Abstract 2060: microRNA-dependent modulation of histone acetylation in Waldenstrom's Macroglobulinemia

Abstract

Abstract Introduction. Epigenetic regulation of gene-expression, including histone acetylation, is commonly deregulated in many malignancies leading to aberrant transcription, but microRNA (miRNA)-dependent modulation of histone acetylation in Waldenstrom's Macroglobulinemia (WM) has not been evaluated yet. Methods. miRNA and gene-expression profiling have been performed on bone-marrow-derived CD19+ WM cells, compared to their normal cellular counterparts. Purified cRNA was hybridized to HG-U133Plus2.0 GeneChip (Affimetrix). A liquid phase Luminex microbead miRNA profiling (Luminex, Austin, TX) has been used for miRNA studies. To further define those miRNAs differentially expressed between groups (patients vs normal), the data were filtered on significance of differences using ANOVA test, (P < 0.01). Microbead-miRNA profiling data were validated data by stem-loop qRT-PCR. To identify specific predicted miRNA-targeted mRNAs, TargetScan, PicTar, and miRanda algorithms were used. Functional studies were performed on precursor-miRNA-9* and anti-miRNA-206-transfected WM cells, as compared to either scramble probe-transfected or non-transfected cells. HDAC activity was determined by using Colorimetric HDAC Activity Assay Kit. Effect on signaling cascades have been evaluated by western blot and immunofluorescence. DNA proliferation, cytotoxicity, cell cycle, and apoptosis were assessed by thymidine incorporation, MTT, PI, and Apo2.7 staining, respectively. Results. WM cells present with a specific miRNA signature characterized by increased expression of miRNA-206 and decreased expression of miRNA-9* (ANOVA;P< 0.01). Predicted targets for miRNA-206 and −9* include histone-deacetylases (HDAC4; HDAC5) and -acetyltransferases (Myst3). We first demonstrated that primary WM cells are characterized by unbalanced expression of HDACs and HATs at gene level with higher level of HDACs and lower level of HATs, responsible for decreased acetylated-histone-H3 and -H4, at protein level and increased HDAC activity. miRNA-206 and −9* played a functional role in regulating histone-acetylation and HDAC activity in WM cells, based on their ability to target HDACs and HATs; leading to induction of toxicity in precursor-miRNA-9* or anti-miRNA-206-transfected cells, as shown by reduced proliferation rate, cell cycle arrest, induction of apoptosis, supported by PARP-, caspase-8- and −9-cleavage. In addition, miRNA-9*-induced autophagy in WM cells by modulating Rab7 and LC3B. Conclusion. These findings confirm that histone-modifying genes and HDAC activity are de-regulated in WM cells; this may be partially driven by the aberrant expression of miRNA-206 and −9* in the tumor clone; and provide the basis for the development of miRNA-based targeted therapies in WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2060.