Growth Inhibition and Synergistic Induction of Apoptosis By Synthetic Mir-125b-5p Mimics and Myc-Targeting Agents in Human Myeloma Cell Lines

MicroRNAs (miRNAs), short non-coding RNAs which tune gene expression at post-transcriptional level, are recently emerging as key players in pathogenesis, progression and drug-resistance of multiple myeloma (MM). In this disease, they can act either with tumor-promoting or tumor-suppressing functions, depending on the nature of target mRNAs. Nowadays, effective strategies are available both to replace or to inhibit the expression of deregulated miRNAs, thus prompting the design of miRNA-based therapeutic strategies.

We have recently demonstrated that miR-125b has tumor suppressor activity in MM and that enforced expression of synthetic miR-125b-5p mimics induces significant anti-MM activity in vitro and in vivo by targeting cell addiction to IRF4/cMyc pro-survival signaling. Moreover, we uncovered a functional feedback loop between cMyc and miR-125b in MM cells, whereas cMyc directly suppresses miR-125b transcription which, in turn, negatively regulates cMyc expression by targeting IRF4 mRNA.

In the present study, we investigated the therapeutic potential of synthetic miR-125b-5p mimics combined with cMyc targeting agents, including the 10058-F4 small molecule inhibitor of cMyc-Max heterodimerization and the BET-bromodomain inhibitor JQ1, which is reported to inhibit cMyc transcription. At this aim, 3 MM cell lines (NCI-H929, SK-MM-1 and RPMI-8226) transfected with either miR-125b-5p mimics or scrambled oligonucleotides (miR-NC) were exposed to 10058-F4 (ranging from 10 to 100 μM) or JQ1 (ranging from 0,1 to 2μM) or DMSO. Effects on cell proliferation were then evaluated by CCK-8 assay at 24h, 48h and 72h time points, while the occurrence of apoptotic cell death was assessed by Annexin V flow-cytometry assay. Importantly, we found that enforced expression of miR-125b-5p mimics significantly and synergistically (synergistic index, SI >1) increases growth-inhibitory and pro-apoptotic activities of both 10058-F4 and JQ1. Similar results were observed in SK-MM-1 cells co-transfected with miR-125b-5p and cMyc siRNAs, while cMyc-defective U266 cells were not sensitized to either 10058-F4 nor JQ1 upon transfection with miR-125b-5p mimics. Furthermore, combinatorial treatments with JQ1 and miR-125b-5p mimics resulted in a stronger downregulation of cMyc protein, as compared to single molecules alone. Indeed, these results confirmed that impairment of cMyc activity/expression mediates the anti-MM synergistic effects between 10058-F4 or JQ1 and overexpression of miR-125b-5p by synthetic mimics.

In conclusion, our data demonstrate a cMyc-mediated synergistic anti-MM activity of synthetic miR-125b-5p mimics with 10058-F4 or JQ1 cMyc targeting agents, providing the rationale for a more advanced preclinical investigations for the design of early clinical trials.