In Vitro and In Vivo Activity of a Novel Locked Nucleic Acid (LNA)-Inhibitor-miR-221 against Multiple Myeloma Cells

Abstract

Background & AimThe miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.Methods In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.Results In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.ConclusionsLNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.

Knock-Down of IL-1Ra in Obese Mice Decreases Liver Inflammation and Improves Insulin Sensitivity

“…To determine whether decreasing systemic IL-1Ra levels in obesity has therapeutic potential, we treated HFD-fed obese mice with antisense oligonucleotides (ASOs) resulting in 50% reduction in the systemic concentration of IL-1Ra, to similar levels measured in age matched, lean normal chow fed mice. The extent of reduction in expression levels is comparable with may other published reports on the use of locked nucleic acid ASOs to inhibit gene expression [30]–[33]. We report for the first time that normalization of IL-1Ra by antisense-mediated knock-down of IL-1Ra expression improves hepatic insulin sensitivity and decreases body weight in diet induced obese mice.…”

A 13 mer LNA-i-miR-221 Inhibitor Restores Drug Sensitivity in Melphalan-Refractory Multiple Myeloma Cells

“…Following informed consent and Istitutional Ethical Comeettee approval, peripheral blood mononuclear cells (PBMCs) and primary CD138+ MM cells from BM aspirates of 3 MM patients, were isolated as previously described (38). LNA-i-miR-221 was designed and synthesized as previously described (35). Melphalan and Bortezomib were purchased from Sigma Aldrich and Selleck Chemicals, respectively.…”

“…Among miRNAs involved in development of drug-resistance, miR-221/222 plays a key role: inhibition of miR-221/222 has been reported to overcome resistance to cisplatin (28), tamoxifene (29), fulvestrant (30), temozolamide (31), tirosin Kinase Inhibitors (32), and TRAIL (33) in a variety of cancers. We recently provided the first evidence that silencing of miR-221/222 by specific inhibitors exerts anti-tumor activity in MM cells bearing t(4;14) translocations in vitro and in vivo (34), and that naked LNA-inhibitors of miR-221 (LNA-i-miR-221) are suitable for systemic delivery in animals (35). Here we investigated the role of miR-221/222 in melphalan-refractory MM, and demonstrate restoration of melphalan-sensitivity in refractory cells after exposure of MM cells to a novel 13 mer LNA-i-miR-221.…”

Small molecules targeting microRNA for cancer therapy: Promises and obstacles

“…55,56 Studies in mice using LNA antimiR have shown the feasibility and high efficiency of this approach. 57,58 Elmen and colleagues examined whether combining LNA antimiR with phosphorothioate modifications could improve delivery of the compounds and silence miR-122 in mice without requiring additional chemical modifications. 59 This research suggests that LNA antimiRs are able to effectively silence their targets at much lower doses than cholesterol-based oligonucleotides.…”