Small RNA Deep Sequencing Highlights the Important Contribution of Mirnas in Regulating IRF4/c-Myc Axis in Myeloma Development

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<0.05) were commonly upregulated, where instead the majority of c-Myc transcriptionally repressed miRNAs, including miR-29s family, were found to be downregulated in the MM-PCs compared to the MGUS-PCs obtained from the same patients. Our data also show that IRF4 expression in MM cells is directly under the control of c-Myc regulated miRNAs, which we found downregulated during the multi-step transformation process of the PCs. We also observed that the use of pan-HDACi's in clinical use, including the recently FDA approved panobinostat (LBH589), in MM cells is able to upregulate the expression of those miRNAs and subsequently downregulate IRF4 at the posttranscriptional level. We also dissected the role of different histone deacetylases to specifically modulate miRNA expression and consequently IRF4-MYC pathway in MM cells.

Conclusions: Collectively, we believe, that these findings support the key role of miRNA aberrant expression in PC transformation. Their role in regulating the expression of IRF4 during myeloma development and lead us to speculate that this explains why IRF4 and c-Myc mRNA levels are higher in newly diagnosed MM patients, without obvious chromosomal abnormalities, compared to MGUS patients. Understanding of the molecular bases of how c-Myc expression can be regulated by a specific histone deacetylase via modulation of miRNA levels will have important impact not only on myeloma therapy, but also other hematopoietic malignancies involving c-Myc deregulation.