change ≥ 1). We filtered genes that were associated with super-enhancers and also significantly overexpressed in NKTL tumors relative to normal tonsils. Using this rigorous strategy, we pinned down a list of 191 genes with high value for further investigation. Pivotal transcription factors regulating NK cell development and function were highly enriched in the list: TOX2, TBX21, EOMES, RUNX2, ID2, GATA3, GFI1, and MAFB. We then scrutinized the SE constituents of these 3 T-box protein family, including TOX2, TBX21 and EOMES, and found that only TOX2 harbored remarkably high SE peaks specifically in all 3 NKTL primary samples, in contrast, only background signals were presented in normal tonsil tissues. TOX2 expression was significantly higher in the primary NKTL samples and cell lines compared with normal NK cells. Most importantly, high expression of TOX2 was association with worse overall survival (Log rank p value: 0.021, hazard ratio: 2.63), demonstrating its prognostic significance (GSE90784). We used two individual TOX2 specific-shRNAs tagged with GFP to decrease TOX2 expression in NKYS cells. FACS analysis of GFP+ cells revealed a markedly decreased GFP+% cells in TOX2-sh1- and –sh2-treated samples compared with scr-shGFP-transduced cells over 11 days post transduction. These results indicate that silencing TOX2 imposes a strong negative selection pressure on NKYS cell growth. Overexpressed TOX2 resulted in significantly higher cell viability of NKYS-TOX2 cells than NKYS-EV in the culture without IL-2, but not in presence of IL-2. CFU assays also demonstrate that TOX2 was effective in enhancing the clonogenic capacity of NKTL cells. Next, we assessed whether the TOX-SE is functional and causative for the TOX2 dysregulation in NKTL. The repression of the TOX2-SE region significantly decreased TOX2 expression in NKYS cells. Repression of the TOX2-SE region also led to impairment of cell viability and cell growth, consistent with an oncogenic function.

Conclusions: Taken together, our integrative approaches defined the landscape of SE and identified high-value SE-associated oncogenes in NKTL. Among them, we demonstrated that TOX2 is a novel tumor driver, which promotes NKTL cell growth and enhances ability of colony formation, as well as protects cell viability under adverse condition. This study not only provides novel insight into the NKTL pathology, but also offer novel, potential therapeutic targets, such as TOX2 for the treatment of NKTL patients.