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

Purpose: The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma. Experimental Design: Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan. Results: miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiplemyeloma cell growth induced by inhibition ofmiR-221/ 222plusmelphalanwas associatedwith amarkedupregulation of pro-apoptotic BBC3/PUMAprotein, amiR-221/222 target, aswell as with modulation of drug influx–efflux transporters SLC7A5/ LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice. Conclusions: Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma. Clin Cancer Res; 1–12. 2015 AACR. Introduction Multiple myeloma is characterized by the abnormal proliferation of malignant plasma cells in the bone marrow (BM; refs.1, 2). Despite recent advances in multiple myeloma biology (3), preclinical models (4–6), and translation of novel agents which have markedly improved the outcome of multiple myeloma patients, the development of drug resistance remains an obstacle to long-term survival (7). Multiple myeloma commonly progresses to drug-refractory end-stage disease (8), and novel therapeutic strategies are urgently needed. For more than 30 years, melphalan has been the mainstay of multiplemyeloma treatment (9). Presently its therapeutic value is in younger patients who undergo high-dose melphalan prior to autologous stem cell transplantation, as well as in nontransplant candidates or elderly patients as a part of first-line combination regimens (10, 11). The use of melphalan combination regimens with new agents, such as bortezomib or lenalidomide, has significantly prolonged progression-free survival and overall survival; however, development of drug resistance leads to relapse of disease (12). Recently, a renewed scientific interest on melphalan is emerging, and major efforts have been devoted to delineate the mechanisms underlying primary or acquired melphalan resistance (13). These efforts have already led to the design of novel regimens to overcome melphalan resistance or to improve its antitumor activity (14). Currently, there is a growing interest for the therapeutic potential of strategies aimed to target miRNAs network. miRNAs are a class of short ncRNA that function as post-transcriptional gene regulators. miRNAs mainly act through complete or partial binding to 30-UTR of their mRNA targets, inducing either mRNA degradation or translational repression (15). By targeting driver genes involved in critical cellular pathways, miRNAs can function as oncogenes or tumor suppressor genes, playing a key role in tumorigenesis, as well as in cancer progression and aggressiveness Department of Experimental and Clinical Medicine, Magna Græcia University, Campus Salvatore Venuta, Catanzaro, Italy. Pathology Unit, "Giovanni Paolo II" Hospital, Lamezia Terme, Catanzaro, Italy. Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Boston, Massachusetts. VA Boston Healthcare System, West Roxbury, Boston, Massachusetts. Center for Biotechnology, Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, Pennsylvania. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Pierfrancesco Tassone, Magna Graecia University, Viale Europa, Catanzaro 88100, Italy. Phone: 39-0961-364-7029; Fax: 39-0961-3647341; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-15-0489 2015 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org OF1 Research. on April 15, 2017. © 2015 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Published OnlineFirst November 2, 2015; DOI: 10.1158/1078-0432.CCR-15-0489