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Non-coding natural antisense transcripts (ncNATs) are regulatory RNA sequences that are transcribed in the opposite direction to protein-coding or non-coding transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.

The nuclear orphan receptor NR4A1 functions as tumour suppressor in aggressive lymphomas by pro-apoptotic genomic and non-genomic effects. Here, we immunohistochemically studied the clinico-pathological relevance of NR4A1 protein expression patterns in a cohort of 60 diffuse large B cell lymphoma (DLBCL) patients and non-neoplastic lymph nodes. We observed a significant association between high cytoplasmic NR4A1 and favourable cancer-specific survival and the germinal centre B cell-like subtype, respectively. Moreover, the percentage of lymphoma cells exhibiting cytoplasmic NR4A1 significantly correlated to those showing cleaved caspase 3. Complementary, functional profiling using gene set enrichment of Reactome pathways based on publicly available microarray data was applied to determine pathways potentially implicated in cytoplasmic localization of NR4A1 and validated by means of semi quantitative real-time PCR. The pathway analysis revealed changes in the ERK1/2 pathway, and this was corroborated by the finding that high cytoplasmic NR4A1 was associated with higher expression of ERK1/2 targets in our cohort. These data indicate that high cytoplasmic NR4A1 is associated with a favourable lymphoma-specific survival and highlights the importance of NR4A1 expression patterns as potential prognostic marker for risk assessment in aggressive lymphomas.

Introduction: The interaction of the chemokine receptor CXCR4 and its ligand CXCL12 appears to be implicated in many important biological processes such as proliferation, survival, migration, and/or invasion. Furthermore, it is important for normal leukocyte trafficking. Deregulation of this axis is frequently observed in several hematologic malignancies. In diffuse large B cell lymphomas (DLBCL), the CXCR4-CXCL12 axis is still largely unexplored and published data are contradictive. Hence, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. Methods: We determined the CXCR4 and CXCL12 expression levels in NGCB- and GCB-DLBCL consisting of primary and transformed follicular lymphomas (n=77 in total), the corresponding bone marrow samples (n=63) and non-neoplastic germinal center-B cells (GC-B, n=5) serving as non-malignant control. To investigate the effects of CXCR4 antagonists in vitro and their function in regulation of important pathways (JNK, ERK1/2 and NF-κB) known to be involved in lymphomagenesis, we treated lymphoma cell lines with three different CXCR4 antagonists, AMD070, AMD3100 and WK1 (a novel nicotinic acid derivative of AMD070 - synthesized by us), followed by functional assays and gene expression profile. Results: CXCR4 was 140-fold higher expressed in DLBCL compared to non-neoplastic GC-B cells. Interestingly, higher CXCR4 expression correlated to a clinically advanced stage, to bone marrow infiltration and worse cancer-specific survival in DLBCL. Further expression analysis by using the corresponding bone marrow biopsies demonstrated that CXCL12 expression correlated to the lymphoma infiltration rate and that CXCR4 expression was reduced in remission under therapy. Moreover, two CXCR4 antagonists - AMD070 and especially WK1 - exerted pro-apoptotic effects on CXCR4 positive lymphoma cells in vitro and induced the expression of certain pro-apoptotic genes in CXCR4 positive cell lines. Remarkably, these effects were more pronounced for the WK1. Finally, WK1 treatment resulted in reduced expression of JNK-, ERK1/2- and NFκB/BCR-target genes. Conclusion: Our data demonstrate that the CXCR4-CXCL12 axis is involved in the pathogenesis of DLBCL. Since CXCR4 antagonists exert pro-apoptotic effects and impact lymphoma relevant pathways, they represent interesting molecules to develop novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Nuclear orphan receptor NR4A1 exerts an essential tumor suppressor function in aggressive lymphomas. In this study, we investigated the hypothesized contribution of the related NR4A family member NR4A3 to lymphomagenesis. In aggressive lymphoma patients, low expression of NR4A3 was associated with poor survival. Ectopic expression or pharmacological activation of NR4A3 in lymphoma cell lines led to a significantly higher proportion of apoptotic cells. In a mouse NSG xenograft model of lymphoma (stably transduced SuDHL4 cells), NR4A3 expression abrogated tumor growth, compared with vector control and uninduced cells that formed massive tumors. Transcript analysis of four different aggressive lymphoma cell lines overexpressing either NR4A3 or NR4A1 revealed that apoptosis was driven similarly by induction of BAK, Puma, BIK, BIM, BID, and Trail. Overall, our results showed that NR4A3 possesses robust tumor suppressor functions of similar impact to NR4A1 in aggressive lymphomas. Cancer Res; 77(9); 2375–86. ©2017 AACR.