Abstract 4789: In vitro and vivo activity against multiple myeloma cells of a novel locked nucleic acid (LNA)-miR-221 inhibitor

Abstract

Abstract Upregulation of miR-221/222 has been found in several solid and hematologic malignancies, including multiple myeloma (MM), and is thought to have oncogenic potential and promote cell proliferation via down-regulation of p27 and/or p57, negative regulators of cell cycle progression. We previously demonstrated that upregulation of both miRNAs occurs in malignant plasma cells (PCs) from multiple myeloma (MM) patients belonging to TC2 and TC4 (traslocation/Cyclin) groups. A rising body of evidence suggests that silencing miRNAs with oncogenic potential could represent a novel approach for human cancer therapy. We previously demonstrated that silencing miR-221/222 exerts significant anti-MM activity and triggers canonical targets in vitro and in vivo (Di Martino et al. Oncotarget, 2013). In the aim to progress to clinical translation of our proof-of-principle findings, we here investigated the anti-tumor activity and the appropriateness for systemic delivery of 10 different, originally designed, miR-221/222 inhibitors which took advantage from locked nucleic acid (LNA) technology and phosphorothioate backbone for increasing the seed sequence binding stability and nuclease resistance. We found that electroporation of t(4;14) MM cells with a novel 13-mer miR-221 inhibitor, named LNA-i-miR-221, significantly inhibited growth and survival of MM cells in vitro. In treated cells, we detected knocking down of miR-221 together with increased levels of p27 mRNA and protein. Specific activity of this LNA-i-miR-221 against mature miR-221 was confirmed by the use of two 3’UTR reporter (luciferase renilla/firefly) constructs containing miR-221 target sites. These constructs were separately co-transfected either with miR-221/222 mimics or LNA-i-miR-221 into MM cells. As predicted, a reduced luciferase activity was detected in miR-221/222 mimics co-trasfected cells, while increase luciferase activity was measured in LNA-i-miR-221 co-transfected MM cells indicating an efficient and stable binding to the miRNA target sequence. Importantly, we evaluated the systemic delivery of the naked LNA-miR-221 inhibitor alone by intraperitoneal or intravenous injection route against MM xenografts in NOD.SCID mice. Significant anti-tumor activity was achieved after 2 weeks of treatment at similar extent by both injection routes. Retrieved tumors from treated animals showed efficient inhibition of miR-221 and increased levels of p27Kip1 in vivo. H&E staining and immunohystochemical analysis showed wide necrosis areas, reduced Ki67 and a significant increase of p27 cytoplasmic expression in retrieved tumors from LNA-i-miR-221-treated mice. No changes in mice behavior or organ toxicity were observed in treated mice. Taken together these findings support the rationale for development of this novel and highly efficient LNA-miR-221 inhibitor as a promising anti-MM drug in subsequent primate toxicology studies. Citation Format: Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Annamaria Gullà, Emanuela Altomare, Eugenio Morelli, Nicola Amodio, Emanuela Leone, Cirino Botta, Niels M. Frandsen, Pierosandro Tagliaferri, Pierfrancesco Tassone. In vitro and vivo activity against multiple myeloma cells of a novel locked nucleic acid (LNA)-miR-221 inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2014-4789