Biological and molecular characterization of in silico identified putative inhibitors of paraspeckle assembly with potential anti-multiple myeloma activity

Introduction: Paraspeckles (PSs) are membraneless nuclear bodies, assembled through the interaction between the lncRNA NEAT1 and PS proteins (PSPs), among which NONO and SFPQ. We reported that NEAT1 is significantly overexpressed in plasma cells of multiple myeloma (MM) patients, and that its silencing leads to PSs disassembly, negatively regulating MM cells proliferation and viability. On these bases we hypothesized that PSs may represent a novel vulnerability in MM, which remains an incurable disease. Here, we assess in vitro the activity of the top 6 hit compounds resulting from an in silico screening of small molecules targeting interaction points of NONO::SFPQ, potentially leading to PSs loss, thus mimicking the effect of NEAT1 silencing. Methods: The biological activity of the 6 small molecules was evaluated in a panel of 6 MM cell lines (HMCLs), 4 haematological non-HMCLs and 6 healthy donors-derived PBMC samples. The IC50 values of the inhibitors were determined from the dose-response curves based on Trypan Blue exclusion counts and confirmed by CellTiter-Glo assay. Confocal microscopy of combined NEAT1 RNA-FISH and NONO IF was used to evaluate modification of PSs number upon treatment. Clonogenic potential modulation was investigated through methylcellulose assay. Cell cycle and apoptosis were investigated by FACS analysis. WB was used to assess PSPs levels. Transcriptome analysis was conducted by means of Clariom™ D arrays. Results: 2 of the 6 compounds led to a significant reduction of the number of viable cells upon treatment in all tested HMCLs but not in non-MM cells, highlighting a specific anti-MM activity. Confocal microscopy analysis showed a ≈45% decrease in the number of PSs/cell in treated HMCLs, validating the expected loss of PS integrity and the on-target activity of both molecules. In line with the reduced viability, cell cycle analysis of treated HMCLs revealed a significant 2-fold increase of the cellular population in the Sub-G0/G1 phase, and a downregulation of the % of cells distributed in the S phase. Treatment also caused a reduced clonogenic potential, with a median of 50 colonies for vehicle vs. 9 and 13 upon treatment with the 2 inhibitors. Flow cytometry analysis revealed a dose-dependent reduction of the percentage of viable cells and a concomitant increase of early and late apoptotic cells in treated HMCLs (2/5-fold increase, depending on the cell line), suggesting a pro-apoptotic effect exerted by both the molecules. Furthermore, WB results highlighted decreased core PSPs levels after treatment in all the HMCLs tested, but not in non-HMCLs. As expected from literature, transcriptomic analysis performed on MM cells of treated HMCLs revealed the negative modulation of pathways associated with tumorigenic processes and circadian rhythm regulation. Conclusions: 2 molecules targeting PSs essential interaction points exhibit anti-MM specific activity, suggesting PS targeting as possible new druggable vulnerability in MM.