The Abnormal Repression of Mir-375 Expression By Overlapping Epigenetic Dysregulations in Myelomas

[Introduction] Multiple myeloma (MM) remains mostly incurable despite major advances in treatment strategies. The complex interplay among various cell-intrinsic and -extrinsic molecular mechanisms confers inter-patients diversity and intraclonal heterogeneity in MM. However, as the universal and relevant therapeutic target molecule against MM, we have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and its major downstream substrate RSK2 were universally active in MM (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014), while we also identified that the repression of miR-375 was found to be the universal underlying mechanism for the overexpression/activation of PDPK1 in MM (Tatekawa S, ASH meeting 2015). In this study, we further extended our study to assess the clinical relevance of miR-375 repression and the molecular mechanisms for the miR-375 repression in MM for the future clinical translation of miR-375/PDPK1/RSK2 signaling axis in the diagnosis and treatment development for MM.

[Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 113 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells; N=10, MGUS; N=30, newly diagnosed MM (NDMM); N=34, relapsed/refractory (RRMM); N=39). The level of miR-375 expression was calculated with 2^-ΔΔCt methods. Human U6 snRNA was examined as the reference and cDNA of RPMI8226 was used as a calibrator sample. The methylation status of miR-375 upstream regions including promoter site were analyzed by methylation-specific PCR (MSP) and bisulfite sequence. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine the histone modification status in miR-375 upstream regions. The circulating plasma RNA samples were converted to cDNA libraries followed by sequencing using the multiplex small RNA library primer set. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent.

[Result] When compared to normal plasma cells, the miR-375 expression was significantly decreased in CD138 positive plasma cells from MGUS (p<0.05), NDMM and RRMM (both p<0.01), and tended to be decreased in HMCL cells (p=0.085). As the causative of miR-375 repression, we focused upon epigenetic deregulation for miR-375 gene. The bisulfite sequence analyses disclosed that CGI-1 and CGI-2, which were CpG-rich regions within 3Kbp from pre-miR-375 gene, were hypermethylated in all three patient-derived myeloma cells of each stage patients examined (MGUS, NDMM and RRMM) and both two HMCLs examined. The abnormal hypermethylation status of CGI-1 and CGI-2 in MM were further conformed with larger number of genomic DNA samples by MSP, i.e., the CGI-1 region and promoter sites (CGI-2) of miR-375 gene were hypermethylated in all 8 HMCLs examined and in 58 patient-derived myeloma cells. Furthermore, the treatment with Trichostatin A, a histone deacetylase inhibitor, upregulated acetylated histones in several fragments including the promoter site of pre-miR375 in HMCLs. Finally, the treatment with SGI-110, a hypomethylating agent, and/or Trichostatin A, markedly increase miR-375 expression in HMCLs, suggesting that the overlapping epigenetic deregulations, such as abnormal DNA hypermethylation or histone deacetylation, are involved in the silencing of miR-375 in MM. In contrast, the expression levels of circulating plasma miR-375 were not significantly different between myeloma patients and healthy donors, indicating that the abnormally repressed miR-375 is the event specific to myeloma cells, but not in normal tissues in MM patients.

[Conclusion and Discussion] This study revealed that the expression level of miR-375 decreased specifically in clonal plasma cells from MGUS to RRMM patients, and the repression of miR-375 was caused by the overlapping epigenetic dysregulations. The abnormal repression of miR-375 which is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM is likely to be involved in the myelomagenesis in MGUS as well as the disease progression in MM, and the epigenetic deregulations may be a novel therapeutic target for MM.