Abstract 120: Epigenetic silencing of miR-17 and miR-20a in chronic lymphocytic leukemia

Abstract

Abstract MicroRNA (miRNA, miR) are an important class of small, non-coding RNAs that function to regulate gene expression through transcriptional repression or degradation of the target transcript. Functionally, miRNAs have emerged as integral components of the oncogenic as well as the tumor suppressor networks, regulating nearly all cellular processes altered during tumor formation. Among these the miR-17-92 cluster has surfaced as a highly over-expressed miRNA with oncogenic properties in several lymphomas and solid tumors, including tumors of the lung, breast colon, pancreas and prostate. In contrast to most solid tumors, two members of this cluster, miR-17 and miR-20a, appear to be under-expressed in specific subtypes of B-cell chronic lymphocytic leukemia (CLL). In this study, we quantified the expression of miR-17 and miR-20a by real-time PCR in lymphocytes from healthy donors (total and CD19+ sorted B-cells), nine tumor B-cell lines, and primary CLL cells (n=83). Compared to normal B-cells, miR-17 and miR-20a were over-expressed in all tumor B-cell lines (3.1 - 33.3 fold). Expression was highest in mantle cell lymphoma cell lines (average ratio 17.2, n=3) as compared to cell lines derived from follicular lymphoma (ratio 10.2, n=1), Burkitt's lymphoma (average ratio 9.0, n=3), and multiple myeloma (average ratio 3.6, n=2). In contrast, miR-17 and miR-20 were significantly under-expressed in primary CLL cells as compared to normal B-cells (average ratios 0.33 and 0.48 respectively). Epigenetic mechanisms mediated by DNA methylation or the action of the histone deacetylases (HDACs) and related gene repressors represent one such mechanism by which miRNA may be silenced. We therefore evaluated the role of the HDACs in silencing the expression of miR-17 and miR-20 in CLL. Chromatin immunoprecipitation (ChIP) assays against HDACs 1-3 revealed that silencing of miR-17 and miR-20a in CLL was associated with increased recruitment of HDAC1 and HDAC2 to the miR-17-92 promoter. Consequently, exposure of primary CLL cells to the HDAC inhibitor panobinostat resulted in the accumulation of the transcriptionally activating chromatin modification, H3K4me3 and re-expression of miR-17 and miR-20a in 22/83 (26%) and 25/83 (30%) cases respectively. Functional evaluation of miR-17/20 revealed that the anti-apoptotic protein Mcl-1 was a target of miR-17 in subset of CLL cases. Over-expression of anti-apoptotic proteins, including Mcl-1, is frequently observed in CLL and is associated with increased apoptotic resistance. Therefore, HDAC inhibitors may therefore offer a therapeutic strategy whereby the aberrant silencing of miR-17/20a in CLL can be reversed so as to target Mcl-1, thus lowering the apoptotic potential of the CLL cell. Microarray studies are currently ongoing to identify other functionally relevant targets of miR-17 in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 120. doi:1538-7445.AM2012-120