Microrna-138 Regulates Osteogenic Differentiation and Its Inhibition Presents a Novel Therapeutic Line to Prevent Bone Lytic Lesions in Multiple Myeloma
Authors: Tsukamoto Shokichi , Salem Karma , Manier Salomon , Reagan Michaela R. , Huynh Daisy , Perilla-Glen Adriana , Sacco Antonio , Zavidij Oksana , Rahmat Mahshid , Takagi Satoshi , Kawano Yawara , Kokubun Katsutoshi , Thrue Charlotte Albæk , Petri Andreas , Dorfman David M. , Roccaro Aldo M. , Capelletti Marzia , Kauppinen Sakari , Ghobrial Irene M. 
Published: October 2019

Abstract
Introduction
The bone marrow (BM) microenvironment in multiple myeloma (MM) plays a pivotal role in tumor growth and bone destructive process. Mesenchymal stromal cells (MSCs) in MM exhibit different genomic and cytokine secretion profiles that ultimately impair their osteogenic differentiation abilities compared to normal MSCs. However, the underlying molecular mechanisms are not fully understood. In the present study, we explored the role of miR-138 in MSCs derived from MM patients (MM-MSCs) and the potential for anti-miR-138 treatment to rescue impaired osteogenic differentiation in MM, both in vitro and in vivo using a human xenograft MM model.
Materials and methods
Primary BM aspirates were obtained from MM patients and normal healthy donors, after obtaining informed consent in accordance with the Declaration of Helsinki. MiR-138 expression in MM-MSCs was measured by quantitative real-time PCR. Publicly available microarray data sets (GSE17306 and E-TABM-508) were analyzed for miR-138 expression in MM cells compared to normal plasma cells. To test the effect of inhibiting miR-138 function, a high-affinity 15-mer locked nucleic acid (LNA)-modified anti-miR oligonucleotide and a corresponding scramble sequence control oligonucleotide were used (In collaboration with Dr. Kauppinen, Denmark). Anti-miR-138 oligonucleotides were transfected into MM-MSCs or normal MSCs co-cultured with MM cell lines and osteogenic differentiation in MSCs was assessed by alizarin red staining. For the in vivo studies, 6-week-old female SCID-beige mice (n=6, each group) were injected intravenously with anti-miR-138 or scramble control oligonucleotides (15 mg/kg) 2 times a week. 3 weeks later, GFP+Luc+ MM.1S cells (3 × 106) were injected into mice. Anti-miR-138 or control oligonucleotides were continued until day 28 after injection of myeloma cells. At day 28, the effect of anti-miR138 was assessed by the number of osteoblastic lineage cell (OBC: Lin-/CD45-/CD31-/CD51+/Sca-1-) from hematopoietic cell-depleted, collagenase-treated crushed bones of mice by flow cytometry.
Results
MiR-138 expression in MSCs from MM patients (n=10) was significantly higher than MSCs from normal donors (n = 4) (P<0.05). In addition, miR-138 expression was significantly higher in MM patient tumor cells compared to normal plasma cells using two independent data sets (GSE17306 and E-TABM-508), (P<0.01 and P<0.01, respectively). In three-dimensional co-culture system of MSCs from normal donors (n=6) with MM.1S cells for 2 weeks (GSE60423), miR-138 expression was increased in 4 out of 6 donors compared to MSCs cultured alone (P<0.05). MM-MSCs (n≥3) transfected in vitro with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation after 3-4 weeks compared to MSCs with scramble control oligonucleotides (P<0.01). Under in vitro two-dimensional co-culture conditions with MM cell lines, normal MSCs transfected with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation compared to MSCs with scramble control oligonucleotides (P<0.001). In an in vivo human xenograft MM model, treatment of anti-miR-138 significantly increased the number of OBCs in the endosteal (Lin-/CD45-) BM stromal fraction of MM bearing SCID-beige mice at day 28 compared to scramble control oligonucleotides (P<0.05).
Conclusions
These findings indicate that miR-138 plays an important role in impaired osteogenic differentiation in MSCs in MM. Inhibition of miR-138 promotes osteogenic differentiation of MSCs in MM and anti-miR-138 treatment holds the potential to prevent MM induced bone loss and lytic lesions. Additional studies are ongoing to further understand the connection between MM cells and MSCs mediated by miR-138.

Disclosures
Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Novartis: Honoraria; Noxxon: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding.