P1226: btg2, a novel target of mir-17-92, regulates b cell receptor signaling in mantle cell lymphoma

Topic: 20. Lymphoma Biology & Translational Research Background: B-cell receptor (BCR) signaling is highly activated and is a bona fide therapeutic target in mantle cell lymphoma (MCL), whereas the mechanism underlying BCR activation has not been fully understood. MicroRNA (miRNA) miR-17-92 cluster, containing miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a, acts as an oncogene and has been shown to be upregulated in MCL; however, the function and target molecules of each miRNA have remained to be clarified. These provoked us to investigate the functional relationship between miR-17-92 cluster and BCR signaling in MCL. Aims: In this study, we aimed to elucidate the mechanism of BCR signaling activation through comprehensive target molecule exploration and functional analysis of miR-17-92 cluster in MCL. Methods: Four MCL cell lines (Jeko-1, JVM2, KPUM-YY1, and Z138) were utilized. Silencing of coding genes and miRNA was performed by the lentiviral shRNA method and antisense oligonucleotide-based miRNA inhibitors, respectively. To comprehensively identify miR-17-92 cluster target genes, we performed pulldown-seq, in which mRNA was captured using the biotinylated miRNA mimics, the complex of captured target mRNA and miRNA was pulled down with magnetic beads-coated streptavidin, and the extracted RNA was analyzed by next-generation sequencing (NGS). Functional effects of silencing miRNAs and BTG2, a candidate of miR-17-92 identified in this study, were examined using assays for cell proliferation and the Ca flux assay, which quantifies the strength of the BCR signal by measuring intracellular Ca2+ concentration. The public database (NCBI GEO; accession number GSE93291) was used to examine the relationship between BTG2 gene expression levels and clinical characteristics of MCL. Results: Using miRNA inhibitors, we revealed that miR-17-92 regulates cell proliferation and BCR signaling activation in MCL cell lines. We hypothesized that SOX11 regulates miR-17-92 transcription based on previous data of ChIP-Seq for SOX11 in MCL cell lines [Kuo et al. Oncogene 2015]. Silencing SOX11 repressed the expression of each miR-17-92 in two of three SOX11-positive MCL cell lines, i.e., Jeko-1 and Z138. Additionally, we analyzed using BRD4 ChIP-seq data of SOX11-negative JVM2 and high BRD4 binding suggesting histone acetylation was found in the MIR17HG enhancer region. The pulldown-seq identified novel miRNA target genes including tumor suppressors such as BTG2 (miR-19b), CDKN2A (miR-17), FCGR2A (miR-17), SYNE1 (miR-20a), TET2 (miR-18, miR-19b, and miR-20a), TNFRSF10A (miR-20a), and TRAF3 (miR-17). Of these genes, BTG2 expression was negatively correlated with the BCR signature gene, and low BTG2 expression was associated with poor overall survival in the publicly available microarray data. Furthermore, the knockdown of BTG2 in MCL cell lines significantly induced the activation of BCR signaling and increased cell proliferation. Summary/Conclusion: This study identified that the overexpressed miR-17-92 cluster promotes BCR signaling activation. Importantly, BTG2 was identified as a novel target of miR-17-92 and found to activate BCR signaling in MCL. Moreover, the BTG2 expression level is associated with the prognosis of patients with MCL. These results may contribute to the prediction of the therapeutic efficacy and improved outcomes of MCL. Keywords: Mantle cell lymphoma