Abstract 4426: A 13 mer LNA miR-221 inhibitor restores drug sensitivity in melphalan-refractory multiple myeloma cells
Authors: Gulla Annamaria , Di Martino Maria Teresa , Gallo Cantafio Maria Eugenia , Morelli Eugenio , Amodio Nicola , Botta Cirino , Pitari Maria Rita , Lio Santo Giovanni , Tagliaferri Pierosandro , Tassone Pierfrancesco 
Published: August 2015

Abstract
The onset of resistance to melphalan (MF) in Multiple Myeloma (MM) patients adversely affects disease outcome. Presently there is increasing evidence on the pivotal role of microRNA (miRNAs) in mediating drug-resistance. We here investigated the effect of an original LNA inhibitor of microRNA-221 (LNA-i-miR-221), in restoring sensitivity to MF in MM cells. LNA-i-miR-221 has been previously demonstrated as anti-tumor agent in preclinical models of MM. We studied the MF-sensitizing effect of LNA-i-miR-221 on MM cells in vitro and in vivo. MiR-221/222 expression inversely correlated with MF-sensitivity in a panel of 6 MM cell lines. Noteworthy, LNA-i-miR-221 overcame MF-resistance in vitro significantly enhancing apoptotic cell death as demonstrated by activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as by PARP cleavage and by histone H2AX (γ-H2AX) phosphorylation. Importantly, the synergistic activity of LNA-i-miR-221 with MF affected cell survival even in the presence of bone marrow stromal cells (BMSCs). Gene expression profiling (GEP) comparing treated and untreated MF-sensitive and -resistant MM cell lines was performed to investigate genes and pathways significantly affected by combinatory treatment. Based on the GEP findings, we confirmed by Western Blot that the growth impairment occurred together with up-regulation of pro-apoptotic BBC3/PUMA protein, a target of miR-221/222 which we validated by luciferase assay in our system. Moreover, GEP analysis showed modulation of drug-influx -efflux transporters such as L-type amino acid transporter 1 (LAT1) and multidrug resistance associated protein 1 (MRP1/ABCC1) which was confirmed by Western Blot analysis. Indeed, LNA-i-miR-222 led to a reduction of the Side Population in MF-resistant cells as assessed by Hoechst dye exclusion assay. Finally, treatment of human MM xenografts in SCID/NOD mice with LNA-i-miR-221 plus MF induced a marked antitumor effect, which again occurred together with BBC3/PUMA and MRP1/ABCC1 proteins modulation in retrieved tumors. Taken together, our findings provide proof-of-concept that LNA-i-miR-221 overcomes MF-resistance of MM cells, providing the rationale for clinical translation of LNA-i-miR-221/MF combination in MM patients.
Citation Format: Annamaria Gulla, Maria Teresa Di Martino, Maria Eugenia Gallo Cantafio, Eugenio Morelli, Nicola Amodio, Cirino Botta, Maria Rita Pitari, Santo Giovanni Lio, Pierosandro Tagliaferri, Pierfrancesco Tassone. A 13 mer LNA miR-221 inhibitor restores drug sensitivity in melphalan-refractory multiple myeloma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2015-4426