Characterization of the biological and molecular relevance of nonoprotein in multiple myeloma

Introduction| Paraspeckles (PSs) are a new class of nuclear ribonucleoprotein organelles, whose relevance inmultiple myeloma (MM) pathogenesis has been well documented. PS assembly relies on the binding of thelncRNA NEAT1 with several PS proteins (PSPs), among which NONO. NONO is a multifunctional protein deregulated in many cancer types. Data concerning NONO involvementin MM are virtually absent. We recently reported its overexpression in CD138+ MM cells as compared tohealthy plasma cells, also highlighting its correlation with poor outcome in MM patients. Moreover, NONOexpression in human MM cell lines (HMCLs) is significantly higher than in other haematological non-HMCLs. Methods| Gymnotic delivery of specific LNA-gapmeR (g#NONO) was used to silence the expression ofNONO in a panel of 4 HMCLs. Dose-effect curves were obtained by Trypan Blue exclusion counts. Cellviability was assessed through CCK-8 assay. Clonogenic potential was evaluated by methylcellulose assay.Cell cycle phases distribution and apoptosis induction were investigated by FACS analysis. PSs integrity wasanalyzed by confocal microscopy analysis of combined NEAT1 RNA-FISH and NONO IF. WB was used tostudy PSPs levels. Results| All the tested HMCLs, albeit at different levels, showed high sensitivity to NONO silencing startingfrom the 3rd day of gapmeR exposure (median IC50 value= 6. 5μM). Growth curves retrieved from CCK-8assay confirmed a significant reduction in the number of viable cells in samples treated with sub-cytotoxicconcentration of g#NONO (5μM) until the 7th day of exposure. Modulation in the proliferative behavior ofNONO-depleted cells (NONO-KD) was confirmed by the 2-fold decreased number of colonies as comparedto controls. In line with FACS results showing an increase of the % of cells distributed in the subG0/G1phase of the cell cycle in NONO-KD samples (>10% for all HMCLs), we demonstrated apoptosis induction from the 4th day of gapmeR treatment (≈2-fold). From a molecular point of view, along with the significantdownregulation of NONO (silencing efficiency >80% for all the HMCLs), we also showed a significantreduction in the expression level of the essential PS scaffold NEAT1 (50-70%, depending on the HMCL).The reduction of both NONO and NEAT1 fluorescence intensity and co-localizing signals was alsoconfirmed by confocal microscopy analysis, demonstrating a strong PSs structure impairment. Of note WBanalysis showed a 2-6-fold increase in the expression levels of two other PSPs, SFPQ and PSPC1, suggestingthe presence of a compensatory mechanism between NONO and other PS elements. Conclusion| Our results clearly demonstrates that NONO silencing in HMCLs leads to PSs structureimpairment and results in an anti-proliferative and pro-apoptotic effect. Overall, a better elucidation ofNONO relevance in MM could highlight it as a therapeutically valuable target for the development of novelpharmacological approaches for this incurable disease.