LNA -Gapmer Antisense Oligonucleotides Targeting the Oncogenic Lncrna MALAT1 Trigger Anti-Multiple Myeloma Activity By Proteasome Blockade

Dysregulation of non-coding RNAs is emerging as a common feature of human cancer. While short non-coding RNAs have been extensively investigated in multiple myeloma (MM), the biological role and the therapeutic significance of aberrantly expressed long non-coding RNAs (lncRNAs) are yet undetermined. A proprietary custom annotation pipeline of microarray data on lncRNA expression in different plasma cell (PC) dyscrasias, including 20 MGUS, 33 smoldering MM, 170 MM, 36 PC leukemia patients and 9 healthy donors, revealed high expression of MALAT1 in MM PCs. On this finding, we investigated the functional significance of MALAT1 by using novel 16-mer LNA gapmeR synthetic ASOs, that trigger selective RNAse-H dependent degradation of MALAT1. LNA-gapmeR ASOs targeting MALAT1 inhibited proliferation and clonogenicity of MM cell lines (n=7) and patient PCs (n=5), triggering ER-stress response and apoptosis, even in the presence of bone marrow stromal cells, while sparing healthy peripheral blood mononuclear cells. Conversely, MALAT1-enforced expression by lentiviral vectors enhanced cell proliferation and migration, reverted the effect of anti-MALAT1 ASOs and activated major oncogenic signal transduction pathways, such as AKT and MAPK. In vivo, i.p. administration of anti-MALAT1 gapmeRs inhibited the growth of human MM xenografts, with no evidence of organ toxicity. Microarray gene expression profiling indicated that MALAT1 depletion downregulated genes encoding for 20S proteasome β-subunits, as evidenced by gene set enrichment analysis (GSEA).Down-modulation of PSMβ2/4/5/7 mRNA and protein by anti-MALAT1 ASOs was confirmed by qPCR and Western blot respectively, and was associated to reduced trypsine-, chymotrypsine- and caspase-like proteasome activities and to the accumulation of polyubiquitylated proteins. Mechanistically, we demonstrated that proteasome inhibition upon MALAT1 knock-down was due to downmodulation of the major transcriptional activators of proteasome gene expression NRF1 and NRF2. At the same time, NRF1 was found to bind to and transactivate MALAT1 promoter, as demonstrated by chromatin immunoprecipitation and reporter assays, thus establishing a positive feedback-loop with MALAT1. Intriguingly, this loop was overactivated in bortezomib-resistant MM cells. Collectively, our findings highlight an unprecedented role of the lncRNA MALAT1 in the regulation of the proteasome machinery, and provide the first preclinical rationale for using ASO therapeutics targeting dysregulated lncRNAs for MM treatment.