Abstract 1077: Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential
Authors: Morelli Eugenio , Biamonte Lavinia , Federico Cinzia , Di Martino Maria Teresa , Amodio Nicola , Gallo Cantafio Maria Eugenia , Frandsen Niels M. , Pitari Maria Rita , Caracciolo Daniele , Gullà Annamaria , Rossi Marco , Tagliaferri Pierosandro , Tassone Pierfrancesco 
Published: July 2016

Abstract
There is emerging evidence that miR-17-92 plays a crucial role in c-Myc driven tumorigenesis of multiple myeloma (MM). We attempted to antagonize its full-oncogenic activity by targeting primary transcripts (pri-miR-17-92) with RNase H-triggering antisense oligonucleotides (LNA gapmeRs). Specifically, 7 different molecules were generated and screened for their ability to inhibit pri-miR-17-92 expression. The most effective molecule, henceforth named miR17-92-i-PT, was selected for furher investigation. As assessed by qRT-PCR, transfection of MM cells with miR17-92-i-PT resulted in downregulation of both pri-miR-17-92 and all 6 mature miRNA transcripts. Importantly, miR17-92-i-PT inhibited MM cell proliferation more effectively than inhibitors targeting each cluster's miRNA. Lack of relevant off-target effects was demonstrated by specifically-designed negative (LNA mixmeRs) and positive (not-phosphorothioated LNA gapmeRs) control oligos, and a dominant-negative genome-edited cell line by CRISPR/CAS9 technology is currently under development. Importantly, treatment of MM cells with naked miR17-92-i-PT resulted in miR-17-92 downregulation. Low micromolar concentrations of miR17-92-i-PT significantly affected survival of MM patient plasma cells (n = 14) and MM cell lines (n = 15), while the viability of CD138+ cells from MGUS patients (n = 3) or PBMCs from healthy donors (n = 3) was not impaired. Further, a synergistic in vitro activity with Bortezomib,Carfilzomib or Melphalan was demonstrated. Molecular perturbations in MM cells exposed to miR17-92-i-PT were firstly investigated by gene expression profiling (HTA 2.0, Affimetrix). Ingenuity pathway analysis (IPA) of differentially expressed genes highlighted alteration of relevant molecular functions, including “cell cycle”, “DNA replication, recombination, and repair”, and “cell death and survival”, which were validated by functional assays. Moreover, impairment of BRD4 activity was indicated by upstream regulator analysis (Zscore&lt;2). Indeed, Hexim1 -the endogenous antagonist of BRD4- was upregulated upon treatment with miR17-92-i-PT and is currently being validated as a direct target of miR-18a, miR-19a and miR-19b cluster members. Consistent with a BET bromodomain inhibitor-like activity, miR17-92-i-PT downregulated c-Myc, thus indicating a novel feedback loop between miR-17-92 and c-Myc. Finally, we found a significant in vivo anti-tumor activity of systemically delivered naked miR17-92-i-PT against human MM xenografts in SCID/NOD mice. Different animal models were utilized, including SCID-hu and mouse orthotopic models, besides classic subcutaneous xenografts. Lack of systemic toxicity was demonstrated both in mice (balb/c) and in monkeys (cynomolgus monkeys). Overall, our results provide the rational framework for development of miR17-92-i-PT-based therapies in MM.
Citation Format: Eugenio Morelli, Lavinia Biamonte, Cinzia Federico, Maria Teresa Di Martino, Nicola Amodio, Maria Eugenia Gallo Cantafio, Niels M. Frandsen, Maria Rita Pitari, Daniele Caracciolo, Annamaria Gullà, Marco Rossi, Pierosandro Tagliaferri, Pierfrancesco Tassone. Targeting oncogenic miR-17-92 primary transcripts by LNA gapmeRs in multiple myeloma: Molecular findings and therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1077.