The biological relevance of upregulated NONO protein in multiple myeloma through the regulation of cell cycle progression and drug treatment response

Background NONO is a multifunctional protein deregulated in many cancer types, involved in several cellular processes among which DNA damage repair and paraspeckles (PSs) assembly. NONO is overexpressed in CD138+ plasma cells isolated from multiple myeloma (MM) patients as compared to healthy donors, and its higher expression levels correlate with worst clinical outcomes in MM patients. Our preliminary results displayed that NONO-KD in 4 human myeloma cell lines (HMCLs) significantly decreases viability and proliferative potential and increases apoptosis. Aims Based on these premises, we aimed to evaluate NONO role in regulating cell cycle progression, and to explore NONO potential involvement in drug resistance mechanisms and its therapeutic implications in MM treatment. Methods Gymnotic delivery of a specific LNA-gapmeR (gNONO) was used to silence NONO expression. Cell cycle phase distribution was analyzed by FACS. Synchronization of the cell cycle was achieved using the SynchroSet Kit. IC50 values and synergistic effects were determined using Compusyn software. Results We silenced NONO in 3 different HMCLs and, after 72 hours, synchronized the cells to arrest cell cycle progression in the G1 phase. After a 16-hours release, we assessed cell distribution across the different cell cycle phases, focusing on the G1/S and G2/M transition phases. Our results showed a significant accumulation of cells in the subG0/G1 phase (mean10%), confirming DNA fragmentation and cell death, as observed in previous experiments. We also evidenced a significant decrease in the percentage of cells in the S phase (mean 20%), further supporting the altered proliferation rate. Additionally, a significant accumulation of cells in G1 phase was highlighted in NONO-KD HMCLs, suggesting that NONO plays a role in regulating the G1/S transition. At the second timepoint, a significant reduction in cells distributed in the G2/M transition phase (mean10%) was observed in NONO-KD HMCLs, indicating that NONO is also involved in the regulation of this phase transition. Importantly, preliminary experiments performed on NONO-KD healthy donors derived PBMCs did not result in any biologic effects. In addition to NONO impact on cell cycle, we demonstrated the synergistic effect of NONO silencing with clinically relevant drugs used for MM treatment, including proteasome inhibitors (BZB and Carfilzomib), alkylating agents (Melphalan), and PARP inhibitors (Olaparib). Finally, we also observed NONO involvement in drug resistance mechanisms. Specifically, two different NONO-KD BZB-resistant HMCLs showed increased sensitivity to bortezomib treatment compared to the corresponding SCR-treated HMCLs (mean IC50 value: BZB-resistant HMCLs=43nM; BZB-resistant NONO-KD HMCLs=31nM). Summary/Conclusion In conclusion, our results highlight the crucial role of NONO in regulating cell cycle progression, particularly in the G1/S and G2/M transitions, and its involvement in drug resistance mechanisms, supporting the need of a deeper molecular characterization. The lack of biological effects in NONO-KD PBMCs from healthy donors further supports the potential of NONO as a therapeutic target in MM.