ALBERT

Description: Introduction: Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Over 50% of patients die within 5 years of diagnosis. The transition from normal PCs to active MM, via premalignant condition (monoclonal gammopathy of undetermined significance; MGUS), consists of many oncogenic events, including upregulation of cyclin D1 and IRF4 genes, as well as activating mutations in NRAS, and KRAS. Despite recent advances in oncogenomics, further studies are needed to identify critical players in MM pathogenesis that could be targeted for pharmacological intervention to improve outcome. Recently, aberrations in epigenetic modifications, including DNA modifications and miRNA expression have been shown to play a crucial role in development and progression of MM. miRNAs are short non-coding RNAs that bind to complementary sequences on target messenger RNA (mRNAs) and downregulate their expression by inhibiting mRNA translation, or inducing its degradation. miRNA analysis, may lead to improved understanding of MM biology and classification, by establishing associations between gene expression changes and MM molecular and clinical features. To assess whether miRNA deregulation plays a critical role in the development of MM we performed small RNA next generation sequencing in the PCs isolated from 3 patients at the MGUS stage and after that they developed active disease, but still untreated. miRNA deregulation was also validated in an independent set of newly diagnosed MM patients (n.34) compared to non-cancer age matched donors (n.6). Mechanisms of transcriptional regulation and biological roles of the differentially expressed miRNAs were also analyzed. Methods: 1x106 CD138+ frozen PCs (purity〉90%) from different 3 donors before and after the disease development were used for the analysis. RNA was extracted with RNA-DNA-protein kit (Norgen Biotek) and 0.8µg of total RNA was used to generate the cDNA libraries using TruSeq Small RNA Library Preparation Kit. The obtained cDNA libraries were sequenced on an ILLUMINA system through the OSU Genomic Shared Resource (GSR). Myeloma cells (MM.1S) were treated with pan-HDACi for 24 hours and total miRNA expression was analyzed by nCounter microRNA array (NanoString Technologies, Inc.). miRNA deregulation upon use of several pan-HDAC'i in clinical use (LBH589, SAHA and AR-42) were validated in 4 different cell lines and in the MM cells of newly diagnosed MM patients. Chromatin immunoprecipitation, silencing RNA for specific histone deacetylase enzymes (HDACI, HDAC2, HDAC3, and HDAC6), western blot analysis, luciferase assays, stem loop RT-PCR, q-RT-PCR and cell proliferation assays were also performed. Results: We found that several miRNAs are commonly deregulated during disease transition. Some of these miRNAs, including miR-223, miR-221, miR-222, miR-92a and miR-93 (p