NONO is required for multiple myeloma cell lines growth through stabilisation of lncRNA NEAT1 and paraspeckle integrity

Background: Paraspeckles (PSs) are nuclear membraneless ribonucleoprotein organelles, with documented relevance in multiple myeloma (MM) pathogenesis. PSs assembly relies on the binding of the essential long non-coding RNA NEAT1 with several PSs proteins (PSPs), among which NONO. NONO is a multifunctional protein found to be deregulated in many cancer types. NONO is overexpressed in CD138+ MM cells as compared to healthy donors-derived plasma cells, and higher expression levels correlate with poor outcome in MM patients. However, mechanistic data on the involvement of NONO in MM pathogenesis are virtually absent. Aims: This study aims at investigating the biological relevance of NONO in MM. Methods: Gymnotic delivery of anti-NONO LNA-gapmeR (g#NONO) was used to silence the expression of NONO in a panel of 4 human MM cell lines (HMCLs). Dose-effect curves were obtained by Trypan Blue exclusion counts. Sensitivity to NONO silencing (NONO-KD) was evaluated through Compusyn software. Cell viability was assessed by CCK-8 assay. Clonogenic potential was evaluated by methylcellulose assay. Cell cycle phases distribution, EdU incorporation, and apoptosis modulation were investigated through flow cytometry. PSs structure integrity was analysed by confocal microscopy combining NEAT1 RNA-FISH and NONO IF. PSPs levels were investigated by WB. Results: All the tested HMCLs, albeit at different levels, showed sensitivity to NONO-KD starting from the 3rd day of g#NONO exposure (mean IC50 value: 8.2μM). Growth curves evaluation confirmed a significant reduction in the number of viable cells in samples treated with sub-cytotoxic concentration of g#NONO (5µM). Modulation of the proliferative behaviour of NONO-KD cells was confirmed by the significant decrease of the number of colonies as compared to controls (mean number of colonies in gSCR vs. gNONO calculated on 4 HMCLs: 22 vs. 5). In line with cytofluorimetric results showing an increase of ≈2-fold of the percentage of cells distributed in the subG0/G1 phase upon NONO-KD (mean percentage of cells in gSCR vs. gNONO: 10% vs. 19.6%), we demonstrated apoptosis induction starting from the 4th day from g#NONO delivery (mean percentage of apoptotic cells calculated on 4 HMCLs in gSCR vs. gNONO: 8.9% vs. 17.7%). Furthermore, a ≈15% fold decreased percentage of Edu+ cells in NONO-KD samples confirmed the reduced proliferative potential highlighted by both flow cytometry and clonogenic assay. RT-PCR results confirmed the significant downregulation of NONO mRNA (silencing efficiency > 80% for all HMCLs), also showing a significant reduction of the expression level of the lncRNA NEAT1 (mean 45%, range 25-70%), thus confirming the essential role of NONO in the stabilization of NEAT1 also in MM cells. Confocal microscopy analysis confirmed the decreased expression levels of NONO protein (mean: 80%, range: 45-90%) and of NEAT1 (mean 50%, range 15-75%), also demonstrating a significant downregulation of the area of co-localizing fluorescent signals (6-fold decrease) and a strong PSs structure impairment. Finally, WB results indicated a 2-6-fold increase in the expression levels of two PSPs, SFPQ and PSPC1. Of note, the finding that NONO-KD did not induce a modulation of SFPQ and PSPC1 mRNA levels led us to exclude its involvement in the transcriptional regulation of both targets. Summary/Conclusion: NONO-KD results in growth arrest and apoptosis in MM cell lines, and correlates with NEAT1 downregulation and PS disassembly. Altogether, these data highlight NONO and PSs as a novel vulnerability in MM and warrant further investigation on the oncogenic role of NONO.