MiR-29b Exerts Anti-Multiple Myeloma Activity by Targeting Key Oncogenic Pathways and Modulating DNA Methylation Profile.
Authors: Amodio Nicola , Martino Maria Teresa Di , Foresta Umberto , Rossi Marco , Leotta Marzia , Leone Emanuela , Lionetti Marta , Gullà Annamaria , D'Aquila Patrizia , Bellizzi Dina , Passarino Giuseppe , Fabiani Fernanda , Fulciniti Mariateresa , Ferrarini Manlio , Morabito Fortunato , Neri Antonino , Munshi Nikhil C. , Anderson Kenneth C. , Tagliaferri Pierosandro , Tassone Pierfrancesco 
Published: October 2019

Abstract
Abstract 2941
The enforced expression of tumor suppressor microRNAs (miRNAs) is a promising strategy for cancer treatment. Among miRNAs frequently deregulated in cancer, miR-29b is of great interest since its overexpression leads to tumor growth inhibition in solid tumors and leukemias. On these bases, we first investigated miR-29b expression pattern in multiple myeloma (MM) cells. miRNA profiling of primary CD138+ MM patient cells (n=55) revealed deregulated expression of miR-29b with almost 60% of cases showing strongly reduced levels. Moreover, as assessed by quantitative real time PCR (qRT-PCR), miR-29b expression was significantly downregulated in a panel of 11 MM cell lines and even further decreased when RPMI-8226 MM cells were co-cultured with bone marrow stromal cells (BMSCs), indicating that the human bone marrow microenvironment (huBMM) modulates miR-29b levels. Either transient expression of synthetic miR-29b mimics through electroporation or stable lentivirus-enforced miR-29b expression induced growth inhibition and apoptosis of MM cells in vitro. In vivo intratumoral or systemic delivery of neutral lipidic formulated-miR-29b mimics in different clinically relevant murine models of human MM, including our recent SCID-synth hu model which recapitulates the huBMM, led to significant tumor growth inhibition together with increased survival of miR-29b-treated animals. By Western blot and qRT-PCR analysis, we observed dramatic reduction of predicted targets of miR-29b including CDK6 and MCL-1, which can thus explain antiproliferative and pro-apoptotic effects triggered by miR-29b expression. Most importantly, we identified Sp1, a transcription factor endowed with oncogenic activity in MM, as a negative regulator of miR-29b expression providing evidence of a novel regulatory loop in MM cells: in fact, enforced expression of Sp1 was able to reduce miR-29b-promoter activity and miR-29b levels, whereas miR-29b expression decreased Sp1 mRNA and protein levels via 3'UTR binding, as assessed in luciferase reporter assays. Moreover, treatment of MM cells with the proteasome inhibitor bortezomib led to Sp1 downregulation and miR-29b upregulation. Strikingly, miR-29b transfection significantly strengthened the in vitro anti-proliferative and apoptotic effects induced by bortezomib, thus highlighting its role in the mechanism of anti-MM activity of this drug. Additional miR-29b targets include de novo DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B). Microarray profiling revealed increased DNMT3A and DNMT3B mRNA levels in cancer cells as compared to normal plasma cells. The integrated analysis of miRNA/mRNA profiling in a panel of 18 MM cell lines highlighted an inverse correlation between miR-29b and DNMT3B levels. Finally, miR-29b was proven to target DNMT3A and DNMT3B and significantly reduced the global DNA methylation levels in MM cells, as assessed by performing an in vitro DNA methylation assay with genomic DNA extracted from MM cells after synthetic miR-29b electroporation. In conclusion, our findings indicate that miR-29b exerts anti-MM activity by targeting relevant oncogenic pathways and by restoring the aberrant methylation pattern of MM cells. These results support a potential therapeutic role for miR-29b mimics in MM as single agent or in combination with the proteasome inhibitor bortezomib and/or demethylating agents.

Disclosures:
Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.