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

Introduction Multiple myeloma (MM) is the second most common haematological malignancy, being characterized by abnormal proliferation of plasma cells (PCs) predominantly within the bone marrow. A new cellular organelle named paraspeckle (PS) has been recently associated with several biological processes, including DNA damage systems regulation and stress response. The assembly of these nuclear bodies requires the presence of the essential long noncoding RNA (lncRNA) NEAT1 and seven PS proteins (PSPs), among which NONO and SFPQ. The relevance of PSs in MM pathogenesis has been well documented. Indeed, the crucial scaffold NEAT1 has been found to be significantly overexpressed in PCs of MM patients with respect to the normal counterpart and its silencing has been shown to affect malignant PCs proliferation and viability, triggering anti-tumour activity, both in vitro and in vivo. On the contrary, NEAT1 transactivation induced by stressful conditions has been associated with increased PSPs levels and enhanced MM cells viability, suggesting a pro-survival and anti-apoptotic role of PSs in MM. Similarly, we recently reported higher NONO expression in primary CD138+ MM cells, correlating with poorer OS and PFS. Aim Because of the genetic complexity of MM cells, it is still difficult to imagine a universal therapeutic approach able to effectively target all the malignant subclones. As a result, patients inevitably relapse and become resistant to subsequent lines of therapies, highlighting the urgent clinical need to identify novel druggable vulnerabilities. Our hypothesis is that PSs could represent a novel specific vulnerability in MM across genetic subgroups. However, clinical translation of lncRNA targeting may be problematic. On the contrary, protein components of PSs are more amenable to drug design. Therefore, we set out to perform an in silico screening of small molecules targeting crucial interaction points between NEAT1, NONO, and SFPQ and supposed to disrupt PSs structure, thus, at least in part, mimicking the NEAT1 silencing strategy. The aim of this study is to assess in vitro the on-target activity of the compounds representing the top 6 hits of the screening. Materials and 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. Dose-effect curves for the determination of IC50 values were obtained by Trypan Blue exclusion cell counts and CellTiter-Glo assay. Alteration of PSs integrity was evaluated by NEAT1 RNA-FISH and NONO IF by confocal microscopy. Clonogenic potential was evaluated through methylcellulose assay. Cell cycle phases modulation and apoptosis induction were investigated by FACS analysis. The levels of PSPs were assessed by means of WB analysis and IF technique. Transcriptome analysis was performed by means of Clariom™ D arrays on Affymetrix platform. Results 2 of the 6 compounds demonstrated a significant biological activity after 5 days of treatment in all the tested HMCLs but not in non-MM cells and healthy donors-derived PBMCs (≈75% vs. 25% of affected cellular fraction, respectively), confirming a specific anti-MM effect. Confocal microscopy experiments showed a ≈45% decrease in the number of PSs per cell in treated HMCLs, validating the expected loss of PS integrity and the on-target activity of both molecules. In line with biological data, cell cycle analysis revealed a perturbation of the cell distribution upon small compounds treatment of HMCLs. Indeed, HMCLs displayed a significant increase of the cellular population distributed in the Sub-G0/G1 phase, and a downregulation of the percentage of cells in the S phase. FACS data were in agreement with clonogenic potential results showing a median of 50 colonies in vehicle-treated samples vs. 9 and 13 upon treatment with the 2 inhibitors. Flow cytometry analysis revealed a dose-dependent increase of apoptotic cells in treated HMCLs (2/5-fold increase, depending on the HMCL tested), suggesting a pro-apoptotic effect of both inhibitors on HMCLs. Additionally, we highlighted a decrease of key and core-localizing PSPs levels after treatment, among which NONO and SFPQ. Remarkably, in line with previous findings highlighting a MM-specific effect of NEAT1 in a panel of HMCLs, none of the above-mentioned results were observed in non-MM cells, confirming the specificity of both inhibitors for HMCLs. Finally, preliminary transcriptomic analysis revealed the downregulation of several pathways associated with tumorigenic processes. Discussion and Conclusions In the present study, we assessed the biological and molecular activity of the top 6 candidates resulted from an in silico screening of commercially available drug-like small molecules potentially affecting PSs structure integrity. We identified 2 molecules that, by specifically targeting NEAT1 essential protein interactors, exert an anti-MM specific activity resulting in PS structural core impairment and apoptosis induction. These promising preliminary results emphasize the need of a better comprehension of the mechanisms underlying the activity of the novel identified small compounds and suggest PS targeting could represent a possible new druggable vulnerability in MM. As a matter of fact, PSs targeting may have a great translational relevance since cytogenetically different cell lines resulted to be responsive to the selected inhibitors. Additionally, stressful conditions promoting PSs assembly are often associated with more aggressive tumor stages and chemoresistance mechanisms, raising the possibility to use this innovative therapeutic strategy in all the subsets of MM patients.